Drug Metabolism

A major problem in all drug discovery programs is to discover compounds with good pharmacokinetics. Although it is possible to examine the metabolism of the drug in animals, it has often been difficult to predict what would happen in man. The obvious implications of drug metabolism are an effect on half-life in vivo and the production of toxic metabolic products. In seeking to establish an effective dose for a new drug, the clinician needs to know what ranges of abilities humans will have to metabolize the drug and what effect the drug will have on the metabolizing enzymes. Failure to metabolize the drug may lead to overdose, whereas rapid metabolism could lead to lack of clinical benefit. Equally, inhibition of the metabolism of another drug could cause problems in a patient receiving several medications.

A large proportion of the metabolizing enzymes are members of the P450 superfamily28 and a large number of these genes have now been cloned and their metabolic potential determined. Increasingly, the enzymes are being expressed in microbial systems, e.g. yeast, where their ability to metabolize the drug can be evaluated. In a few years, it would not be surprising if all new drugs were “typed” for their complete P450 metabolism profile.

Equally, their metabolic products can be identified and their biological activity and toxicity determined. An additional application is likely to be the P450 genotyping of patients. As “poor metabolizers” become recognized in the population, the problem is often found to be mutations in one or more of their P450 genes. Once identified, such mutations are easily screened for and it is entirely likely that some degree of P450 “profiling” will take place for patients in the future. Armed with knowledge on the metabolic fate of new drugs, the physician will then be able to prescribe the best drug for an individual depending on their P450 profile. This individualization of drug therapy based on genetic information is known as pharmacogenomics. There is a massive effort currently underway to identify and characterize polymorphisms in a wide variety of genes, including drug receptors and effectors, in addition to drug metabolizing enzymes. It is hoped that the correlation of these polymorphisms with clinical outcomes and drug effects across a population will allow for the

prediction of the safety, efficacy and toxicity of both established drugs and new drugs in development and, thereby, a reduction in the size and expense of clinical trials.

Receptors

Molecular recognition refers to the affinity of biological molecules to their receptor and is basis for biological processes. The physical basis for association between drug and their receptors (affinity) is important for the drug action, hence for drug design. The drug-receptor interactions (molecular recognitions) are key for the biological responses. The molecular interaction pattern (affinity) determines the molecular function as agonist and antagonist.

The macromolecular protein entities (sensing elements) interacting with the endogenous chemical messengers (e.g., acetylcholine, histamine) as well as exogenous molecules (e.g., paracetamol) are known as receptors. The term receptors include cellular macromolecules such as regulatory proteins (enzymes) trans-membrane glycoproteins (ion channel), mucopolysaccharides and nucleic acids. The tissue (functional elements) is composed of the lipid layers sandwiched between protein. These proteins adopt different conformations in bio-phase and acts as a receptor. The molecular interaction between the endogenous compounds and receptor (target) coordinates the cellular functions. The receptors with no defined pharmacological activity is known as orphan receptor.

Receptor Types

The receptor mediated cellular communication may produce rapid (synaptic transmission), intermediate time scale (adrenergic action) and prolonged (thyroid action) effects. The functional variation is mainly dependent on the receptor molecular structure. Based on these mechanisms, receptors are grouped into four

sub-families.

1. G-protein coupled receptors (GPCRs)

2. Ion channels

3. Transmembrane catalytic receptors

4. Nuclear receptors

G-protein Coupled Receptors (GPCRs) G-protein coupled receptors (GPCR) are large membrane-bound proteins, which are known as metabotropic/7-transmembrane (7-TM) receptors. These receptors transduce their bio-chemical signal through activating guanine nucleotide binding proteins (G-protein). G-proteins are constituted by á, â and ã subunits (hetero trimeric). The subunits are anchored to the receptor through prenylation reaction. The subunit of trimer has binding affinity for guanine nucleotides (GTP and GDP). In resting state (before bonding to receptor), it has affinity for GDP and hold in it.

GPCR mediated adenylyl cyclase (AC) activation and resulting biological contraction.

The receptor extracellular region has amino terminus and loops. This region is called as ligand binding site. The carboxylic end of receptor exists intracellularly, which has binding sites for G-proteins. The ligand binding activates the receptor (induces conformational changes). The binding of ligand to the extracellular ligand binding site facilitates the recognition of G-protein binding site by G-protein. G-protein (trimer) binds to the receptor in between the sixth and seventh trans-membrane region (serine and threonine rich region).

The conformational changes on trimer upon binding to the receptor triggers the dissociation of GDP molecule from á-subunit and replacement of GTP (GDPGTP exchange). The GTP binding to the trimer (abg) favours the dissociation of trimer into monomeric unit bound with GTP and the dimer (bg). The released a_GTP monomer binds to the particular target enzyme and produces activation or inhibition of the target function. The a-subunit has GTP hydrolysing potential and converts a-subunit bound GTP into GDP through dephosphorylation reaction. The a-subunit bound with GDP molecules reunion with bg dimer. The cycle repeats and elicits the biological response (Fig.).

GPCRq mediated phospholipase-C (PLC) activation and resulting biological contraction.

These receptors are grouped into four families.

1. GPCRs: The a_GTP binding to the enzyme (target) facilitates the second messenger synthesis. The activated adenylyl cyclase (AC) generates cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and activated guanylyl cyclase (GC) generates cyclic guanosine monophosphate (cGTP) from guanosine triphosphate (GTP). The resulting cAMP and cGMP activates the intracellular protein kinase and favours phosphorylation mediated cellular function.

2. GPCRi: The aGTP binding to the enzyme (target) inhibits the functions of AC and GC. This binding stabilizes the conformation of the enzyme and no catalysis for the generation of cAMP and cGMP.

3. GPCRq: The aGTP binding activates the enzyme phospholipase-C (PLC). The activated enzyme catalyzes the generation of second messengers phosphatidylinositol (PI2) and diacylglycerol (DAG) from inositol triphosphate (IP3) (Fig.).

4. GPCR12 This receptor is expressed in renal convoluted tubules (GPR12):and facilitates the Na+/H+ exchange function.

Ion Channels

Ion channels undergo conformational switch with reference to the physical and chemical changes (electro chemical gradient) in their environment. The conformational changes decides the transport of ions through the channels (Fig.). The different types of ion channels present in the bio-phase are as follows

1. Ligand (transmitter)-Gated Ion Channels (LGICs)

2. Second-messenger-Gated Ion Channels (SGICs)

3. Voltage-Gated Ion Channels (VGICs)

Structural depiction for the ion transport through ion channels.

1. Ligand-Gated Ion Channels (LGICs): It contains four membrane-spanning domains. These channels are also known as ionotropic/metabotropic receptor. The neurotransmitters namely, gamma-amino butyric acid (GABA), glutamate, N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl isoxazole-4propionic acid (AMPA), glycine and serotonin are the substrates for the LGICs. The binding of selective ligands to the ion channels favours the opening of ion channel and influx of particular ions.

2. Second-messenger-Gated Ion Channels (SGICs): The second messengers namely cAMP, cGMP, IP3 and DAG influences the opening and closing of calcium (Ca²⁺) and potassium (K⁺) channels.

3. Voltage-Gated Ion Channels (VGICs): The influx of sodium (Na⁺), calcium (Ca²⁺) and efflux of potassium (K⁺) ions are responsible for the generation of depolarization and action potential. The electro chemical gradient of intra and extracellular regions facilitates the movement of selective ions through the ion channels. The channels permits the movement of ions. The brief depolarization introduced by the glutamate receptors favours the opening of NMDA receptor and produce long depolarization.

Drugs: Local anaesthetics, anti-convulsants and calcium channel blockers.

Changes in membrane (chemical) potential regulates the opening of the channels. Voltage gated ion channels open, when cell membrane is depolarized. Depolarization caused by the opening of Na⁺ influx initiates the opening Ca²⁺ channels. This kind of channel opening is short-lasting even if the depolarization is maintained. It produces influx of Ca²⁺ and initiates oxidative phosphorylation.

Transmembrane Catalytic Receptors / Enzyme-Coupled Receptors

These receptors contain three domains namely, extracellular ligand binding domain, a single membrane-spanning domain and intracellular effector domain with enzymatic activity. Peptide hormones (insulin, leptin), epidermal growth factors, platelet-derived growth factors and arterial natriuretic factors activate the functions of their receptors. The ligand binding to the receptor promotes the receptor dimerization (association of cellular effector domain) and permits the auto phosphorylation of receptor tyrosine residues. The phosphorylated tyrosine residues are binding sites for intracellular proteins (adapter proteins).

(a) The activated catalytic receptors results in cellular functions such as opening of ion channels and gene expression.

(b) In case of enzyme-coupled receptors, signal transducers and activators of transcription (STATs) bonds to the phosphorylated receptors. The enzyme Janus Kinase (JAK) phosphoralytes the STAT and translocates into nucleus (Fig.). This initiates the gene transcription, which is essential for the cell growth, cell division and cell differentiation.

Biochemical pathway for transmembrane catalytic receptor.

Nuclear Receptors

Nuclear receptors are composed of a three functional domains namely modulator protein binding domain (heat-shock protein-90, HSP-90), DNA binding domain and ligand binding domain. The thyroid receptor (TR), Vitamin D receptor (VDR), retinoic acid receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) belongs to this nuclear receptor category. The natural ligands include nitric oxide, steroid hormones, thyroid hormones, retinoic acid and vitamin D. The lipophilic nuclear receptor ligands readily cross the cell membrane. The binding of ligands to the nuclear receptor favours the dissociation of heat-shock protein-90 (HSP-90). This ligand bound receptor diffuses into the nucleus and binds with DNA response element. This promotes the gene transcription (RNA polymerase function) and biological functions (Fig.).

Biochemical pathway for nuclear receptor.

Receptor Theories

The difference in the biological potential difference of molecules can be explained through the different drug-receptor theories (hypothesis). These theories explain about the pattern of drug-receptor binding. Paul Ehrlich proposed side-chain theory for the molecular (dye) interactions. The theory states that all cells have side chains for binding with molecules (with specific functional groups). The nutrient-capturing structures (side chains) of cells bind to molecule (endogenous and exogenous) through specific groups and elicit characteristic biological response. The cell extends more side chains after the initial binding with fewer molecules. This statement is valid for the release of antibodies (side chains) with respect to the antigens. These side chains are later identified as amino acids and are named as receptors. This hypothesis is primer for drug-receptor theories.

The different drug receptor theories are listed below and explained in the following section.

1. Occupancy theory

2. Rate theory

3. Induced fit theory

4. Macromolecular-perturbation theory

5. Activation-aggregation theory

Occupancy Theory

The occupancy theory states that the receptor mediated biological response is directly proportional to the number of receptors occupied. A relationship between drug concentration and the proportion of receptors occupied was explained through occupancy theory. The earlier hypothesis of Clark and Gaddum were basis for the evolution of occupancy theory.

Clark (1933), proposed that only a small population of the receptor (affected tissue) could be occupied by the drug.

Gaddum (1937) explained the binding of two mutually exclusive drugs with opposite functions at the same receptor. This is known as drug antagonism theory.

A tissue response is dependent on the fraction of specific receptors occupied by the drug. In many instances a very small portion of tissues (receptors) are occupied by the drug. The maximum response results when more receptors are occupied by a drug.

Human organism is made up of 3 × 10²³ molecules.

1 mole (molecular weight = 100 Dalton) of drug contains 6.023 × 10²³ molecules. 1 mg of drug (molecular weight = 100 Dalton) contains

Therefore each drug molecule would act on 10⁶ molecules of the receptor (3 × 10²³ / 3 × 10^–19).

The law of mass action describes the receptor occupation. The interaction between a drug (D) and a receptor (R) is responsible for the drug effect (E), which can be expressed as

k_a = rate constant for association; k_d= rate constant for dissociation;

D = drug; R = receptor;

DR = drug-receptor complex; E = biological effect

The dissociation of drug from the complex terminates the therapeutic effect.

The structural features of the molecules decide their molecular affinity towards the receptor and initiates pharmacological function.

Structurally specific drugs: The structural difference (even minor) among the analogues alters their biological effects (activity and potency). These drugs are called as structurally specific drugs. The natural alkaloid morphine has analgesic 

potential. But the N-methylmorphine produces skeletal muscle relaxant effect. This reflects the influence of chemical structure in their receptor specificity.

Structurally non-specific drugs: The physicochemical properties (not structural) of drugs are also determinant factor for their pharmacological function. These drugs are called as structurally non-specific drugs. Gaseous general anesthetics (e.g., halohthane and isoflurane) and antacids (e.g., aluminium hydroxide and calcium carbonate) are examples. The halothane and isoflurane accumulate (not by binding with receptor) in the membranes and elicit their general anesthetic function. The antacids produce their effect by acid neutralization function.

Schild ‘Dose Ratio’ Concept The number of drug-receptor complexes responsible for the observed biological response is concentration dependent.

Ariens-Stephenson Concept: The occupancy theory provides qualitative description of drug-receptor interaction and lacks quantitative descriptions. The independent observation of Ariens and Stephenson explains that the ability of the

molecules to induce conformational change in the receptor is responsible for the activity.

