Transcript lect13a

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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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
-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
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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
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 5,00010,000 related compounds that were synthesized/tested
-process has been made systematic through structureactivity relationships (SARS)
-SARS: the determination by synthesis and screening of
which groups on a lead compound are important for drug
function
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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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-typically QSAR depends
on the chemical properties
of a variety of subsituents
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-pK values, van der Waals
radii, H-bonding energy,
and conformation
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
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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-manycompounds-and-see-what-they-do approach
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-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 5
CA 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
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-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 paminobenzoic acid (PABA),
essential for bacterial growth
5,6,7,8-tetrahydrofolate
-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
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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®
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