COMPUTER- AIDED DRUG DESIGN

CONTENTS

1. INTRODUTION TO COMPUTER-AIDED DRUG DESIGN

*Introduction

*How drugs are discovered

2. QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP (QSAR)

*Parameters

*Quantitative models

3. USES OF COMPUTER GRAPHICS IN COMPUTER-ASSISTED DRUG DESIGN

*Molecular modeling

*Molecular mechanics

*Quantum mechanics

4. IMPORTANT TECHNIQUES FOR DRUG DESIGN

*X-Ray crystallography

*NMR spectroscopy

5. APPLICATIONS

6. CONCLUSION

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7. REFERENCES

INTRODUCTION TO COMPUTER-AIDED DRUG DESIGN

INTRODUCTION

Although the phrase computer-aided drug design may seem to imply that drug
discovery lies in the hands of the computational scientists who are able to manipulate
molecules on their computer screens, the drug design process is actually a complex and
interactive one, involving scientists from many disciplines working together to provide
many types of information. The modern computational and experimental techniques that
have been developed in recent years can be used together to provide structural
information about the biologically active molecules that are involved in disease
processes and in modulating disease processes.

HOW DRUGS ARE DISCOVERED

Occasionally new drugs are found by accident. More frequently they are
developed as part of an organized effort to discover new ways to treat specific diseases.
The discovery of new pharmaceutical agents has gone through an evolution over the
years and has been adding new technologies to this increasingly complex process1.

1. Screening for new drugs

The traditional way to discover new drugs has been to screen a large number of
synthetic chemical compounds or natural products for desirable effects. Although this
approach for the development of new pharmaceutical agents has been successful in the
past, it is not an ideal one for a number of reasons.

The biggest draw back to the screening process is the requirement for an
appropriate screening procedure. Although drugs are ultimately developed in the clinic, it
is usually inappropriate to put chemicals of unknown efficacy directly into humans.
Consequently, other systems have to be developed. Normally a battery of screens is used
to select potential new drug candidates, with activity in initial, rough screens feeding

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compounds into later, more sophisticated screens. Initial screens are often in vitro tests
for some fundamental activity, such as the ability to kill bacteria in solution. Ultimately,
however, more applicable in vivo screens are needed. This second level of screening is
normally carried out using animal model systems for the disease.

Screens have inherent limitations2. Primary screens are used for large number of
chemicals to choose which compounds should be further tested with more sophisticated
tests. If the primary screen does not select for an appropriate activity, however, an active
structure will appear to be inactive and will not be discovered. Secondary screening in
animal model systems has additional problems, such as

1. The animal model may not accurately reflect the human disease

2. The chemical may be extensively metabolized to a different compound in the

animal before it reaches its target

3. The chemical may not be absorbed or distributed as it is in humans.

In each of these cases, the active structure potentially will not be identified.

Another serious problem with the screening process is that, because of its
random nature, it is inherently repetitious and time-consuming just to find a chemical
with the desired activity.

Furthermore, chemical compounds discovered by this approach commonly do not have

optimal structures for modulating the biological process. This in turn may require
administration of larger quantities of the drug and increase the risk of unwanted side
effects.

The major advantage of screening is the larger amount of information that

is not needed to carry out the process. One does not need to know the structure of the
drug being sought. Nor does one need to know the structure of the target upon which the
drug will act. Most importantly, one does not need to know about the underlying
mechanism of the disease process itself.

2. Modifications for improvements

Once an active (lead) compound has been identified and its chemical structure
determined, it is usually possible to improve on this activity and/or to reduce side effects

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by making modifications to the basic chemical structure. Modifications to improve
performance are often carried out using chemical or biofermentative means to make
changes in the lead structure or its intermediates. Alternatively, for some natural
products, the gene itself may be engineered so that the producer organism synthesizes the
modified compound directly.

The process of developing drugs via modification of active lead compounds

requires the structure of the compound to be known. One still does not need to know the
structure of the target on which the drug works. Likewise, no information about the
underlying disease process is required.

As with screening, the process of modification is often based on a primarily trial-

and-error approach. Because more information is known, however, this process can be
carried out with much greater probability of success than a purely random process. A
prime example of the power of this approach is in the anti-infective area where
modifications of the original first generation cephalosporins have led to second and now
third generation offspring with substantially improved characteristics3.

The limitations of this process are inherent to the fact that one is using a single lead
compound as the basis for further drug design. Improvements are likely however, no
major breakthrough in developing new chemical entities (NCEs) is probable. Further, if
the original lead compound fails to generate a desirable drug, one must start the process
over again by finding a new lead molecule.