• Ariens (1954) introduced the term intrinsic activity to explain about the strength of drug response.

• Stephenson (1956) introduced to the term efficacy to explain about the strength of drug affinity with receptor.

Agonist and antagonist molecules possess complementary structural features (desirable affinity) for the receptor. The agonist molecules upon binding with receptor produce stimulant action, whereas the antagonist produces no biological response. The agonist molecules have capacity to induce the conformational change in the receptor upon binding, whereas antagonists do not induce the conformational change. The binding affinity difference among the agonists is reason for their biological potential variation.

Chaerniere Theory: Rocha Silva (1969) stated that the agonists and antagonists establish reversible binding on the receptor. The theory proposes that pharmacological receptor has two sites for binding with drugs.

(a) Specific site (critical): The receptor sites which interacts with pharmacophore group.

(b) Non-specific site (non-critical): The receptor sites which interact to the non-polar groups of antagonists.

The antagonist competes with agonist and the interactions are stronger (through hydrophobic, van der Waals and charge transfer interactions). According to the hypothesis, the agonist (even in high concentrations) cannot displace the antagonist due to their stronger interactions.

Rate Theory

Paton (1961) suggested that the rate of association between the drug and receptor as an important factor in the drug response. The rate of association and dissociation of drug-receptor complex rather than number of receptors occupied is responsible for the observed pharmacological response. The therapeutic effects of molecules are very much influenced by their rate of association and dissociation. The activation of receptor is proportional to the rate of drug-receptor association in unit time. The drug-receptor complex strength has very less influence on its biological effect. (a) Agonists: In case of agonists, the rate of dissociation is relatively faster than the rate of association (high dissociation rate). (b) Antagonists: In case of antagonists, the rate of association is faster than the rate of dissociation (low dissociation rate). The drug dissociation from the drug-receptor complex can be estimated through dissociation constant.

K_D = dissociation constant; D_f = concentration of free drug; R_f = concentration of free receptor; DR = drug-receptor complex;

Induced Fit Theory

The active binding site of the receptor is more flexible and can be altered by the interactions of ligands. The binding of ligand to polymeric protein induces conformational changes in one of the subunit. In several cases, the binding of one molecular unit to the receptor binding pocket facilitates the binding of another group by altering the receptor shape.

del Castillo and Katz (1957) suggested that the drug exhibits its activity through two step mechanism. In the first step, the drug generates intermediate drug-receptor conformation and in the next step, it converts into active conformation. This active conformation determines the drug response.

A receptor will undergo conformational change as the ligand approaches it. In the induced conformation, receptors will expose catalytic groups for binding to drug. Thus, the receptor will initiate the biological response and returns to its original conformation once the drug is released.

Macromolecular Perturbation Theory

Belleau (1964) described about the conformational adoptability of the muscarinic (cholinergic) receptor. This hypothesis is known as macromolecular perturbation theory. The molecular interaction between the drug and macromolecule produce specific conformational perturbation (SCP) and non-specific conformational perturbation (NSCP) in receptors.

1. Specific conformational perturbation (SCP): The molecular perturbation occurs through the van der Waals interaction. The agonists with intrinsic activity favor the SCP.

2. Non-specific conformational perturbation (NSCP): It involves hydrophobic interactions. The molecules with no intrinsic activity (antagonists) generate NSCP. A partial agonist will induce both SCP and NSCP.

Activation-Aggregation Theory

Stephenson (1956) proposed that the drug-receptor interaction can be viewed as graded activation and the two-state model.

(a) Graded activation: Agonist molecules induce different conformations in their receptor; hence produce different degree (strength) of biological function.

Receptor conformational perturbation : specific conformational perturbation (SCP) induced by agonist and non-specific conformational perturbation (NSCP) induced by antagonist.

Activation-aggregation theory: Illustration for graded activation.

(b) Two-state model: The receptor exhibits two-state namely active conformation (R*) and resting state conformation (R). The receptors exhibit dynamic equilibrium between active and inactive form even in resting state. The agonist molecules bind to active form of the receptor and produce biological response. But the antagonist binds to inactive form of the receptor and antagonizes (blocks) biological function. The partial agonists preferentially bind to active form but also bind to inactive form; hence, produces mixed response (less biological response).

Activation-aggregation theory: Illustration for two-state model.

Receptor Promiscuity

Molecules (agonists, inverse agonists, antagonists) exhibit entirely different biological response in different bio-phase, even though the binding receptor is same. This phenomenon is called as receptor promiscuity and can be exemplified by the action of H₁ and H₂ blockers on histamine receptors in different locations of the biological system.

Receptor Topology

The distances between two consecutive turns of á-helix of proteins is 5.38 Å. The adjacent two peptide bonds are separated by the distance of 3.61 Å. The chemical groups of several drugs keep one or another of these distances or multiple of them. 1. In case of local anesthetics (procaine), adrenergic blockers (piperoxan), cholinergic agents (acetylcholine), spasmolytics (adiphine) and antihistamines (chlorpheniramine) the bond distance between X and N atoms are 5.5 Å. 2. The distance between acetyl choline nitrogen atom and carbonyl group is 5.16 Å. In cholinergic (carbachol) and anticholinergic (ipratropium bromide) drugs the distance between carbonyl group and nitrogen group is 7.2 Å (multiple of 3.61 Å). Three-Dimensional Arrangement In the chiral environment, the enantiomers of a drug behave differently and display different chemical and pharmacologic behavior. The R-isomer of the drug will not bind in the similar pattern, compared with the corresponding S-isomer. In the illustration given below, the groups (B, C and D) present in active enantiomer (eutomer) interact with their respective binding sites (b, c and d) and produces beneficial effect (Fig. 2.12). The difference in three dimensional (3D) arrangement of groups present in the inactive enantiomer (distomer) offers no binding or weak binding and results in no therapeutic effect.

Illustration for the three-dimensional (3D) attachment for drug to the receptor.

Three point attachment: In asymmetric molecule of R-(-) epinephrine, the quaternary nitrogen, aromatic group and alcoholic hydroxyl groups (â-hydroxyl group) are involved in the receptor binding. The p-and m-hydroxyl groups (catechol group) determines the intensity of attachment (binding). The alcoholic hydroxyl group of S-(+) epinephrine is in opposite orientation, compared with R-(-) epinephrine (Fig. 2.13). The correct orientation of alcoholic hydroxyl is important for the adrenergic activity.

Three point attachment of epinephrine to the receptor.

Xenobiotics

Xenobiotics are chemicals or compounds that are foreign to a biologic system. Exposure to xenobiotics may occur via the air, water, diet, bedding, caging, and/or equipment, or may be pharmacologic agents intentionally introduced as part of the routine conditioning or experimental procedure. The effect or toxicity of a xenobiotic is based on the dose and disposition. Absorption, distribution, biotransformation, and excretion all affect xenobiotic disposition. In addition, host barriers, i.e., the skin, lungs, and alimentary tract, and the physical and chemical composition of the xenobiotic also affect its toxicity. The xenobiotic or its metabolites may cause physiologic alterations in the animal and thus affect the outcome of the experiment by altering immune function, and by acting as a mutagen and/or a teratogen. Examples include aflatoxins; phytoestrogens; endocrine disruptors; heavy metals such as lead, mercury, and cadmium; organochlorine insecticides; and commonly administered anesthetic agents.

Inter-animal or inter-colony response variability to xenobiotics may be attributable to differences in the intestinal microbiome of individual animals or colonies as microbiotia may affect chemical metabolism by altering biotransforming enzymatic activity, enterohepatic circulation, absorption, direct chemical activation, the bioavailability of antioxidants and environmental chemicals from feed, as well as gut motility, epigenetic mechanisms, as well as genetic polymorphisms, gender and age.

Substances and sources of xenobiotics

Xenobiotics include plant components, pharmaceutical drugs, pesticides, cosmetic products, added food flavors, fragrances, etc. At higher concentrations in environmental matrices, naturally occurring substances (endobiotics) may also be considered as xenobiotics. Xenobiotics are categorized as pesticides, pharmaceutical chemicals, personal care products, illicit narcotic drugs/substances, industrial/commercial goods, and nuclear waste and can be present in various environmental matrices. Xenobiotics are used by people and directly or indirectly penetrate in the different environmental matrices generating various metabolites and secondary products (some are even more toxic). Finally, plants, algae, and aquatic organisms take up xenobiotics leading to bioaccumulation, further causing biomagnification. One of the main obstacles to the sustainable water availability in urban systems is the presence of xenobiotics in aquatic ecosystems. In addition to the greater diversity of enzymes present in complex and varied community of microflora, the chemical distinctions between human and microbial transformations of ingested chemicals result from different selection processes that cause these activities. While host metabolism aids in the body’s elimination of xenobiotics, microbial changes to these substances and their human metabolites frequently promote microbial development by supplying nutrients or producing energy.

The amount of xenobiotics found in environmental matrices can be varied from ng/L to g/L. In both humans and animals, long-term chronic exposure to even little doses of xenobiotics may cause toxic, mutagenic, carcinogenic, or teratogenic effects. These compounds may block the enzyme’s active site or affect it in an allosteric way. Some xenobiotics including chlordecone, dichlorodiphenyltrichloroethane (DDT), and dichlorodiphenyldichloroethylene (DDE) show tendency to bioaccumulate, and even their low-level chronic exposures can potentially have an adverse impact on cell signaling pathways. In order to create safer molecules for use in human environment, knowledge of enhanced molecular designs may be useful along with mechanism of xenobiotics’ action.

Biotransformation

Biotransformation is the metabolic conversion of endogenous and xenobiotic chemicals to more water-soluble compounds. Generally, the physical properties of a xenobiotic are changed from those favoring absorption (lipophilicity) to those favoring excretion in urine or feces (hydrophilicity). An exception to this general rule is the elimination of volatile compounds by exhalation.

Chemical modification of a xenobiotic by biotransformation may alter its biological effects. Some drugs undergo biotransformation to active metabolites that exert their pharmacodynamic or toxic effect. In most cases, however, biotransformation terminates the pharmacologic effects of a drug and lessens the toxicity of xenobiotics. Enzymes catalyzing biotransformation reactions often determine the intensity and duration of action of drugs and play a key role in chemical toxicity and chemical tumorigenesis.

The metabolism of xenobiotics involves two sequential steps known as phase I and phase II reactions.

Sequential steps of drug biotransformation. After uptake by the cell, a phenyl ring of a xenobiotic undergoes first a functionalization reaction (oxidation, phase I). The hydroxyl metabolite is then conjugated by addition of a sulfate group (phase II), before being exported from the cell by transporters (phase III) and excreted. P-450, cytochromes P-450; ST, sulfotransferases.

During phase I, a functionalization reaction of the xenobiotic is achieved. New polar groups such as CO2H, OH or NH2 are introduced or unveiled from pre-existing functions through oxidative, reductive or hydrolytic reactions. The polar group created serves then as an anchor point for the second metabolic step. The phase II reactions, known as conjugation reactions, link an endogenous, generally hydrophilic moiety, either to the original drug (if polar functions are already present) or to the phase I metabolite. Common endogenous groups are glucuronic acid, various amino acids, the tripeptide glutathione, or sulfate. The water soluble conjugate is finally eliminated from the cell by transport proteins (organic anion transporters, multidrug resistance associated proteins), and finally excreted via the renal or the bile route. This transport step is considered as phase III of drug metabolism.

The global result of phase I and II transformations should normally be the inactivation and detoxication of the xenobiotic. However, innumerable examples exist of metabolic activation. Phase I metabolite will possess its own activity which will be similar, higher or different from that of the parent drug. Phase 2 metabolites are generally less subject to activation. Metabolic precursors can even be designed to intentionally release the active species only in vivo upon transformation. Such compounds are called prodrugs. Other metabolites, such as electrophiles, may be highly reactive entities able to bind covalently to circulating or intracellular proteins (formation of adducts), to enzymes (mechanism-based, irreversible inactivation), or to DNA (mutagenic and carcinogenic compounds).

In this context, it is important to predict, at an early stage of a drug’s development, the metabolic pathway, the type of metabolites formed and their potential side/toxic effects. This challenge requires better knowledge, at a molecular level, of the enzymes that are implicated in drug biotransformation and the elucidation of the reaction mechanisms.

Drug biotransformation is catalysed by large families of enzymes (also known as phase I and II drug metabolizing enzymes). These proteins are also implicated in the metabolism of endogenous compounds. This situation, in which the drug or the xenobiotic and the natural substrate compete toward the same protein, may lead to cellular dysfunction and toxicity. Structural study of drug metabolizing enzymes is an increasing area of research. With the development of sophisticated technologies (genetic engineering: cloning, expression of cDNAs encoding these proteins, site-directed mutagenesis), protein chemistry, molecular modelling, X-ray crystallography, NMR etc., the organization of the active site can be elucidated and the amino acids that play a crucial role for catalysis and in the substrate recognition are identified. This chapter will illustrate the reaction mechanisms that have been established and which account for the biotransformation of drugs. Such information may help in predicting drug metabolism and provide a rational basis for the design of safer drugs.

