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Lecture #13: Enzyme Mechanisms-III: Techniques for Drug Discovery

A. Drug Design: Improvements in medical care are largely attributed to development of wide variety of drugs including:

-antibiotics, anti-inflammatory agents, analgesics and anesthetics, antidepressants, antipsychotics,

immunosuppressants, cancer drugs, cardiovascular and stroke drugs, etc.

B. Techniques of Drug Discovery: -most drugs act by modifying the function of a particular receptor in the body or an invading pathogen

-receptor: enzyme, channel or signalling protein

-compounds that modify the receptor function = agonists

-compounds that bind without modifying function, but block agonist binding = antagonists

-biochemical and physiological effects of a drug and its mechanism of action are called pharmacodynamics

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-as the number of drug candidates is reduced then testing in animals are employed

-a drug candidate that exhibits a desired effect is called a lead compound

-a good lead compound binds to its target with a Kd < 1 M

-high affinity is necessary to minimize nonspecific binding to other macromolecules to ensure that low doses are required

Re: for enzyme inhibitors the Kd = KI

1. Complexity of Drug Discovery

-most drugs were discovered by screening large numbers of synthetic compounds and natural products for the desired effect-natural products are discovered by fractionation of the organism in which they occur and isolating the “active ingredient”-in vitro screens are initially used such as the binding of a drug to a target enzyme implicated in the disease

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2. SARS and QSARS

-lead compound is the starting point to design more efficacious compounds

-even minor modifications to a drug candidate can result in major changes in its pharmacological

properties

-might place methyl, chloro, hydroxyl, or benzyl groups at various places on the lead compound

-most successful drugs today are the result of 5,000-10,000 related compounds that were synthesized/tested

-process has been made systematic through structure-activity relationships (SARS)

-SARS: the determination by synthesis and screening of which groups on a lead compound are important for drug function

Measures of a Drug’s effectiveness IC50: the [inhibitor] at which an enzyme exhibits 50% of its maximal activity (vi/vo = 0.5) ED50: the effective dose of a drug to produce a therapeutic effect in 50% of a test sample TD50: the toxic dose of a drug to produce a particular toxic effect in animals LD50: mean lethal dose of drug required to kill 50% of a test sample therapeutic index: is the TD50/ED50 ratio

Drugs that have high therapeutic indices are the best

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QSAR-quantitative structure-activity relationship

-there is a simple mathematical relationship between the biological activity of a drug and its physiochemical properties

-e.g., if the hydrophobicity of a drug is important for its biological activity then changing the substituents on the drug to alter its nonpolar character will affect its activity

-hydrophobicity is measured by P (partition coefficient) between two immiscible solvents where

P = [drug]oct/[drug]H20

-if C is [drug] to achieve a specified level of biological function then activity may be expressed as 1/C

-plot of 1/C vs log P for a series of derivatives having a small range of log P values often indicates a linear relationship

log(1/C) = k1logP + k2

where k1 and k2 are constants whose optimum values in QSAR can be determined by computerized curve fitting

-for compounds with a larger range of logP values, the plot of log1/C vs log P will have a maximum value and will be described by a quadratic equation

log(1/C) = k1(logP)2 +k2logP + k3

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3. Structure-based drug design

-also called rational drug design uses the structure of a receptor in complex with a drug candidate to guide the development of more efficacious compounds

-method is possible due to dramatic advances in the speed and precision with which a macromolecular structure can be determined by X-ray crystallography and NMR

-structures reveal positions of H-bonding donors/acceptors in receptor’s binding site and cavities where substituents may be placed on a drug candidate to increase its binding affinity

-may be supplemented with molecular modelling tools

-typically QSAR depends on the chemical properties of a variety of subsituents

-pK values, van der Waals radii, H-bonding energy, and conformationV&V:F15-29

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such as energy minimization, quantum mechanical calculations, docking simulations

-process is iterative: structure of receptor in complex with improved properties is determined to further improve its properties

4. Combinatorial Chemistry and High-Throughput Screening

-recent advances in combinatorial chemistry to rapidly and inexpensively synthesize large numbers of compounds and development of robotic high-throughput screening techniques has facilitated this make-many-compounds-and-see-what-they-do approach

-previously, investigations of a hydrophobic substituent might have involved ethyl, propyl, benzyl derivatives

-through combinatorial synthesis perhaps 100 different groups may be added to a single position

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C. Introduction to Pharmacology

-in vitro development of an effective drug candidate is only the first step in the development process

-a useful drug must also be delivered in sufficiently high concentration to the target receptor in the human body without causing unacceptable side effects

1. Pharmacokinetics

-refers to the ways in which a drug interacts with various cell barriers

-most convenient form of drug delivery is orally

-drugs must pass a series of barriers

-chemically stable in the acidic pH of stomach and not be degraded by digestive enzymes

-must be absorbed into the bloodstream and pass several membranes

-must not bind too tightly to other substances in the body

-must survive modification by a battery of enzymes (mainly in the liver)

-avoid rapid excretion by the kidneys

-must pass from capillaries to the target tissue

-if target is in brain then must pass blood-brain barrier

-if target is intracellular then must pass through the plasma membrane

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-bioavailability of a drug (extent to which it reaches its site of action) depends on both the dose given and its pharmacokinetics