3. Mechanism-based drug design

As still more information becomes available about the biological basis of a disease,
it is possible to begin to design drugs using a mechanistic approach to the disease
process. When the disease process is understood at the molecular level and the target
molecule(s) are defined, drugs can be designed specifically to interact with the target
molecule in such a way as to disrupt the disease1-6.

Clearly a mechanistic approach to drug design requires a great deal of knowledge.

Furthermore, processing this knowledge in such a way that a scientist can use the
knowledge to develop a new drug is a formidable task. The major breakthroughs in drug
design in the future are most likely to come via the use of this approach7. Because of the
massive amount of information that must be harnessed to develop drugs by this

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technique, it is in this area where computer-aided drug design will have its greatest
impact.

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4.Combining technique

The various techniques for finding new drugs, it is important to remember that drug
discovery is both a cumulative and a reiterative process8. Potential drugs developed by
modifying a lead structure are certain to be sent through selective screening processes to
confirm activity and select for the best candidate to go on for further development.
Likewise, drugs developed mechanistically will likely be both screened and later
modified in order to produce the best candidate drug.

Furthermore, every new chemical entity that affects the disease process whether
found by accident, screening, modification, or mechanistic design provides useful
information for developing still better compounds. This is true whether the chemical has
positive or negative effects on the disease process9. Each new chemical increases the data
base of information about the disease-target-drug interaction. This in turn is the basis for
rational drug design10.

THE BASICS OF MECHANISTIC DRUG DESIGN

Most diseases affecting man have been identified by their clinical manifestations.
Thus we are familiar with medical conditions such as hypertension, cancer, infections,
etc. Modern biological techniques now have enabled researchers to study such diseases
at the molecular level and to identify the processes or molecules responsible for
producing the clinical effects.

A. Defining the disease process

The first step in the mechanistic design of drugs to treat diseases is to determine
the biochemical basis of the disease process. Ideally, one would know the various steps
involved in the physiological pathway that carries out the normal function. In addition,
one would know the exact step(s) in the pathway that are altered in the diseased state.
Knowledge about the regulation of the pathway is also important. Finally, one would
know the three-dimensional structures of the molecules involved in the process.

B. Defining the target

There are potentially many ways in which biochemical pathways could become
abnormal and result in disease. Therefore, knowledge of the molecular basis of the

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disease is important in order to select a target at which to disrupt the process. Target for
mechanistic drug design usually fall into three categories: enzymes, receptors and
nucleic acids.

1.Enzymes as targets:

Enzymes are frequently the target of choice for disruption of a disease. If a

disease is the result of the overproduction of a certain compound, then one or more of the
enzymes involved in its synthesis can often be inhibited, resulting in a disease in
production of the compound and disruption of the disease process. This is the theoretical
basis behind the design of both the angiotensin-converting enzyme inhibitors and the
rennin inhibitors. Inhibition of either of these enzymes, which are in the same
biochemical pathway, decreases the production of angiotensin II and consequently
reduces blood pressure. In other instances specific enzymes may be required for
pathogenic micro organisms or cancerous cells to live and grow, thereby causing disease.
Inhibition of such enzymes would prevent the growth of these microbes or cells and
hence reverse the disease. Such is the case with the enzyme dihydrofolate reductase.

Enzymes are usually the targets of choice because they are relatively small,
aqueous-soluble proteins that often can be isolated for study. When enough of the
enzyme is difficult to obtain from its natural source, genetic engineering techniques are
frequently utilized to provide material for conducting X-ray crystallography, NMR
spectroscopy and enzyme kinetics. Ultimately the data obtained by these techniques
allow one to determine the

three-dimensional structures of the enzyme molecule in its active conformation. These

structures provide a starting point for the design of new effector molecules by computer
graphics and molecular modeling techniques.

RECEPTORS AS TARGETS:

Sometimes a disease can be modulated by blocking the action of an effector

at its cellular receptor. A classic example of this is the well-known inhibition of the
gastric histamine-2 receptor by the drug cimetidine which decreases acid secretion in the
stomach and reduces ulcer formation. Unlike enzymes, which often circulate in the body
and can be isolated and studied outside their biological environment, cellular receptors
consist of proteins imbedded in a surface membrane. Consequently these targets are
difficult to isolate and thus it is difficult to determine their structures. Nonetheless,

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molecular biological techniques are beginning to produce these macromolecules in larger
amounts. Structural information will soon be available for many of them, using the same
experimental techniques used for determing enzymes structures.