Phase I reactions

Oxidations. Most oxidative processes take place in liver microsomes and are catalysed by mono-oxygenase enzymes known as mixed-function oxidases. These processes require reduced nicotinamide-adenine dinucleotide phosphate, molecular oxygen and a complex of enzymes in the endoplasmatic reticulum. The terminal oxidizing enzyme is cytochrome P450, a hemoprotein. The notation ‘P450’ refers to the ability of the reduced (ferrous) form of the hemoprotein to react with carbon monoxide, yielding a complex with absorption peak at 450 nm. For each molecule of substrate oxidized, one molecule oxygen is consumed; one oxygen-atom is introduced into the substrate, and the other is reduced to form water. The P450s represent a superfamily of enzymes. Initially it was believed that there were only two forms, termed P450 and P448. Nowadays more than 30 different P450s have been identified in humans. To unify the nomenclature, P450s are grouped in families, designated by an arabic number, within which the amino acid sequence homology is higher than 40%. The majority of P450s involved in drug metabolism belong to three distinct families, CYP1, CYP2 and CYP3. Each P450 family is further divided into subfamilies, designated by capital letters, which in mammals contain proteins that share more than 55% amino acid sequence homology. In each subfamily, specific enzyme are denoted by an Arabic number. Each isoenzyme has more or less distinct substrate specificity requirements. Only six of the numerous cytochromes P450 play a major role in the metabolism of drugs in common clinical use. Prominent among them in regard to the number of substrate drugs are CYP3A4 and CYP2D6, with smaller numbers of drugs metabolized by CYP2C9, CYP2C19, CYP1A2 and CYP2E1. Some selected substrates are listed in Table. Cytochrome CYP1A2 is particularly involved in the metabolism of environmental chemicals but also of drugs. CYP3A4 accounts for 30% of total P450 enzyme in the liver and is clinically the most important isoenzyme present in the liver. It is also substantially expressed in the mucosal epithelium of the intestine. Nearly 50% of all clinically used medications are metabolized by CYP3A4. This explains to a large extentwhy this enzyme is involved in many important drug interactions. Sometimes a single substrate is metabolized by a single P450 enzyme, while other substrates can beoxidized to varying degrees by multiple P450 enzymes.

Phase I reactions

In addition to cytochrome P450s, hepatic microsomes contain another class of mono-oxygenases, the flavin containing mono-oxygenases (FMO). These enzymes catalyse oxidation at nucleophilic nitrogen, sulfur and phosphorus atoms rather than oxidation at carbon atoms, e.g. for phenothiazines, ephedrine, norcocaine and the mono-ether and carbamate-containing pesticides. Some oxidations are mediated by hepatic enzymes localized outside the microsomal system. Alcohol dehydrogenase and aldehyde dehydrogenase, which catalyse a variety of alcohols and aldehydes such as ethanol and acetaldehyde, are found in the soluble fraction of the liver.  Xanthine oxidase, a cytosolic enzyme mainly found in the liver and in small intestine, but also present in kidneys, spleen and heart, oxidizes mercaptopurine to 6-thiouric acid. Monoamine oxidase, a mitochondrial enzyme found in liver, kidney, intestine and nervous tissue, oxidatively deaminates several naturally occurring amines (catecholamines, serotonin), as well as a number of drugs.

Nonexhaustive list of substrates for, and inducers and inhibitors of some human liver cytochrome P450 isoenzymes

Reduction. Reduction, for example azo- and nitro-reduction, is a less common pathway of drug metabolism. Reductase activity is found in the microsomal fraction and in the cytosol of the hepatocyte. Anaerobic intestinal bacteria in the lower gastrointestinal tract are also rich in these reductive enzymes. A historical example concerns ProntosilR, a sulfonamide prodrug. It is metabolized by azo-reduction to form the active metabolite, sulfanilamide. Sulfasalazine is also cleaved by azoreduction by intestinal bacteria to form aminosalicylate, the active component, and sulfapyridine. Chloramphenicol is metabolized by nitro-reduction to an amine in bacteria and in a number of tissues.

Hydrolysis. Hydrolysis of esters and amides is a common pathway of drug metabolism. The liver microsomes contain non-specific esterases, as do other tissues and plasma. Hydrolysis of an ester results in the formation of an alcohol and an acid; hydrolysis of an amide results in the formation of an amine and an acid. The ester procaine, a local anaesthetic, is rapidly hydrolysed by plasma cholinesterases and, to a lesser extent, by hepatic microsomal esterase. An example of an amide which is hydrolysed, is the antiarrhythmic drug procainamide. Enalapril, a prodrug, is hydrolysed by esterases to the active metabolite enalaprilate, which inhibits the angiotensin-converting enzyme.

Phase II reactions

Compounds having polar constituents such as — OH, —NH2 or —COOH, or acquiring them by a phase I reaction, may undergo a phase II or conjugation reaction. The major conjugation reactions are listed in Table.  The reactive group of the drug interacts with endogenous compounds such as glucuronic acid, sulfate, glycine, acetate or glutathione. Glucuronidation and sulfation are the most common conjugation process. Knowledge of the function, biochemistry, and molecular biology of the responsible enzymes, namely the UDP glycosyltransferases (UGTs) and sulfotransferases (STs), has increased extensively in recent

years. UGTs are membrane-bound enzymes and are located in endoplasmatic reticulum, while STs are present in the cytosol. Both UGTs and STs comprise a superfamily of enzymes; in humans at least eight UGTs and two STs have been identified.

Conjugation reactions may involve an active, high energy form of the conjugating agent, such as uridine diphosphoglucuronic acid (UDPGA) and acetyl CoA, which in the presence of the appropriate transferase enzyme combines with the drug to form the conjugate. For other conjugating reactions, the drug is activated to a high-energy compound that then reacts with the conjugating agent in the presence of a transferase enzyme. Glutathione, for example, reacts via the enzyme glutathione-S-transferase with reactive electrophilic oxygen intermediates of certain drugs, such as paracetamol. Conjugates are usually pharmacologically inactive; they are more hydrophilic than the parent compounds and are easily excreted by the kidneys or the bile. Some conjugates, e.g. morphine-6-glucuronide and acetyl procainamide, are pharmacologically active.

Stereoselective metabolism

Synthesis of a drug with an asymmetrical or chiral centre usually results in two enantiomers, mirror images that cannot be superimposed. Such a 50:50 mixture is called a racemate. The enantiomers of a racemic drug often differ in their pharmacodynamic and/or pharmacokinetic properties as a consequence of stereoselective interaction with optically active biological macromolecules. Stereoselective metabolism of chiral xenobiotics is well recognized. Both phase I and phase II metabolic reactions are capable of discriminating between enantiomers. Stereoselective metabolism of chiral drugs implies the preferential enzymatic removal of one enantiomeric form over the other. Stereoselective drug metabolism may be divided in three groups: substrate stereoselectivity, product stereoselectivity and substrate-product stereoselectivity.

1. Substrate stereoselectivity is characterized by the preferential enzymatic metabolism of one enantiomer; metabolism can occur with retention or with loss of stereoisomerism. Most examples of stereoselective metabolism belong to this group. Several chiral nonsteroidal anti-inflammatory agents of the 2-aryl propionic acid group undergo an unusual metabolic reaction whereby R-enantiomers are inverted to the active Santipodes. The extent of inversion varies considerably depending on the drug, but is also species-dependent.

2. Product stereoselectivity is observed when a prochiral drug is preferentially metabolized to one or more chiral products. There are only a few examples of this type of stereoselectivity, e.g. the 5-hydroxylation of phenytoin and the 4-hydroxylation of debrisoquine, both with preferential formation of the S-enantiomer of the hydroxylated product.

3. Substrate-product stereoselectivity: the enantiomers of a drug which possess both asymmetrical and prochiral characteristics can undergo stereoselective metabolism, whereby a second chiral centre is introduced. Examples are the hydroxylation of perhexiline and the ketoreduction of warfarin.

Phase II reactions or conjugation reactions

Stereoselective metabolism is the most important process responsible for the stereoselectivity observed in pharmacokinetics. Verapamil has received considerable attention as an example of substrate stereoselective pharmacokinetics in humans. After oral administration, the drug undergoes an important stereoselective first pass metabolism, so that (-)-verapamil, the active enantiomer, has a two to three times lower bioavailability than its antipode. The (-)/(+) plasma concentration ratio is therefore higher after intravenous than after oral administration.

Phase 3 Transporters

Once xenobiotics have been converted into low-toxicity and water-soluble metabolites by the combination of Phase 1 and Phase 2 reactions, these metabolites must be transported against a concentration gradient out of the cell into the interstitial space between cells, and then into the bloodstream for filtration by the kidneys. The biggest hurdle is the transport of these hydrophilic metabolites out of the cell. Charged Phase 2 metabolites will be effectively “ion-trapped” in the cell, as the cell membrane is highly lipophilic and is an effective barrier to the exit, as well as entry, of most hydrophilic molecules. In addition, failure to remove the hydrophilic products of conjugation reactions can lead to toxicity. Consequently, an array of multipurpose membrane-bound transport carrier systems has evolved which can actively remove hydrophilic metabolites and many other low-molecular-weight drugs and toxins from cells. Thus, the term of Phase 3 metabolism has been applied to the study of this essential arm of the detoxification process. Efflux transporters transport hydrophilic substrates out of cells into interstitial fluid, blood, kidneys, and the GIT. Influx transporters transport hydrophilic substrates into cells from the bloodstream.

Membrane transport plays an important role in the pharmacokinetic (administration, distribution, metabolism and excretion) profiles of drugs. Membrane transporter interaction leads to changes in transporter function and have effects on the other co-administered drugs. Membrane transporters are proteins that govern the mechanism by which drugs get into and out of cells in order to reach system circulation as well as their sites of metabolism, storage and action.  Membrane transporters are found especially in epithelial tissue of the liver, intestines and kidneys as well as the blood-brain barrier, testes and the placenta.

Mechanism of Membrane transport

There are five different mechanisms by which drugs are transported across membranes.

Passive diffusion

Depends solely upon a gradient concentration across the membrane and the lipophilic character of both drug (unionized form) and the membranes.

Facilitated transport can be facultative or active.

Facultative transporters: Membrane proteins that move drug molecules down a concentration gradient without the use of a separate energy source.

Active transporters: Membrane proteins that require an energy source (usually ATP) to move drug molecules down and against a concentration gradient.

Paracellular transport

Drug molecules cross membranes by passing through the space between cells.

Transcytosis

The transport of extracellular drug molecules by their inclusion into cell surface vesicles for transport across the interior of a cell.

Efflux transporters

Move drug molecules out of cells usually by an active mechanism.

ATP Binding Cassette (ABC) Transporters and inhibitors

Solute Carrier (SLC) Transporters and inhibitors

Cytochromes P450

Cytochromes P450 (CYP) are by far the most important xenobiotic- and endobiotic-metabolizing monooxygenases. They represent up to 25% of the total microsomal proteins and more than 50 cytochromes P450 monooxygenases are expressed by humans. They are

involved in three main processes:

(1) drug metabolism;

(2) steroid metabolism (production of steroid hormones;

(3) haem degradation (conversion of haem into biliverdin and bilirubin).

Cytochromes P450 contain a molecule of haem, protoporphyrin IX, and a variable protein of MW ,50 kDa. Cytochromes P450 form a very large group of haemoproteins encoded by the CYP gene superfamily and classified in families and subfamilies. The major xenobiotic metabolizing cytochromes P450 in humans are found in family 1 (CYP1A1 and CYP1A2), family 2 (CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2D6 and CYP2E1), subfamily 3 (CYP3A), and family 4 (CYP4A9, CYP4A11 and CYP4B1). CYP3A4 is the most abundant and the most clinically important cytochrome in humans, as it metabolizes up to 50% of the available drugs. Cytochromes P450 belong to the haem-thiolate proteins in which the haem iron fifth ligand is a thiolate group, generally a cysteine residue. Such protein exhibits a Soret absorption band at 450 nm in the CO-difference spectrum of a dithionite-reduced form.

The mechanism of the cytochrome P450 redox system is represented in Fig. In microsomes, the two electrons necessary for monooxygenation are transferred by NADPH cytochrome P450 reductase, the second electron in some cases coming from NADH-cytochrome b5 reductase and cytochrome b5. In mitochondria, which also contain cytochromes P450 devoted to the formation of steroid hormones from the hydroxylation of cholesterol or to the biosynthesis of bile acids and vitamin D, the electrons are supplied by the electron transfer chain composed of ferredoxin (adrenoxin) and ferredoxin reductase (adrenoxine reductase).

In the resting state, the central iron atom of protoporphyrin IX is in a hexacoordinated, ferric form. The substrate R — H binds reversibly to the enzyme and the complex undergoes a reduction to the ferrous state. This allows molecular oxygen to bind as a third partner. Following the second reduction step, molecular oxygen is ultimately reduced to a hydroperoxide which is cleaved with liberation of H2O and formation of a monooxygen known as oxene. The oxene, which is electrophilic and quite reactive, can act on the substrate in a manner which depends on the reactivity of the substrate itself. Thus, the oxene can either:

(1) be transferred directly to the substrate (oxygen insertion or addition);

(2) remove an electron, or more frequently;

(3) pull a hydrogen radical away from the substrate and transfer back a formal HO8 radical (a reaction known as oxygen rebound).