-Lipinski’s rule of five states that a compound is likely to exhibit poor absorption or permeation if:

-its molecular weight is greater than 500 Da

-it has more than 5 H-bond donors

-it has more than 19 H-bond acceptors

-its value of log P is greater than 5CA Lipinski, Adv. Drug Del. Rev. 1997, 23, 3

-most drugs are a compromise: they are neither too lipophilic nor too hydrophilic

-pK values usually in the range of 6 - 8

2. Toxicity and Adverse Reactions Eliminate Most Drug Candidates

-final criteria for a drug candidate are that its use be safe and efficacious in humans

-tests initially carried out in animals but since humans and animals often react quite differently then ultimately the drug must be tested in clinical trials

-in US, clinical trials are monitored by the FDA and have 3 phases

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a. Phase I

-designed to test the safety of a drug candidate but also to determine the dosage range and optimal dosage method

-carried out on a small number of normal, healthy volunteers except for highly toxic drugs (e.g., a cancer chemotherapeutic agent) where it is carried out on volunteer patients with the target disease

b. Phase II

-tests the efficacy of the drug against the target disease in 100 to 500 volunteer patients but also refines the dosage range and checks for side effects

-usually tested via single blind tests in which the patient is unaware of whether he/she has received the drug or a placebo (an inert substance)

c. Phase III

-monitors adverse reactions from long-term use as well as confirming efficacy in 1000 to 5000 patients

-tests the drug candidate against control substrances through statistical analysis of carefully designed double blind tests in which neither the patients or the investigators know whether a given patient has received the drug or a control substance

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3. Drug Candidate Statistics

-5 drug candidates in 5000 that enter preclinical trials reach clinical trials

-1 of the 5 is approved for clinical use

-40% of candidates pass Phase I trials

-50% of those passing Phase I trials pass Phase II trials

-most drugs that enter Phase III pass this trial

-preclinical portion of drug discovery process averages 3 years

-successful clinical trials usually require 7 – 10 years

-becoming increasingly expensive to bring a drug to market, on average the cost is $300 million USD

-not uncommon for a drug to be withdrawn some months or years later when it is found to have caused unanticipated life-threatening side effects in as few as 1 in 10,000 individuals

4. Cytochrome P450 Metabolize Drugs

-why is it that drug that is well tolerated by most patients can pose a danger to others?

-differences in reactions to drugs arise from genetic differences , different disease states, other drugs that they are taking, age, sex, and environmental factors

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-cytochrome P450 role is to detoxify xenobiotics and assist in metabolic clearance of drugs

-humans express ca. 100 isoenzymes that catalyze the general reaction:

RH + O2 + 2H+ + 2e- ROH + H2O

-many cytochrome P450 in humans are polymorphic with several common alleles

-gives rise to tremendous variation in drug metabolism among individuals

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-acetaminophen is a widely used analgesic and antipyretic (fever reducer)

-safe in therapeutic doses (1.2g/day for an adult) but in large doses (> 10g) is highly toxic

-usually 95% of acetaminophin is enzymatically glucuronidated or sulfated at its OH group to form conjugates which are readily excreted

-remaining 5% is converted by cytochrome P450 (CYP2E1) to acetimidoquinone which is conjugated with glutathione

-upon large doses of acetaminophin, the glucuronidation and sulfation pathways become saturated and the cytochrome P450-mediated pathway becomes more significant

-if hepatic glutathione is depleted faster than it can be replaced, acetimidoquinone, a reactive compound, instead conjugates with the sulfhydryl groups of cellular proteins causing often fatal hepatotoxicity

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5. Many Drugs are Enzyme Inhibitors

-modern drug therapy is based on concepts of enzyme inhibition

-toxicity is unavoidable because, with the exception of cell wall synthesis in bacteria, most drugs that kill tumors, viruses, and bacteria also kill normal eukaryotic cells within the host

-relatively short generation time of the microbial organisms and tumor cells can be exploited

-these cells will be more sensitive to metabolic inhibitors than the host eukaryotic cells

a. Sulfa Drugs-sulfanilamide is an antibacterial

agent because it completes (competitive inhibitor) with p-aminobenzoic acid (PABA), essential for bacterial growth

-bacteria can’t absorb folic acid but must synthesize it

-bacterial dihyropteroate synthase is tricked into making an intermediate containing sulfaniliamide that can’t be converted to folate

-bacteria is starved of the required folate and can’t grow or divide

-since humans obtain folate from dietary sources, the sulfanilimide is not harmful at doses that kill the bacteria

5,6,7,8-tetrahydrofolate

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b. Viagra®-an unexpected outcome in a drug-design program

-sometimes the outcome of a drug design program is unanticipated

-e.g., penicillin discovery by Fleming

-relaxation of smooth muscle cells of blood vessels is controlled by intracellular [Ca2+] as triggered by [cGMP]

-cGMP is hydrolyzed by phosphodiesterases (PDE) to form 5’-GMP and let muscles contract again

-Pfizer designed PDE inhibitor against PDE5 isotype, a dominant form in human vascular tissue

-no significant benefits for angina or hypertension but some men in clinical trials reported penile erection

-apparently, Viagra® causes an increase in [cGMP] in penile vascular tissue allowing vascular muscle relaxation, improved blood flow and erection. A drug was born!

5’-GMP

Viagra®