Receptors that are easily isolated are the most amenable to rational design of
effectors. An illustrative use of this concept is in the three-dimensional structural
determination of rhinoviruses, which then can serve as a receptor-type target for the
design of antiviral drugs.

Nucleic acids as targets:

Diseases can also potentially be blocked by preventing the synthesis of

undesirable proteins at the nucleic acid level. This strategy has frequently been employed
in the antimicrobial and antitumor areas, where DNA blocking drugs are used to prevent
the synthesis of critical proteins. Since the microorganisms or tumor cells cannot grow
and/or replicate, the disease process is effectively blocked.

Eaxmples include the use of the DNA intercalating drug adriamycin to treat
certain forms of cancer.

C. DEFINING THE RECEPTOR

Effector molecules are compounds that can occupy an active site of a target
molecule. As used in this context, they can be substrates, natural effectors that regulate
the target I positive or negative ways or drugs. Effector molecules and their targets
interact with each other via a lo0ck and key type of mechanism, in which the target
enzyme or receptor is the lock and the effector is the key. Implicit in this concept is that
the two fit together in a physically complementary fashion. Therefore, it should be
possible to determine the shape of the mutual contact surface of either by knowing the
three-dimensional conformation of the active portion of one.

In reality the relationship between the effector and target is more complex. The
natural effect or molecule fit into the effective site of enzyme or the binding site of the
receptor in a manner that maximizes the complementarity’s of the two molecules. In
addition, this complementarity not only recognized as a function of shape, that also
includes the interaction of charged regions, hydrogen bonding hydrophilic interactions,
etc. Because of the interactions between effector and its target are so complex , the best
information for designing drugs is obtained when one can determine the three-
dimensional structure of both the target and effector molecules. However, since effector

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molecules are often much smaller and are more readily available than their targets, they
are ususally more amenable to structural analyses. Again the information obtained from
experimental techniques provides the spatial coordinates that are utilized in the
computerized analyses of effectors structure.

D. DESIGNING NEW DRUGS TO EFFECT TARGETS

To make a good drug, a compound should exhibit a number of useful

characteristics. In addition to producing the desired effect, it should be sufficiently potent
that large amounts donot have to be administered. It should have low toxicity and
minimal side effects. Drugs that have to be given for chronic conditions should have
considerable residence time in the body(half life) so that continuous administration is not
needed. Oral administration of the drug is the preferred route in order to encourage
patient compliance.

In the normal condition, natural effectors interact with their targets to carry
out a needed physiological function. The natural effectors for a target thus often
represent an optimal structure for the complex formed. These natural molecules are not
often used as drugs, however, for a number of reasons. The body generally has the ability
to produce these effectors, when ever they are needed to modulate a physiological
process. Once they have fulfilled their functions, they are rapidly removed via.,
metabolic and elimination mechanisms. Natural effectors also generally are not orally
active. The metabolic instability built into the molecule to facilitate natural inactivation.
Often allows it to be degraded by enzymes in the gastrointestinal tract. Even when
natural effector survive this process, they typically donot have the properties necessary to
pass through the gastrointestinal mucosa. Additionally, endogenous effectors frequently
interact with similar targets in a variety of systems. Thus, they tend to cause substantial
unrelated side effects under conditions of high-level or long-term administration.

On the other hand, natural effectors molecules are often used as the starting
point for the development of new drugs, since they generally have selectivity and
potency for the desired target. By careful manipulation of the native structure, one can
frequently retain the binding characteristics of the effector. While designing in other
desirable characteristics. Examples of drug design with natural effectors as the starting
point include the use of the structure of luteinizing hormone-releasing hormone in the
design of LHRH receptor agonists such as the anticancer drug Leuprolide and the use of

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the structure of the Enkephalins in the design of opioid receptors agonists as potential
analgesics.

There are other sources for complimentary structures for enzyme and receptor
targets, which can also be used as a starting point, or to provide additional structural
information, for designing new drugs. If the natural effector is unavailable, similar
effectors from a different host may be used.

Example, the structure equine angiotensinogen was used in the development of

early human rennin inhibitors. Natural products, particularly those obtained from
microbes, often provide novel structures that are potent effectors.

Fro example, Pepstatin, a natural product produced by an actinomycete, is a

potent inhibitor of aspartic proteinases and therefore was useful in the design of rennin
inhibitors.

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 QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP (QSAR)

In 1968 Crum-Brown and Fraser published an equation which is considered to the

first general formulation of QSARs. In their investigation on different alkaloids they
recognized that alkylation of the basic nitrogen atom produced different biological
effects of the resulting quaternary ammonium compound, when compared to the basic
amines11. Therefore they assumed that biological activity must be the function of the
chemical structure.