The latter is the mechanism by which RR0R00C — H is oxidized to RR0R00C — OH. After release of the product, the regenerated cytochrome P450 is ready for a new cycle. As illustrated below, the substrates to be oxidized are structurally unrelated, the oxidation involving C, Si, N, P, S, Se and other atoms (Fig.). The most common reaction catalysed by P450 is hydroxylation. However, it is also involved in a wide spectrum of reactions including epoxidation, O-, N-, and S-dealkylation, deamination, desulfuration, dehalogenation and peroxidation.

Step 1. The resting [Fe3+-P450] complex binds reversibly with a molecule of the substrate (RH) displacing the distal water resulting in a complex resembling enzyme-substrate complex [Fe3+-P450*RH]. The binding of the substrate triggers/facilitates the first one-electron reduction step from NADPH.

Step 2. The substrate complex of [Fe3+-P450*RH] undergoes reduction to a [Fe2+-P450*RH] substrate complex by an electron originating from its redox partner by the flavoprotein [NADPHP450 reductase FNMH2/FADH complex].

Step 3. The reduced [Fe2+-P450*RH] substrate complex readily binds dioxygen as the sixth ligand of Fe2+ to form a [dioxy-Fe2+-P450*RH] substrate complex.

Step 4. The [dioxy-Fe2+-P450*RH] complex rearranges by resonance because of the strong

electronegativity of O2 to form the [Fe3+-P450*RH-superoxide anion] complex.

Step 5. The [Fe3+-P450*RH-superoxide] complex undergoes further reduction by accepting a second electron from NADPH-P450 reductase to form the equivalent of a two-electron reduced [peroxy-Fe3+-P450*RH] (hydroperoxide anion) complex (the step where O2 is split into an oxygen atom). If the electron is not delivered rapidly, this complex dissociates and is aborted (uncoupled) from subsequent substrate hydroxylation at this step by xenobiotics which can cause release of superoxide, which decomposes to hydrogen peroxide and dioxygen with regeneration of the starting point of the cycle, the Fe3+-P450 complex.

Step 6. The unstable [Fe3+-peroxy-P450*RH) complex undergoes heterolytic cleavage of peroxide anion upon protonation to form water and a highly electrophilic porphyrin-radical cation intermediate (ferryl oxenoid species, Fe4+=O) (Compound I, the more favorable oxygen-cysteineporphyrin radical cation resonance-stabilized complex). The Fe4+=O species represents the catalytically active oxygenation species. One role of the cysteine sulfur ligand is thought to be electron donation (push) that weakens the peroxide O-O bond, causing peroxide bond scission to produce a highly reactive and strong oxidizing intermediate (Compound I).

Step 7. Abstraction of a hydrogen atom (HAT) from the substrate (RH) by the electrophilic

[Fe4+=O*RH] species to produce either a carbon-centered radical, radical addition to a π-bond, or single electron transfer (SET) from a heteroatom to form a heteroatom-centered radical-cation ferryl intermediate. Subsequent radical recombination with the ferric-bound hydroxyl radical [Fe4+ *OH] (step 7/8, oxygen rebound) or single electron-transfer (SET, deprotonation) yields the carbon oxidized hydroxylated product (Compound II) and the regeneration of the initial Fe3+-P450 enzyme complex.

The simplified cytochrome P450 redox cycle

Major reactions of oxygenation catalysed by cytochrome P450.

Glutathione-S-Transferase (GST)

Glutathione (GSH, g-glutamyl-cysteinyl-glycine) is a thiol-containing tripeptide of capital significance in the detoxication and toxication of drugs and other xenobiotics. Glutathione reacts with endogenous and exogenous compounds in a variety of ways. First, the nucleophilic properties of the thiol group make it an effective conjugating agent. Second, glutathione can act as a reducing or oxidizing agent depending on its redox state (i.e. GSH or GSSG). Furthermore, the reactions of glutathione can be enzymatic (e.g. conjugations catalysed by glutathione-S-transferases, and peroxide reductions catalysed by glutathione peroxidases) or non-enzymatic (e.g. some conjugation and various redox reactions).

The glutathione transferase (GST) comprises multifunctional proteins coded by a multigene family. These enzymes are both cytosolic and microsomal and function as homodimers and heterodimers. They exist as four classes in mammals. The human enzymes comprise the following dimers: A1-1, A1-2, A2-2, A3-3 (alpha class), M1a-1a, M1a-1b, M1b-1b, M1a-2, M2-2, M3-3 (mu class), P1-1 (pi class), T1-1 (theta class), and three microsomal enzymes (MIC). The GST A1-1 and A1-2 are also known as ligandin when they act as binding or carrier proteins, a property also displayed by M1a-1a, M1b-1b and also by GSH (pi class).

GST are powerful detoxifying enzymes. The overexpression of GST that has been demonstrated in some human cancer cells, such as breast cancer cells, is associated with the multidrug resistance that impairs the efficacy of anticancer drugs. In this regard, GST inhibitors or substrate competitors, such as the diuretic drug ethacrynic acid, have been proposed as an adjuvant for cancer chemotherapy, thus enhancing the cytotoxicity of alkylating drugs in cancer cell lines. The nucleophilic character of glutathione is due to its thiol or rather thiolate group. In deed if the thiol group of GSH (pKa , 9) is largely protonated at physiological pH, the binding to the enzyme is associated with the loss of the proton and to the electrophilic stabilization of the thiolate group. A hydroxyl group of a serine (GST theta class) or of a thyrosine residue (GST alpha, mu, pi classes) acts as hydrogen donor to the sulfur of GSH whereby lowering the pKa of the thiol, leading to the presence of a predominantly ionized form at physiological pH.

As a result, GSTs transfer glutathione to a very large variety of electrophilic groups (R — X, see Fig.) in nucleophilic reactions categorized as either substitutions or additions. With compounds of sufficient reactivity, these reactions can also occur nonenzymatically. Once formed, glutathione conjugates (GS-R) are seldom excreted as such, but usually undergo further biotransformation. Cleavage of the glutamyl moiety by glutamyl transpeptidase and of the cysteinyl moiety by cysteinylglycine dipeptidase or aminopeptidase M leaves a cysteine conjugate (Cys-S-R) which is further N-acetylated by cysteine-S-conjugate N-acetyltransferase to yield an N-acetylcysteine conjugate (CysAc-S-R). The latter type of conjugates are known as mercapturic acids. Thesemay be either excreted or further transformed, since cysteine conjugates can be substrates of cysteine-Sconjugate b-lyase to yield thiols (R-SH). These in turn can rearrange or be S-methylated and then S-oxygenated to yield thiomethyl conjugates (R-S-Me), sulfoxides (R-SO-Me) and sulfones (R-SO2-Me).

Factors Affecting Metabolism

Drug therapy is becoming oriented more toward controlling metabolic, genetic, and environmental illnesses rather than short term therapy associated with infectious diseases. In most cases, drug therapy lasts for months or even years, and the problems of drug-drug interactions and chronic toxicity from long-term drug therapy have become more serious. Therefore, a greater knowledge of drug metabolism is essential. Several factors influencing xenobiotic metabolism include:

Genetic polymorphism. Genetic polymorphisms of drug metabolizing enzymes give rise to distinct subgroups in the population that differ in their ability to perform certain drug biotransformation reactions. Polymorphisms are generated by mutations in the genes for these enzymes, which cause decreased, increased, or absent enzyme expression or activity by multiple molecular mechanisms. Individual differences in drug effectiveness (drug sensitivity or drug resistance), drug-drug interactions, and drug toxicity can depend on racial and ethnic characteristics impacting the population frequencies of the many polymorphic genes and the expression of the metabolizing enzymes. Pharmacogenetics focuses primarily on genetic polymorphisms (mutations) responsible for interindividual differences in drug metabolism and disposition. Genotype-phenotype correlation studies have validated that inherited mutations result in two or more distinct phenotypes causing very different responses following drug administration. The genes encoding for CYP2A6, CYP2C9, CYP2C19, and CYP2D6 are functionally polymorphic; therefore, at least 30% of P450-dependent metabolism is performed by polymorphic enzymes. For example, mutations in the CYP2D6 gene result in poor (PM), intermediate (IM), or ultrarapid (UM) metabolizers of more than 30 cardiovascular and central nervous system (CNS) drugs. Thus, each of these phenotypic subgroups experiences different responses to drugs extensively metabolized by the CYP2D6 pathway ranging from severe toxicity to complete lack of efficacy.

Physiologic factors. Age is a factor, as both very young and old have impaired metabolism. Hormones (including those induced by stress), sex differences, pregnancy, changes in intestinal microflora, diseases (especially those involving the liver), and nutritional status can also influence drug and xenobiotic metabolism. Because the liver is the principal site for xenobiotic and drug metabolism, liver disease can modify the pharmacokinetics of drugs hepatically metabolized. Liver disease affects the elimination half-life of some drugs but not of others, even if they all undergo hepatic biotransformation. Some studies have shown that the capacity for drug metabolism is impaired in chronic liver disease, which could lead to unintentional drug overdosage. Because of the unpredictability of drug effects in the presence of liver disorders, drug therapy in these circumstances is complex, and more than usual caution is needed. Protein deficiency leads to reduced hepatic microsomal protein and lipid metabolism, and oxidative metabolism is decreased due to an alteration in endoplasmic reticulum (ER) membrane permeability affecting electron transfer. Protein deficiency would increase the toxicity of drugs and xenobiotics by reducing their oxidative P450 metabolism and clearance from the body.

Pharmacodynamic factors. Dose, frequency, and route of administration, plus tissue distribution and protein binding of a drug, affect its metabolism.

Environmental factors. Competition of ingested environmental substances for metabolizing enzymes and poisoning of enzymes by toxic chemicals such as carbon monoxide can alter drug and other xenobiotic metabolism. Induction of enzyme expression (in which the number of enzyme molecules is increased, while the activity is constant) by other drugs and xenobiotics is another consideration. Environmental factors can change not only the kinetics of an enzyme reaction but also the whole pattern of metabolism, thereby altering bioavailability, pharmacokinetics, pharmacological activity, and/or toxicity of a xenobiotic. Species differences in response to xenobiotics must be considered in the extrapolation of pharmacological and toxicological data from animal experiments to predict effects in humans.

Enzymes as drug targets

Several families of drugs do not act on receptors and their therapeutic properties are attributed to inhibition or activation of enzyme activities. A number of drugs in clinical use exert their effect by inhibiting a specific enzyme present either in tissues of an individual under treatment or in those of an invading organism. The basis of using enzyme inhibitors as drugs is that inhibition of a suitable selected target enzyme leads to a build-up in concentration of substrate and a corresponding decrease in concentration of the metabolite, which leads to a useful clinical response. The type of inhibitor selected for a particular target enzyme may be important in producing a useful clinical effect. Enzyme inhibiting processes may be divided into two main classes, reversible and irreversible, depending upon the manner in which the inhibitor is attached to the enzyme. Reversible inhibition occurs when the inhibitor is bound to the enzyme through a suitable combination of Van der Vaals’, electrostatic, hydrogen bonding, and hydrophobic attractive forces. Reversible inhibitors may be competitive, noncompetitive, uncompetitive, or of mixed type. During irreversible inhibition, after initial binding of the inhibitor to the enzyme, covalent bonds are formed between a functional group on the enzyme and the inhibitor. This is the case, for example, for the active-site-directed inhibitors (affinity labelling).

The inhibitors used in therapy must possess a high specificity towards the target enzyme, since inhibition of closely related enzymes with different biological functions could lead to a range of side-effects. There is a very large area of enzyme targets as illustrated in Table. For example, dihydrofolate reductase (DHFR) catalyses the NADPH-linked reduction of dihydrofolate to tetrahydrofolate. The tetrahydrofolate are cofactors for the biosynthesis of nucleic acids and aminoacids. The reduction of their level induces a limitation of cell growth. Thymidylate synthase methylated the deoxyuridylate into thymidylate using 5,10-methylenetetrahydrofolate as a cofactor. This reaction is the rate-limiting step in the de novo synthetic pathway to thymidine nucleotide. Antitumoral effects are obtained with antifolate compounds.

A functional HIV protease is required for the production of infective virions; this key role of the protease in the viral life cycle makes the inhibition of this enzyme a potential way for therapeutic intervention in the treatment of AIDS. New strategies for the development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule are under investigation.

Drugs influencing synaptic transmission by: (1) inhibiting enzymes that synthesize neurotransmitters; (2) preventing neurotransmitter storage in synaptic vesicles; (3) blocking the release of neurotransmitter into the synapse; (4) blocking enzymes that degrade neurotransmitters; (5) blocking neurotransmitter (or metabolite) re-uptake; (6) binding to the receptor and either mimicking or blocking neurotransmitters; and (7) interfering with second messenger activity.

Angiotensin-converting enzyme (ACE) inhibitors are used for the treatment of high blood pressure, and were designated using the carboxypeptidase structure as a model for Zn²⁺ protease action. Captopril is a small, potent, orally available, dipeptidyl inhibitor of ACE. Acetylcholinesterase (AchE) hydrolyses the neurotransmitter acetylcholine and yields acetic acid and choline. AchE is a serine hydrolase inhibited by organophosphorus poisons, as well as by carbamates and sulfonyl halides which form a covalent bond to a serine residue in the active site. AchE inhibitors are used in the treatment of various disorders.  In conclusion, the modification by drugs of a precise function can be achieved in several different ways, acting on the receptors of mediators, on enzymes or transmembrane exchange processes. This view is illustrated in Fig. showing a synapse. Future development of drug targets will be based on the diversity of biological targets, according to the definition of the human genome, increasing the selectivity of drugs. Recent development in gene drug delivery systems73 and in antisense oligonucleotide technology might also be crucial factors in drug development.

Selective illustration of enzymes inhibitors

Protein Kinase Inhibitors

Protein kinases belong to the family of group transfer phosphorylating enzymes that transfer a phosphate group from ATP onto amino acid residues of proteins. It is estimated that there are over 500 of these kinases encoded by the human genome (the kinome), and this kinase reaction when coupled with a phosphatase (dephosphorylation) reaction, i.e., reversible phosphorylation of proteins, offers cells a precise regulatory mechanism to control its differentiation, maturation, proliferation, apoptosis, and other cellular functions. The substrate amino acid residues on proteins that are phosphorylated belong largely to the hydroxyl bearing amino acids such as serine, threonine, and tyrosine. Hence, these kinases are referred to as either serine/threonine kinases or tyrosine kinases. Similarly, the phosphatases are referred to as serine/threonine or tyrosine phosphatases of which the tyrosine phosphatases predominate. Since these proteins are involved in regulatory functions of the cell, it becomes intuitive that mutations or aberrations of expression of these proteins can lead to dysregulation of cellular functions giving rise to tumors and cancers and other diseases. Indeed, research has shown that of the 518 kinase genes present in the human genome, 244 map to disease loci and cancer. Moreover, targeting these enzymes with inhibitors would be a way to selectively target cancers without the noxious side effects seen with conventional anticancer drugs such as the alkylating agents or antitumor antibiotics. Research over the last decade has provided this as a rationale to develop selective (targeted) anticancer therapy where such kinases which manifest themselves in certain cancers have been specifically targeted for inhibition resulting in dramatic declines of cancer cells and greater survival times for the patient. Examples of such targeted anticancer drugs have involved primarily the development of the tyrosine kinase inhibitors (TKIs) several of which are FDA approved; however, more recently, inhibitors of serine/threonine kinases have entered the clinical market while inhibitors of the tyrosine phosphatases are in development.

Tyrosine Kinase Inhibitors

The tyrosine kinases are a group of enzymes responsible for signal transduction and intracellular signaling functions many of which are involved in cell differentiation and proliferation. They can be divided into two major types depending upon where they act in the cell, viz. receptor tyrosine kinases (RTK), a membrane spanning protein having an extracellular ligand binding domain and an intracellular catalytic (kinase) domain involved in the transduction of extracellular signals from the membrane to the cytoplasm, and the nonreceptor tyrosine kinases involved in cytosolic signaling events. Inhibitors for these have been developed for both types and have shown excellent and selective activity in cancers manifested by aberrant expression of these proteins. The design for selective inhibitors has been based on determining important binding regions to the protein. The kinase domain has a C-terminal domain linked via a “hinge region” to the N-terminal domain, and structural studies have indicated that ATP is known to bind to the backbone of this hinge region (ATP binding pocket). These kinases all have an “activation loop” which contains a tyrosine residue (Tyr393), the major phosphorylated residue that allows switching the kinase from inactive to active forms and allowing for ATP binding. The large majority of the TKIs bind to this region via H-bonding. Areas of the protein that have been identified as important for the function of kinases include a glycine-rich loop (G-loop), ATP binding pocket, a gate-keeper residue (an amino acid preceding the hinge region), and the DFG activation motif. Most of the inhibitors that have been developed tend to bind to the ATP binding pocket and have been classified based on their binding motif. The Type I inhibitors bind the “DFGin” motif of the activation loop (the active form of the kinase, where the kinase is poised for the phosphoryl transfer) and is a more conserved region, while the Type II inhibitors bind the inactive (DFG-out) motif which is a region of less conserved residues but would allow for greater specificity. Type III and Type IV inhibitors bind to regions outside of the ATP binding pocket (distal sites) and are classified as allosteric inhibitors. The kinase-ligand interaction fingerprints and structures (KLIFS) is a useful database that identifies the binding pocket for Type I to Type IV inhibitors which includes the gatekeeper residues for various kinases and is often used to aid medicinal chemists in designing new kinase inhibitors.

The development of a tyrosine kinase inhibitor (TKI) to selectively treat a cancer, chronic myelogenous leukemia, CML, was the impetus leading to the large number of presently available TKIs. CML in the majority of patients is due to a reciprocal translocation of chromosomes 9 and 22 resulting in a fusion of the abl (Abelson leukemia virus) gene of chromosome 9 to the bcr (breakpoint cluster) gene of chromosome 22 leading to the bcr-abl fusion gene (the Philadelphia chromosome). While the abl gene normally produces a nonreceptor tyrosine kinase whose activity is highly regulated, the bcr-abl fusion gene produces a tyrosine kinase that is constitutively active and whose activity is required for the transformation of cells to become malignant. The knowledge of this direct correlation between expression of the abnormal fusion protein and CML allowed for the development of specific inhibitors for this protein and other such dysregulated kinases that are overexpressed in many cancers. Using a high-throughput screening program to develop inhibitors for receptor tyrosine kinases as a possible treatment for such cancers, 2-phenylaminopyrimidine (PAP) became a lead compound. Further structure activity optimization and refinement of this lead led to Imatinib, the first targeted drug for treatment of CML. Note, the structure optimization to imatinib included addition of a pyridine, a methyl as well as a benzamide to enhance the potency of the basic PAP nucleus. The piperazinyl functionality helped to increase water solubility allowing for better “druglike” properties. Imatinib also proved to be inhibitory in many other cancers with overexpressed kinases, such as gastrointestinal stromal tumors (GIST) (which overexpress c-kit), myelodysplastic diseases associated with plateletderived growth factor receptor (PDGFR) as well as in Philadelphia chromosome-positive adult lymphoblastic leukemia. X-ray crystallographic studies with imatinib co-cyrstallized with the TK expressed from abl showed that imatinib binds to the ATP binding site of the protein in its inactive conformation (DFG-out form—Type II), and this binding prevented the kinase from achieving its productive binding conformation with ATP. Studies showed that with the protein-bound imatinib, Tyr393 was not phosphorylated and the conformation of this activation loop in the nonphosphorylated protein changed to that resembling substrate (ATP) being bound to the kinase. In this way, the altered geometry brought about by imatinib binding to the protein prevented the protein from binding its true substrate, ATP.

Resistance develops to imatinib due to mutations (in the hydrophobic pocket, gate keeper residue being mutated to a larger residue) that prevent access of imatinib to the protein in the off state, thus allowing for the kinase to bind ATP and cancer to progress. TKIs dasatinib and bosutinib have also been developed to bind the kinase in its “on” (active conformation, Type-I) where the drugs can access hydrophobic regions in the ATP binding pocket as well as drugs that can bind both “on” and “off” forms—dual mode inhibitors which are more potent than imatinib.

It has been more than 15 years since the introduction of imatinib, and today many other TKI drugs have been developed for related tyrosine kinases such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR). The inhibitors make use of differences in the variable region of the protein surrounding the ATP binding pocket which allows for specific binding interactions with the various functionalities present on the individual inhibitors. Resistance to these inhibitors manifest themselves due to mutations to these variable regions on the protein as well as to cellular efflux pumps being activated. While the vast majority of these clinically used drugs are reversible inhibitors of the Type I and II, more recently Type III inhibitors have been introduced in the clinic.

Furthermore, irreversible inhibitors of Type I which employ the “Michael acceptor” functionality to irreversibly alkylate an active site cysteine residue have also been introduced. Examples of these inhibitors such as dasatinib (Type I for imatinib resistance), sunitinib (Type I, inhibitor of VEGFR), gefitinib (Type I, inhibitor of EGFR), trematinib (Type III, inhibitor of MEK), and the irreversible Type I inhibitor, Ibrutinib with the α, β unsaturated system (Michael acceptor) are shown in Figure.

Serine/Threonine Kinase Inhibitors

The serine/threonine kinases are a family of enzymes that phosphorylate the hydroxyl groups of serine and threonine present on proteins. There are a number of such serine/threonine kinases that are important regulators of cell proliferation and survival and whose dysregulation often lead to cancer and tumorigenesis. Protein kinase C is perhaps the most well-studied system, whose activation results in the formation of diacylglycerol (DAG), phosphatidylinositol-3,4,5-triphosphate (PIP3), and concomitant increase in intracellular calcium, leading to various signal transduction events in the cell through activation of the mitogen-activated protein kinase (MAPK) family. Furthermore, other serine/threonine kinase proteins such as PI3K (phosphoinositide-3-kinase), Akt (protein kinase B), and mTOR (mammalian target of Rapamycin) are part of an intracellular signaling pathway that is important for regulation of cell proliferation, migration, and survival. Initial events leading to cell proliferation via the PI3K-Akt-mTOR have been shown to begin with growth factor or hormonal activation of a receptor tyrosine kinase which leads to activation of PI3K. This activation results in the release of PIP3 which in turn leads to phosphorylation and further activation of Akt and mTOR. Inhibitors of mTOR include rapamycin (sirolimus) and its derivatives, everolimus and temsirolimus. Rapamycin is a macrolide originally isolated from a microbe on Easter Island and primarily used as an immunosuppressant drug while its derivatives, everolimus and temsirolimus, have been used to treat renal cell carcinoma.

Additional development of molecules to specifically inhibit the PI3K-Akt pathway include copanlisib, which was recently introduced to treat follicular lymphoma, and duvelisib, a first-in-class inhibitor of PI3K that has recently received FDA new drug application (NDA) status to treat chronic lymphocytic leukemia (Fig.). Additional serine/threonine kinase function is also observed during mitosis, which is a highly regulated process with multiple checkpoints encountered during the chromosomal segregation stage of cell division. The phosphorylation of specific serine/threonine residues by these mitotic kinases (also known as Aurora A and Aurora B kinases) serves as important checkpoints during mitosis. These Aurora kinases interact with many proteins, including tumor suppressors and activators from the mitotic entry stage all the way to cytokinesis. It is not surprising then that they are overexpressed in many tumors (breast, colon, gastric, ovarian, and pancreatic) and thus have become an attractive target for anticancer drug development. Examples of some of these inhibitors include alisertib, danusertib, and a phosphate-based prodrug currently under development, barasertib (Fig.).

Other serine/threonine kinases include the RAF proteins which are involved in the MAPK activation cascade during cell growth. B-RAF is one member of the RAF family where mutations (specifically the V600E) have resulted in dysregulation of the cascade leading to many cancers. Specific clinically used inhibitors of the V600E B-RAF mutated protein include vemurafenib and dabrafenib (Fig.) used to treat metastatic melanoma.

ATPase Inhibitors

The transmembrane proteins establishing these gradients are transporters such as the Na+–K+ ATPase pumping three Na+ out and two K+ into the cell while consuming one ATP molecule. Other transporters are the Ca2+-ATPases pumping Ca2+ out of the cell or into the endoplasmatic reticulum, and secondary active transporters such as the Na+–Ca2+ exchanger not using energy themselves, but exploiting the ion gradients created by the ATPases. The transporters typically move 0.1–10 ions/ms each, they show saturation kinetics like enzymes, and they slowly build up the ion gradients.

The sesquiterpene lactone, thapsigargin which is structurally unrelated to ryanodine, also interacts with an intracellular calcium mechanism. Thapsigargin has become the key pharmacological tool for the characterization of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Thapsigargin effectively inhibits this ATPase, causing a rise in the cytosolic calcium level which eventually leads to cell death. Although the SERCA pump is essential for all cell types, attempts to target thapsigargin toward prostate cancer cells have been made based on a prodrug approach.

Gastroesophageal reflux disease (GERD) is one of the most common GI disorders and affects a significant number of people in the United States and worldwide. GERD is typically associated with the esophageal mucosa being continuously exposed to gastric secretions. This often occurs when the lower esophageal sphincter relaxes and the duration of esophageal acid exposure is considerably prolonged. Typically, when gastric secretions enter the esophagus, innate clearing mechanisms limit the duration of exposure by rapidly pushing the refluxed content into the stomach. Additionally, bicarbonate-rich secretions by esophageal glands help neutralize the residual acid trapped within the mucosa. Since GERD is associated with gastric acid reflux and associated symptoms, most treatment options are directed toward reducing the acidic nature of the refluxate. An effective therapeutic option should heal esophageal damage while providing symptomatic relief. Current therapy for acid reflux–related GI diseases focuses on inhibiting biological pathways that directly or indirectly play a role in stomach acid secretion. Two such pathways involve inhibition of the histamine H2 receptor and inhibition of the proton pump (H+/K+-ATPase) in the stomach. Selective muscarinic receptor antagonists and prokinetic agents can also be used for the treatment of GERD.