BA=f[C]

Richet discovered that toxicity of organic compounds inversely follows their

water solubility. Such relationship shows that changing the biological activity (∆BA)
corresponds to the change in the chemical and physiological properties ∆C.

∆BA=f (∆C)

All the QSAR equation corresponds to equation2, because only the difference in
BA are quantitatively correlates with changes in lipophilicity and/or other
physiochemical properties of the compound under investigation.

QSAR involves the derivation of mathematical formula which relates the

biological activities of a group of compounds to their measurable physiochemical
parameters. These parameters have major influence on the drug’s activity. QSAR derived
equation take the general form

Biological activity=function {parameters}

Biological activity of a drug is a function of chemical features (i.e., lipophilicity,

electronic and steric) of the substituents and skeleton of the molecule. For example

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lipophilicity is the main factor governing transport, distribution and metabolism of drug
in biological system. Similarly electronic and steric features influence the metabolism
and pharmacodynamic process of the drug.

PARAMETERS

The various parameters used in QSAR studies are

1. Lipophilic parameters: Partition coefficient, chromatographic parameters and π-

substitution constant.

2. Polarizability parameters: Molar refractivity, Molar volume, Parachor

3. Electronic parameters: Hammett constant, Field and resonance parameters, parameters

derived from spectroscopic data, Charge transfer constant, Dipole moment, Quantum
chemical parameter.

4. Steric parameters: Taft’s steric constant, Vanderwaal’s radii.

5. Miscellaneous parameters: Molecular weight, Geometric parameters, Conformational

entropies, Connectivity indices, other topological parameters.

LIPOPHILIC PARAMETERS

Lipophilicity is defined by the partitioning of a compound between an aqueous

and a non-aqueous phase. Two parameters are commonly used to represent lipophilicity,
namely the partition coefficient (p) and lipophilic substitution constant (π). The former
parameter refers to whole molecule, while the latter is related to substituted groups.

PARTITION COEFFICIENT

A drug has to pass through a number of biological membranes in order to reach

its site of action. Partition coefficient is generally given as

P= [C]org

[C]aqu

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It is a ratio of concentration of substance in organic and aqueous phase of a two
compartment system under equilibrium conditions.

P= [C]org

[C]aqu (1-α)

α = degree of ionization.

The nature of the relationship between P and drug activity depends on the range of P
values obtained in the compounds used.

Log1/c=K1 logP+K2

Where

K1 and K2 are constants.

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Chromatographic parameters
When the solubility of a solute is considerably greater in one
phase than the other, partition coefficient becomes difficult to determine experimentally.
Chromatographic parameters obtained from reversed phase thin layer chromatography
are occasionally used as substituent for partition coefficient. Silica gel plate, being
coated with hydrophobic phases, is eluted with aqueous/organic solvent system of
increasing water content. The Rf values are converted into Rm value, which are the true
measure of lipophilicity from the following equation.

Rm = log (1/ Rf-1)

Rm value has been used as a substitute for partition coefficient in QSAR investigations.
The determination of Rm values offers many important advantages, as compared to the
measure of logP values.

• Compounds need not be pure.

• Only trace of materials needed.

• A wide range of hydrophilic and lipophilic congeners can be investigated.

• The measurement of practically insoluble analogs possesses no problem.

• No quantitative method for concentration determination needed.

• Several compounds can be estimated simultaneously.

The main disadvantages are

• Lack of precision and reproducibility.

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• Use of different organic solvent system renders the derivation of π and f
related scales are impossible.

POLARIZABILITY PARAMETERS

Molar refractivity

The molar refractivity is a measure of both the volume of a compound and

how easily it is polarized.

MR= (n2-1)M

(n2+2)d

Where

N is the refraction index

M is the molecular weight and

d is the density.

The term Mw/d defines a volume, while the term (n2-1) / (n2+1) provide a correction
factor by defining how easily the substituent can be polarized. This is particularly
significant if the substituent has a π electron or lone pair of electrons.

The significance of molar refractivity terms in QSAR equation of some ligand-

enzyme interaction could be interpreted with the help of 3D structure. These
investigation shows that substituent modeled by MR bind in polar areas, while
substituents modeled by π, bind in hydrophobic space. The positive sign of MR in QSAR
equation explains that the substituent binds to polar surface, while a negative sign or
nonlinear relationship indicates steric hindrance at the binding site.