Proton Pump Inhibitors

A second class of drugs that are commonly and more frequently used in the treatment of peptic ulcer is PPIs. This class of drugs covalently inhibits the gastric H+/K+-ATPase that is responsible for secretion of stomach acid in parietal cells of the gastric mucosa. The gastric proton pump is very similar to Na+/K+-ATPase, and also similar in structure and function to the H+/K+-ATPase found in osteoclasts, which play an important role in bone resorption. PPIs enable healing of peptic ulcer, erosive esophagitis, GERD, GERD-related laryngitis, Barrett’s esophagus, and Zollinger-Ellison syndrome, as well as the infection caused by H.pylori, the latter in combination with antibiotics. Since the H+/K+-ATPase–mediated process is the final step in gastric acid secretion, inhibiting this enzyme is considered the most effective approach to acid suppression. PPI discovery began with the early investigation of timoprazole. This compound is a pyridyl methyl sulfinyl benzimidazole, which is the conserved pharmacophore for subsequently developed PPIs. Based on its acid dependent activity, it was identified as an acid-activated prodrug. Omeprazole was later synthesized and, in 1989, became the first drug of this class available for clinical use. Other clinically available PPIs include lansoprazole, dexlansoprazole, esomeprazole, pantoprazole sodium, and rabeprazole sodium (Fig.). Dexlansoprazole and esomeprazole are enantiomerically pure forms of lansoprazole and omeprazole, respectively. Omeprazole and lansoprazole are both also marketed as racemic mixtures.

Structural Features and Mechanism of Action of PPIs

The 2-pyridylmethylsulfinylbenzimidazole motif is conserved in all members of the PPI family, as it is necessary for bioactivity through acidcatalyzed decomposition to the reactive sulfenic acid and sulfenamide structures. Typically, the structural modifications are done on the pyridine ring or at the benzo component of the benzimidazole to generate drugs with differing levels of acid stability, which impacts duration of proton pump inhibiting action. The investigational drug, tenatoprazole is slightly different in that it has an imidazopyridine isostere, instead of the traditional benzimidazole scaffold. PPIs are weak bases with a pyridine pKa between 3.8 and 4.9, which enables them to selectively accumulate through ion trapping in the stimulated parietal cell (pH∼1.0). This acidic environment-selective accumulation of PPIs is an important property that contributes to their selectivity and activity. As mentioned, PPIs are prodrugs, and acidcatalyzed activation generates the active form of the drug. The active form has electrophilic sulfenic acid and sulfenamide sulfur atoms capable of reacting with the thiol group of a Cys residue of the ATPase, forming a covalent adduct. Covalent inhibition of the proton pump results in inactivation of the catalytic function of this enzyme. The chemical mechanism of activation of omeprazole and subsequent covalent interaction is described in Figure.  All PPIs follow a similar mechanism of action. The imidazole ring N3, an exceptionally weak base with a pKa <0.8, is first protonated under the very low pH of the parietal cell through proton transfer from pyridine N1. Nucleophilic attack by the unionized pyridine nitrogen at benzimidazole C2 (made electron deficient by cationic N3) generates a highly electrophilic and unstable electrophilic spiro intermediate, which rearranges to generate the sulfenic acid intermediate that preferentially forms the inactivating disulfide bond with the proton pump Cys (Fig). The following step involves loss of water and the formation of a reactive sulfenamide intermediate, which can also covalently modify the Cys thiol of the proton pump.

Within the proton pump, several different Cys residues are potential sites for covalent modification. The different substituents on the pyridine or benzimidazole ring of PPIs determine which Cys residue the drug preferentially reacts with, which subsequently determines the permanency of the covalent attachment, as discussed below. Electron donating groups on the C5-position of benzimidazole enhance reactivity by increasing the extent of benzimidazole N3 protonation and the strength of the δ+ charge on the carbon at the C2 position of benzimidazole moiety. Electron-donating groups at the C4 position of the pyridine ring increase the nucleophilicity of pyridine nitrogen, enhancing the rate at which N1 attacks electrophilic benzimidazole C2. Conversely, electron-withdrawing groups at either site have been shown to decrease reactivity.

All PPIs can react with the readily accessible Cys813 of the ATPase. It hasbeen observed that activated molecules of omeprazole can bind to either Cys813 or Cys892 of the proton pump. Similarly, lansoprazole is known to covalently modify Cys813 or Cys321. Pantoprazole, the most sluggish-reacting of the PPIs, can react with either Cys813 or the more deeply recessed and less accessible Cys822. Although PPIs are covalently bound to the proton pump, the interaction with accessible Cys residues can be reversed by reduced glutathione (GSH). The disulfide bond formed between the drug and receptor can be cleaved by this endogenous reducing agent, regenerating the essential Cys thiol of the pump and prompting the excretion of the drug fragment as a glutathione conjugate. Due to low concentration of GSH in the body, regeneration of proton pumps inhibited through Cys813 is incomplete. Pantoprazole binding is found to be less susceptible to GSH-mediated removal. These observations suggest that modification of Cys813 is more easily reversed, providing a fast phase of recovery, while modification of Cys822 is difficult to reverse, as GSH may not easily reach this site.

Since the activation and rearrangement of PPIs take place at a strongly acidic pH, all of the oral formulations of PPIs have been developed to be acid-stable. This allows for better dissolution and absorption of PPIs (typically enteric-coated formulations) from the intestines. Both lansoprazole (enteric-coated) and omeprazole (either enteric coated or formulated with NaHCO3) are available in granular form. This allows for the drug to be absorbed readily with minimal drug destruction in the stomach. Rearrangement selectively occurs in the acidic environment of the canaliculus within parietal cells after the drug is absorbed from the intestine and delivered to that target site. Typically, the half-life of PPIs is relatively short (∼1 hr); however, the duration of action is long (∼20-48 hr) because of their ability to irreversibly inhibit the proton pump.

Benzimidazole N1 is typically nonionizable at physiological pH and considered neutral in nature. However, as noted in Figure, benzimidazole N1 within PPIs is slightly acidic due to the electron withdrawing property of the attached sulfinyl moiety. Due to this change in acidity, PPIs can be made into salt forms when benzimidazole N1 ionizes (loses proton) through treatment with strong base and becomes paired with the resultant metal cation (e.g., Na+ or Mg2+). Due to this property, some of the PPIs are marketed in the salt form, which providesimproved aqueous solubility.

Although the proton pump inhibitors, such as omeprazole, lansoprazole, pantoprazole, rabeprazole, esomeprazole and tenatoprazole, were not originally developed as prodrugs, they provide a good example of site-selective prodrugs. Due to the basic pyridine group (pKa of omeprazole is 3.97), these drugs are protonated and accumulated in the acidic secretory parietal cells (Figure). In the acidic conditions of parietal cells (pH of 1–2), prazoles undergo spontaneous chemical reaction to their active sulfenamide metabolites followed by their irreversible binding to a cysteine group of H+/K+ ATPase. This irreversible binding inhibits the ability of parietal cells to secrete gastric acid. The fact that proton pump inhibitors are only effective on H+/K+ ATPases, which contain highly acidic compartments that nongastric H+/K+ ATPases lack, corresponds their excellent safety profiles. Therefore, proton pump inhibitors are converted to their active species only under highly acidic conditions at their site of action.

Binding Site and Molecular Interactions

The binding site provides a base for the interaction of two molecules and describes the ability of the receptor to form bonds with other substances. Based on the bound molecule, the binding site can be protein–protein, protein–nucleic acid, protein–carbohydrate, protein–lipid, and protein–small molecule binding sites. The binding of the drug molecule to plasma proteins (albumin, lipoproteins, and globulins) is a major determinant of drug distribution.

Protein–Drug Interactions

DNA and protein are molecules that show interaction with other small molecules such as substrate, drug, or other ligands. Proteins are an important molecule in all living cells and play an essential role in various cellular processes in the form of enzyme, hormone, and receptor. Each protein performs a specific function that is governed by its 3D structure. Protein–ligand interactions are vital for all biological processes that occur in living organisms. The function of a protein depends upon the specific sites that are designed to bind with a specific ligand molecule. Ligand-binding interactions can alter the protein conformations and its function. To perform its function properly, binding of a protein with other molecules should be very specific. A drug is a small organic molecule, which binds to the receptor and forms a protein–drug complex and controls the function of biological receptors. Binding can be of two types: intracellular or extracellular. Based on the drug binding mechanism, drug binding may be of reversible or an irreversible type.

Reversible Binding

In reversible binding, usually, the drug binds the proteins with weaker chemical bonds such as hydrogen bonds, hydrophobic bonds, ionic bonds, and van der Waals interactions. The binding of drugs to plasma protein is a reversible process.

Irreversible Binding

In the case of irreversible binding, a drug or inhibitor permanently binds with the binding site of the drug target. Irreversible binding of drugs rarely takes place. As a result of covalent bonding or strong force of interaction between drug and target protein, the event of carcinogenicity or cellular toxicity takes place.

Factors Affecting Protein–Drug Binding

(a) Drug-related factors: It includes physicochemical characteristics of the drug, concentration of drug in the body, and affinity of a drug.

(b) Protein/tissue-related factors: It includes physicochemical characteristics of protein or drug and concentration of protein or drug.

(c) Drug interactions: It contains allosteric changes in a protein molecule, competition between drugs to occupy the binding site, and competition between drug and biological components.

(d) Patient-related factors: It includes the age of the patient, inter-subject variabilities such as due to genetics, environmental factors, and disease states.

Altogether, more protein binding disturbs the absorption and also decreases the distribution and metabolism of drugs.

Role of Water Molecules

In the last 10–15 years, the significance of water molecules in drug design and protein structures has become of extensive interest. Traditionally, water molecules play two crucial roles in ligand binding. Water molecules stabilize a protein–ligand complex by contributing hydrogen bond interaction between a ligand and a protein. The second role is that water can be displaced by ligands on binding with the target protein. The role of the water molecule in binding interaction of a ligand with the active site of the target protein can be studied using the molecular dynamics simulation. Slight changes in water-based hydrogen bonding networks affect ligand–protein interaction energies and show the effect of solvation or water molecule on the binding. Water molecules also determine the binding or rejection of ligand to the binding site of proteins. Water molecules can mediate to direct interactions or may cause an effect of electrostatic screening.

Drug–Nucleic Acid Interactions

Nucleic acids are the carrier of genetic information, and hence they are an important molecule for disease prevention. Nucleic acids are targeted for various diseases, including various types of cancer. DNA, the carrier of genetic information in humans, is mutated in various diseases, which often result in gene expression alteration. The structures of DNA can be used for designing small molecules to regain the gene expression pattern. The small molecules which bind to DNA can be categorized into two major classes: (1) covalent binder and (2) non-covalent binder. The non-covalent binders can further be classified into major groove binders, minor groove binders, and intercalators. On the other hand, RNA itself regulates various activities from catalysis to gene expression, which makes RNA a suitable target for binding. Since the structure of RNA is highly variable, designing a small molecule against them is a challenging task. The advancement in technology and growth in the structures of protein–nucleic acid complexes help the computational tools to predict the binding of small molecules with nucleic acids.

Receptor Pharmacology

1. Agonist: The drug which show affinity for the receptor and activates the receptor to produce regular physiological function are called as agonist. Agonist molecules mimics the effects of endogenous mediators.

(a) Full agonist: The drug with similar pharmacological potential (similar affinity strength) compared with the natural ligand is called as full agonist.

(b) Partial agonist: The drug with less pharmacological potential compared to the natural ligand (irrespective of concentration) is called as partial agonist.

2. Inverse agonist: The drug showing affinity for the receptor and producing opposite pharmacological activity is called as inverse agonist. The molecule beta-carboline and diazepam shows similar affinity for the benzodiazepine receptor. But, beta-carboline produce excitation of the receptor and diazepam produces inhibitory effect.

3. Antagonist: The drug which offer affinity for the receptor, but do not activates the receptor are called as antagonist.

(a) Competitive antagonist: The antagonist drug, which binds to the binding site of the natural ligand with similar affinity (blockade of natural ligand) and produce no biological response is called as competitive antagonist.

(b) Non-competitive : The antagonist drug which binds to the receptor on the site other than the binding site specific for agonist (allosteric binding site) is called as non-competitive antagonist. It induces the newer conformational change (different from the agonist conformation), hence natural ligand and agonist molecules do not find receptor (no biological response).

Kinetic analysis of ligand–receptor interactions

Theoretical background

Considerable evidence suggests that 7TM receptors can exist in two different coexisting conformational states: an ‘inactive’ state R and an ‘active’ state R^*. The relative proportion of R and Rp will depend on both the actual receptor–effector system and the presence of ligands (L) having affinity for the receptor, i.e. able to form complexes with R (LR) and R^* (LR^*). Only R^* and AR^* can couple with G-proteins to induce a cellular response (e.g. increase in cAMP levels). If, to simplify, the reversible interaction between the receptor and the G-protein is omitted, the minimal scheme depicting the relationship between A, R, R^*, LR and LR^* can be written as follows:

Scheme 1

where:

(1) g is an intrinsic allosteric constant (g = R^*/R), reflecting the relative proportions of R and R^* forms which coexist in the absence of any ligand L.

(2) Ka and a Ka are the association constants of L for R and R^*, respectively. These constants actually measure the affinity of the ligand L for the R and R^* forms of the receptor. The factor a denotes the differential affinity of L for R^*. This means that L, along with g, can modify the relative proportions of R, R^*, LR and LR^* (see Scheme 1).

Two main experimental situations can be observed (Fig.):

(1) In the absence of any ligand L, a cellular response can be easily measured. In such constitutively active systems (e.g. native or mutated receptors, cells highly transfected with a particular cloned receptor) a sufficient proportion of R^*, spontaneously present, binds to the G-protein to induce a cellular response (high basal level). In this case, ligands L are classified according to the value of the a constant, as full agonists and partial agonists (a > 0), neutral antagonists (a = 0) and inverse agonists (a < 0) (Fig.). Thus, the higher the absolute value of the a constant, the higher the cellular response. The a constant actually measures the intrinsic efficacy—positive, null or negative — of a particular ligand L for a given receptor–effector system. In this respect, a neutral antagonist with zero efficacy, does nothing to the receptor but bind to it and therefore preclude activation of the receptor by an agonist. As pointed out by Kenakin8 efficacy is ‘the property of a drug that modifies subsequent interaction of the 7TM receptor with other membrane proteins’. The inverse agonism patterns depicted in Fig.  have been observed with b-adrenergic ligands on basal adenylyl cyclase activity mediated by a constitutively active mutant of the b2-adrenergic receptor9 or on Sf9 cells overexpressed with b 2 wild-type receptors as well.

(2) A low basal level of activity is measured in the absence of any ligand L. In such quiescent systems (e.g. in general native wild-type receptors present on cellular or tissue membranes) inverse agonists cannot be revealed since they behave as neutral antagonists. In contrast, as in the former situation, full and partial agonists will induce a cellular response the magnitude of which depends on their efficacy (Fig).

Evolution of the cellular response as a function of the ligand and the receptor–effector complex. (A) Constitutively active systems. (B) Quiescent systems.

Quantitative measurement

As far as affinity and efficacy of drugs are concerned in their therapeutic activity in humans, the correct measurement of these parameters using well-chosen and reliable methods is highly important for drug discovery. These methods mainly, but not exclusively, concern radioligand-binding techniques and specific functional assays.

Radioligand binding

The simplest assumption about the reversible formation of a ligand–receptor complex (see left part of Scheme 1) is that it may be expressed according to the mass-action law, as the following chemical reaction:

where L is the ligand, k1 and k-1 are the rate constants for association and dissociation of the LR complex. Square brackets signify the concentration of the entity they enclose. At equilibrium (or steady state), the rates of association and dissociation of the complex LR are equal, and:

The equilibrium binding constant may then be defined as dissociation constant (kd) such as:

The total number of receptors (RT) is the maximum number of receptors on which the drug is able to bind specifically. This finite number, also termed Bmax, is the sum of the receptors engaged in forming the complex (LR), plus the free receptors (R), such as:

From equations (3) and (4) after rearranging:

If we now define LR as bound ligand = B, and L as free ligand = F, from equation (5):

Equation (7) is the Scatchard equation. Receptor labelling studies involve binding of a radioactive form of the ligand to receptor preparations of target tissues. Radiolabelled ligands — ideally neutral antagonists that are devoid of efficacy for the receptor used — must be pure, stable and have a high radiochemical specific activity. The most commonly used isotopes are tritium [³H] and radioactive iodine [¹²⁵I]. Radioactive ligand and receptor preparations are incubated in appropriate conditions: receptor concentration, temperature, pH and ionic strength. At the end of the incubation period, while the binding reaction is at equilibrium, bound and free ligands are promptly separated using filtration or centrifugation.

Experimental determination of kd and Bmax for a given radioligand and receptor preparation requires incubation of various concentrations of the radioligand with a fixed concentration of receptors: this is the so-called saturation experiment. Such experiments can be performed in the absence (control) or presence of various concentrations of non-radioactive (‘cold’) ligand or drug — also termed displacer — able to compete with the radioligand for the same receptor. Finally, interaction of displacers with a given receptor can also be studied by incubating various concentrations of the displacer with fixed concentrations of radioligand and receptors: this is the so-called displacement experiment (Fig.). In all cases, the non-specific binding is measured by the bound radioactivity remaining in the presence of a large excess of cold ligand. In most cases, data generated from a saturation experiment are analysed according to equation (7). When the radioactive ligand interacts with a single population of non-interacting receptors, the Scatchard-plot, i.e. B/F vs B, leads to a linear curve. Kd is estimated as the negative reciprocal of the slope of the line of best fit, and Bmax by the abscissa intercept of the line (Fig.). The reciprocal of Kd measures the association constant Ka (see Scheme 1) of the radioligand for the receptor. Thus, for a given ligand–receptor pair, the smaller the Kd (generally 0.1–10 nmol l^-1 or nM) the higher is its affinity. Bmax is expressed as pmol or fmol per mg tissue or protein. A downward curvilinear Scatchard plot may indicate that there is more than one type of binding site, and that the different types differ in their dissociation constants for binding with the radiolabelled ligand under investigation. Alternatively, the binding of ligand to its receptors may inhibit further binding and hasten dissociation from the receptor (e.g. insulin receptor). This latter phenomenon is called negative cooperativity. Positive cooperativity also occurs, i.e. the binding of ligand facilitates the binding of subsequent molecules to the receptors (e.g. nicotinic receptor). In this case, the Scatchard plot is upward concave. Cooperativity generally implies that the ligand behaves as an allosteric modifier that induces a conformational change in the structure of the receptor protein.

Analysis of ligand–receptor interactions. (A) Scatchard plot in absence (control) or presence of two different displacers. X and Y are competitive and non-competitive displacers, respectively. (B) Displacement curve.

When the saturation experiment is performed in the presence of a displacer, the line of best fit of the Scatchard plot can be modified in a manner that depends on the type of receptor interaction exhibited by the displacer. Two main cases exist: (1) decreased slope and unchanged Bmax, the displacement is of the competitive type; (2) unchanged slope and decreased Bmax, the displacement is of the noncompetitive type (Fig.A). Intermediate cases where both the slope and Bmax are modified also exist. Data generated from a displacement experiment are generally fitted by a sigmoid curve termed displacement or inhibition curve, i.e. percentage radioalabelled ligand specifically bound vs log [Displacer] (in mol 1^-1 or M) (Fig. B). The abscissa of the inflexion point of the curve gives the IC50 value, i.e. the concentration of displacer that displaces or inhibits 50% of the radioactive ligand specifically bound. IC50 is a measure of the inhibitory or affinity constant (Ki) of the displacer for the receptor. IC50 and Ki are linked as follows: if the displacement is of the competitive type, then,

Forces involved in drug-receptor complexes

According to the receptor theory of drug action, the common event in the initiation of pharmacological responses is the formation of a complex between the drug molecule and its site of action. Since most pharmacological responses are mediated through receptors, recognition of the more mobile drug molecules by the cellular receptor is the critical element determining the specificity of the response. There must be some forces that not only attract the drug to its receptor, but also hold it in combination with the receptor long enough to initiate the chain of events leading to the effect. Thus the combination of various chemical bonds is of great interest to drug potency.

·       Hydrophobic binding plays an important role in stabilizing the conformation of proteins and in the association of hydrophobic structure between the drug and the receptor.

·       Hydrogen bonding, which is strongly directional, has considerable importance in stabilizing structures by intramolecular bond formation. The formation of such bonds between a drug and a receptor can result in a relatively stable and reversible interaction. Such bonds are also involved in the maintaining of the tertiary structure of the receptor macromolecule and are thought to be involved in the specificity and selectivity of drug–receptor interaction.

·       Drug–receptor interaction often involves charge transfer complexes formed between electron-rich donor molecules and electron-deficient acceptors.

The angiotensin-converting enzyme (ACE) inhibitor, captopril interacts with ACE through electrostatic interactions.

(a) The carboxylic acid group (negatively charged, acidic) of the captopril binds to positively charged glutamate and lysine.

(b) The amide group of captopril establishes dipole-dipole interaction (hydrogen-bond) with histidine.

(c) The mercapto group makes dipole interaction with glycine.

(d) The ACE tyrosine residue bonds with carboxylic group of captopril through hydrogen-bond.

Electrostatic interaction between the captopril and angiotensin-converting enzyme (ACE).

·       Ionic bonds which are very ubiquitous are of importance in the actions of ionizable drugs since they act across long distances. The formation of an ionic bond results from the electrostatic attraction that occurs between oppositely charged ions. Most receptors have a number of ionizable groups (COO2, OH2, NH3þ) at physiological pH that are available for the binding with charged drugs.

Non-covalent interaction: Interaction between the adrenaline and adrenergic receptor through electrostatic, hydrogen-bond, van der Waals and hydrophobic interactions.

Electrostatic interactions are grouped based on the type of charge present in the molecules/atoms.

1. Charge-charge interaction

2. Charge-dipole interaction (ion-dipole)

3. Dipole-dipole interaction

4. Hydrogen bond interaction

5. Van der Waals interaction

6. Hydrophobic interaction

Charge-Charge Interactions

The deprotonation of acidic side chain groups present in aspartic acid and glutamic acid provides anionic environment. The protonation of basic side chain groups of lysine, arginine and histidine provides cationic environment.

Bond Strength: The bond strength of this type of interaction is in the range of -20 to -40 kJ/mol.

Salt bridge: The carboxylic group of glutamic acid and the ammonium group of lysine (charge centers are separated by 4 Å) makes ionic interaction. This interaction is known as a salt bridge and is essential for the bio-active conformation of the receptor. The ligand bound receptor establishes salt bridge.

Charge-Dipole (Ion-Dipole) and Dipole-dipole Interaction

Dipole moment: It indicates the movement of electrons along the bond. The higher electronegative atom draws electrons towards it and becomes electron rich centre (bears partial negative charge; d^˜). The other atom experiences electron deficiency and bears partial positive charge (d⁺).

The permanent dipoles of the carbonyl and amide groups of the peptide are structural determinants. The dipole-dipole (permanent) interactions are much weaker than charge-charge interactions. A dipole on one group induces dipole on neighbouring group and establishes much weaker dipole-induced dipole interaction.

Bond Strength: The partial charge of dipole is less than that of an ion; hence charge-dipole and dipole-dipole interactions are weaker than ionic bonds. In most cases these interactions provides a DG of –2 to –20 kJ/mol.

Hydrogen Bond Interactions

A strong electrostatic interaction between the hydrogen atom attached with a hetero atom and a hetero atom (or electro negative atom) is called as hydrogen-bond. The hetero atoms include oxygen, nitrogen and sulphur. The electronegative atoms namely fluorine and chlorine also behave as hetero atoms. The interaction between weakly acidic (partial positive) hydrogen bond donor (HBD) group and a hydrogen bond acceptor (HBA) group (partial negative) generates hydrogen bond.

The hetero atom attached with hydrogen (one or two) are known as hydrogen bond donors (HBDs). The hydrated hetero atom such as oxygen, nitrogen and sulphur are examples. A protonated tertiary (3°) nitrogen acts as a strong hydrogen bond donor (HBD). In biological systems these HBD and HBA are highly electronegative nitrogen (N) and oxygen (O) atoms (occasionally sulphur atoms). The heteroatom attached with no hydrogen is known as hydrogen bond acceptor (HBA). The tertiary (3°) nitrogen can act as a strong hydrogen bond acceptor (HBA). Each HBD forms interaction with two HBA (reverse is also true) and are responsible for the macromolecular structure. The hydrogen-bonding is a type of dipole-dipole interaction. The polarisable p electron system of aromatic system acts as weak acceptor and relatively weak acidic groups (-CH) acts as weak donor groups.

Bond strength: The association energies of hydrogen bonds range from -12 to -20 kJ/mol. The distance between the HBD and HBA groups are normally in between 2.7 to 3.1 Å.

·       Intramolecular hydrogen bond: The hydrogen-bond between different parts of a single molecule are known as intramolecular hydrogen bond. The intra molecular hydrogen bond generally form six membered ring structures (rigid) and increases the hydrophobicity behaviour of molecules. These types of bonds are known as bifurcated hydrogen bond. The multiple inter molecular hydrogen bonds introduce the conformational stability.

·       Intermolecular hydrogen bond: The hydrogen-bond between different molecules is known as intermolecular hydrogen bond. This kind of hydrogen -bond enhances the drug aqueous solubility.

Van der Waals Interactions

The non-covalent interactions between induced dipoles in electrically neutral molecules are collectively called as van der Waals force. The transient dipole of non-polar molecules induces dipole in the neighbouring group. The two non-polar molecules approach each other (close) due to their temporary polarizability. The attractive forces that hold non-polar molecules together in liquid phase are known as London dispersion forces. This type of forces is most common in interiors of protein (buried) and influences the conformation of the proteins.

Bond strength: The bond strength in the range of –2 to –4 kJ/mol (weaker) and is inversely proportional to the 7^th power of distance. In high molecular weight molecule the summation of these forces provides significant bonding.

Hydrophobic Interactions

It describes the tendency of non-polar molecules to transfer from aqueous phase into an organic phase. The association of non-polar drug molecule with the biological receptors results through hydrophobic interactions. A non-polar molecule cannot be solvated by water. The water molecules surrounding non-polar molecules associate to form quasicrystalline structure (ice-bergs). This facilitates the contact of non-polar part of drug molecules to the receptor. Bond strength: The association energies of hydrophobic interactions range from 0.1 to 0.2 kJ/mol.

Aryl-aryl interaction (p-p stacking): The stacking arrangement of an electron-poor and electron-rich aromatic ring offers charge transfer.

·       The covalent bonds are less important in drug–receptor interaction. Since bonds of this type are so stable at physiological temperature, the binding of a drug to a receptor through covalent bond formation would result in the formation of a long-lasting complex. Although most drug–receptor interactions are readily reversible, some drugs, such as anticancer nitrogen mustards and alkylating compounds form reactive cationic intermediates (i.e. aziridinium ion) that can react with electron donor groups on the receptor. Covalent bonds are also seen for example in the case of penicillin, which acylates a transpeptidase enzyme that is essential to bacterial cell-wall synthesis. In this case a long-lasting inhibition of bacteria replication is needed. Most drugs, however, induce a brief formation of a reversible drug–receptor complex.

Types of molecular interactions and their distance strength.

Recombinant versus In Situ Assays

The last decades have had a profound impact on how receptor pharmacology is performed. As Mentioned in the introduction, receptor cloning was initiated in the mid-eighties and today the vast majority of receptors have been cloned. Thus, it is now possible to determine the effect of ligands on individual receptor subtypes expressed in recombinant systems rather than on a mixture of receptors in, e.g., an organ. This is very useful given that receptor selectivity is a major goal in terms of decreasing side effects of drugs and development of useful pharmacological tool compounds which can be used to elucidate the physiological function of individual receptor subtypes. Furthermore, recombinant assays allow one to assay cloned human receptors which would otherwise not be possible. Most receptors have very similar human and rodent sequences, but due to the small differences in primary amino acid sequence there have been cases of drugs developed for rats rather than for humans, because the compounds were active on the rat receptor but not on the human receptor.

It should be noted that the use of organ and whole animal pharmacology is still required. As previously noted, the cellular effect of receptor activation depends on the intracellular contents of the proteins involved in, e.g., the signaling cascades. These effects can only be determined when the receptor is situated in its natural environment rather than in a recombinant system. In most situations, both recombinant and in situ assays are thus used to fully evaluate the pharmacological profile of new ligands. Furthermore, once a compound with the desired selectivity profile has been identified in the recombinant assays, it is important to confirm that this compound has the predicted physiological effects in, e.g., primary nonrecombinant cell lines, isolated organs, and/or whole animals.

Binding versus Functional Assays

Binding assays used to be the method of choice for primary pharmacological evaluation, mainly due to the ease of these assays compared to functional assays which generally required more steps than binding assays. However, several factors have changed this perception: (1) biotechnological Functional assays have evolved profoundly and have decreased the number of assay steps and increased the throughput dramatically, (2) functional assay equipment has been automated, (3) ligand binding requires a high-affinity ligand, which for many targets identified in genome projects simply does not exist, (4) binding assays are generally unable to discriminate between agonists and antagonists, (5) binding assays will generally only identify compounds binding to the same site as the radioactively labeled tracer. One important aspect of binding assays is the ability to determine ligand–receptor kinetics (on-rate, off-rate, and ligand residence time) which are important pharmacological properties affecting drug efficacy in vivo.

The Fluorometric Imaging Plate Reader (FLIPR™) illustrates this development toward functional assays. Cells transfected with a receptor coupled to increase in intracellular calcium levels (e.g., a Gαq-coupled GPCR or a Ca2+ permeable ligand-gated ion channel) are loaded with the dye Fluo-3 which in itself is not fluorescent. However, as shown in Figure, the dye becomes fluorescent when exposed to Ca2+ in the cell in a concentration-dependent manner. In this manner, ligand concentration–response curves can be generated on the FLIPR very fast as it automatically reads all wells of a 96-, 384-, or 1534-well tissue culture plate. Many other functional assays along these lines have been developed in recent years. Importantly, the majority of these assays can be applied on both recombinant and native receptor expressing cell lines.

(a) Relation between Ca2+ concentration and relative fluorescence intensity of the fluorescent probe fluo-3. (b) The 5-HT2B receptor subtype belongs to the superfamily of G protein-coupled receptors and is coupled to increase in inositol phosphates and intracellular Ca2+. Cells expressing 5-HT2B receptors were loaded with fluo-3 and the fluorescence was determined upon exposure to the endogenous agonist 5-HT (•) and the partial agonists MK-212 (◦) and 2-Me-5-HT (◾) on a FLIPR™.

Partial and Full Agonists

Agonists are characterized by two pharmacological parameters: potency and maximal response. The most common way of describing the potency is by measurement of the agonist concentration which elicit 50% of the compound’s own maximal response (the EC50 value). The maximal response is commonly described as percent of the maximal response of the endogenous agonist. The maximal response is also often described as efficacy or intrinsic activity which were defined by Stephenson and Ariëns, respectively. Compounds, such as 2-Me-5-HT and MK-212 in Figure, show a lower maximal response than the endogenous agonist and are termed partial agonists. The parameters potency and maximal response are independent of each other and on the same receptor it is thus possible to have, e.g., a highly potent partial agonist and a low potent full agonist. Both parameters are important for drug research, and it is thus desirable to have a pharmacological assay system which is able to determine both the potency and the maximal response of the tested ligands.

Antagonists

Antagonists do not activate the receptors but block the activity elicited by agonists and accordingly they are only characterized by the parameter affinity. The most common way of characterizing antagonists is by competition with an agonist (functional assay) or a radioactively labeled ligand (binding assay). In both cases, the antagonist concentration is increased and displaces the agonist or radioligand, which are held at a constant concentration. It is then possible to determine the concentration of antagonist, which inhibits the response/binding to 50% (the IC50 value). The IC50 value can then be transformed to affinity (K) by the Cheng–Prusoff equation:

Functional assay:

K = IC50/(1 + [Agonist]/EC50)                                 (1)

where

[Agonist] is the agonist concentration

EC50 is for the agonist in the particular assay

Binding assay:

K = IC50/(1 + [Radioligand]/KD)                              (2)

where

[Radioligand] is the radioligand concentration

KD is the affinity of the radioligand

It is important to observe that the Cheng–Prusoff equation is only valid for competitive antagonists.

The Schild analysis is often used to determine whether an antagonist is competitive or noncompetitive. In the Schild analysis, the antagonist concentration is kept constant while the agonist concentration is varied. For a competitive antagonist, this will cause a rightward parallel shift of the concentration–response curves without a reduction of the maximal response (Figure-a). The degree of right-shifting is determined as the dose ratio (DR), which is the concentration of agonist giving a particular response in the presence of antagonist divided by the concentration of agonist that gives the same response in the absence of antagonist. Typically, one will chose the EC50 values to calculate the DR. In the Schild analysis, the log (DR-1) is depicted as a function of the antagonist concentration (Figure-b). When the slope of the curve equals 1, it is a sign of competitive antagonism and the affinity can then be determined by the intercept of the abscissa. When the slope is significantly different from 1 or the curve is not linear, it is a sign of noncompetitive antagonism, which invalidates the Schild analysis.

Schild analysis of the competitive antagonist S16924 on cells expressing the 5-HT2C receptor.

(a) Concentration–response curves of the agonist 5-HT were generated in the presence of varying concentrations of S16924. Note the parallel right shift of the curves and the same level of maximum response.

(b) Dose ratios are calculated and plotted as a function of the constant antagonist concentration generating a straight line with a slope of 1.00 ± 0.012. These results and the observations from (a) are in agreement with a competitive interaction and the antagonist affinity can thus be determined by the intercept of the abscissa; K = 12.9 nM.

As shown in the example in Figure, five concentration–response curves are generated to obtain one antagonist affinity determination, illustrating that the Schild analysis is rather work-intensive compared to, e.g., the transformation by the Cheng–Prusoff equation where one inhibition curve generates one antagonist affinity determination. However, the latter cannot be used to determine whether an antagonist is competitive or noncompetitive, which is the advantage of the Schild analysis.

When testing a series of structurally related antagonists one would thus often determine the nature of antagonism with the Schild analysis for a couple of representative compounds. If these are competitive antagonists, it is reasonable to assume that all compounds in the series are competitive and thus determine the affinity of these by the use of the less work-intensive Cheng–Prusoff equation.

Constitutively Active Receptors and Inverse Agonism

Most receptors display no or only minor basal activity but some receptors display increased basal activity in the absence of agonist, which has been referred to as constitutive activity. Interestingly, it has been shown that inverse agonists can inhibit this elevated basal activity, which contrast antagonists that inhibit agonist-induced responses but not the constitutive activity (Figurea). Examples of important constitutively active receptors include the human ghrelin receptor and several viral receptors that display constitutive activity when expressed in the host cell. This latter group includes the ORF-74 7TM receptor from human herpesvirus 8 (HHV-8), which show a marked increased basal response when expressed in recombinant cells (Figure 12.13b). ORF-74 is homologous to chemokine receptors and does indeed bind chemokine ligands. As shown in Figure 12.13b, chemokines display a wide range of activities on the receptor from full agonism (e.g., GROα) to full inverse agonism (e.g., IP10), which correlates with the angiogenic/angiostatic effects of the chemokines. Constitutive activity can also be caused by somatic mutations. Known examples include constitutively activating mutations in the thyrotropin receptor and the luteinizing hormone receptor which leads to adenomas, and the rhodopsin receptor which leads to night blindness.

(a) The nomenclature of ligand efficacies and schematic illustration of their concentration dependent effects on constitutive activity. (b) Ligand regulation of the constitutively active ORF-74 receptor from human herpesvirus 8 (HHV8). ORF-74 is a G protein-coupled receptor coupled to phosphatidylinositol (PI) turnover, which is regulated by a variety of human chemokines ranging from full agonism by GROα to full inverse agonism by IP10.

Allosteric Modulators

Allosteric modulators can both be stimulatory or inhibitory (noncompetitive antagonists) and typically these compounds bind outside the orthosteric binding site (Figure). Allosteric modulators have a number of potential therapeutic benefits compared to agonists and competitive antagonists which has led to significant increased pharmaceutical interest in recent years. This increased interest has also been fueled by the development of functional high-throughput screening assays which has made it possible to screen for allosteric modulators.

The allosteric modulators mentioned below act through allosteric mechanisms as evident from the fact that they do not displace radiolabeled orthosteric ligands. Furthermore, their activity is dependent on the presence of agonists as they do not activate the receptors by themselves. The fact that they bind outside of the orthosteric ligand binding pocket often leads to increased receptor subtype selectivity. Evolutionary pressure has led to conservation of the orthosteric binding site at different subtypes, as radical mutations would severely impact the binding properties. Thus, it is often seen that the orthosteric binding site is much more conserved than the remaining part of the receptor and accordingly, ligands binding to an allosteric site have a higher chance of being selective. Likewise, the allosteric ligands will have a different pharmacophore than the endogenous ligand which might improve, e.g., bioavailability. For example, ligands acting at the orthosteric site of the GABAA receptor need a negatively charged acid function and a positively charged basic function which greatly impairs the transport through biomembranes, whereas allosteric ligands such as the benzodiazepine diazepam does not have any charged groups and show excellent bioavailability. It is well known that many agonists, particularly full agonists, lead to desensitization and internalization of receptors (Fig). Unlike agonists, the positive modulators should prevent the development of tolerance (as seen for, e.g., morphine), because they merely potentiate the endogenous temporal receptor activation pattern and avoid prolonged receptor activation leading to desensitization and internalization. The fact that the receptors are stimulated in a more natural way by positive modulators rather than the prolonged receptor activation caused by agonists may also lead to a difference in physiological effects which may or may not be an advantage.

Schild analysis of the noncompetitive antagonist fenobam on cells expressing the metabotropic glutamate receptor subtype mGluR5. Concentration–response curves of the agonist quisqualate were generated in the presence of varying concentrations of fenobam. In contrast to the Schild analysis shown in one before previous Figure, a clear depression of the maximal response is seen with increasing antagonist concentrations. This shows that the antagonist is noncompetitive. The localization of the orthosteric and allosteric binding sites is depicted in previous Figure.

Negative Allosteric Modulators (Noncompetitive Antagonists)

As noted in the previous section, the Schild analysis is very useful to discriminate between competitive and noncompetitive antagonists, and an example of the latter is shown in Figure. Fenobam is a selective antagonist at the mGluR5 receptor, and the Schild analysis clearly demonstrates that the antagonism is noncompetitive due to the depression of the maximal response (compare Figures). As noted previously, glutamate binds to the large extracellular amino-terminal domain whereas fenobam has been shown to bind to the extracellular part of the 7TM domain. Fenobam does not hinder binding of glutamate to the extracellular domain, but hinder the conformational change leading to receptor activation.

Positive Allosteric Modulators

Positive allosteric modulation can be achieved through several mechanisms. For example, benzodiazepines positively modulate the GABAA receptor by increasing the frequency of channel opening. Positive modulation can also be obtained by blocking receptor desensitization as exemplified by cyclothiazide.