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Parachor

The parachor [p] is molar volume V which has been corrected for forces of
intermolecular attraction by multiplying the fourth root of surface tension γ .

[p] = Vγ1/4 = M γ1/4

Where

M is molecular weight

D is the density

ELECTRONIC PARAMETERS

The distribution of electron in a drug molecule has a considerable influence on

the distribution and activity of the drug. In general, non-polar and polar drug in their
unionized form are more readily transported through membranes than polar drugs and
drugs in their ionized form. If the drug reaches the target site, the distributed electron
will control the type of bond that it forms with the target site, which in turn affects its
biological activity.

The Hammett constant (σ)

The distribution of electrons within a molecule depends on the nature of the

electron withdrawing and donating group found in the structure. Hammett used this
concept to calculate what now known as Hammett constant.

Hammett constant is defined as

σx= log KBX

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 KB

i.e., σx= log KBX- log KB

And so as pKa = -logKa

σx = p KB-pKBX

Where KB and KBX are the equilibrium constants for benzoic acid and mono substituted
benzoic acid respectively.

Hammett substitution constant (σ) is a measure of the electron withdrawing or

electron donating ability of a substituent. A negative value of σx indicates that the
substituent is acting as an electron donor and the positive value indicates that it is acting
as electron withdrawing group. Hammett constant takes into account both resonance and
inductive effect. Hammett constant suffer from the disadvantage that they only apply to
substituents directly attached to benzene ring.

Taft’s substituent constant

Taft’s substituent constant (σ*) are a measure of the polar effects of substituent
in aliphatic compound when the group in question does not form part of a conjugated
system. They are based on the hydrolysis of ester and calculated from the following
equation

σ* = 1/2.48 [log (k/ko)B - log(k/ ko)A]

Where

k represents the rate constants for the hydrolysis of the substituted compound

ko those of methyl derivative.

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The bracketed term with subscript B represent basic hydrolysis and A as acid hydrolysis
respectively. In Taft’s substituent constant only methyl group is the standard for which
the constant is zero. However, that can be compared with other constant by writing the
methyl group in the form CH2 – H and identifying it as the group for H. Taft’s and
inductive substituent constants are related as

σ*= 2.51σ i

STERIC SUBSTITUTION CONSTANT

For a drug to interact with an enzyme or to receptor, it has to approach to the

binding site. The bulk, size and shape of the drug may influence on this process. A steric
substitution constant is a measure of the bulkiness of the group it represents and its effect
on the closeness of constant between the drug and the receptor site.

Verloop steric parameter

Verloop steric parameter is called as sterimol parameter, which involves a

computer programme to calculate the steric substituent values from standard bond
angles, Vander Waals radii, bond length and possible conformation for substituents. It
can be used to measure any substituents.

For example the Verloop steric parameters for carboxylic acid group are demonstrated. L
is the length of the substituent while B1- B4 are the radii of the group.

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Charton’s steric constants

The principal problem with Vander Waal’s radii and Taft’s Es value is the
limited number of groups to which these constants have been allocated. Charton
introduced a corrected Vander Waal’s radius U in which the minimum Vander Waal’s
radius of the substituent group (rv(min) ) is corrected for the corresponding radius for
hydrogen (rvH), as defined by equation. They were shown to be a good measure of steric
effect by correlation with Es values.

U= rv(min) - rvH = rv(min) – 1.20

OTHER PARAMETERS

Molecular weight was used by Lein to improve the fit of parabolic Hansch
equation. A more appropriate use of MW was demonstrated in QSAR study of multidrug
resistance of tumor cells, where the MW term stands for the dependence of biological
activities on diffusion rate constant. The relationship between MW and volume implies
that 3√MW corresponding to linear dimension of size should be better than log MW.

Indicator variables sometimes known as dummy variables or de-nova

constant are used in linear multiple regression analysis to account for certain features,
which can not be described by continuous variables. It is used to account for other
structural features like intra molecular hydrogen bonding, hydrogen donor and acceptor

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properties, ortho effects, cis/trans isomers, different parent skeleton, different test
models etc.

QUANTITATIVE MODELS

To draw the QSAR equation with these parameters, it is simple to draw a

QSAR model with such property. But biological activity of most of the drug is related to
combination of physiochemical properties. Various methods are used to draw the QSAR
model. One among these models is Hansch analysis.

Hansch analysis (The extra thermodynamic approach)

This is the most popular mathematical approach to QSAR introduced by

Corwin Hansch. It is based on the fact that the drug action could be divided into two
stages.

• Transport of drug to its site of action.

• The binding of drug to the target site.

Each of these stages depends on the chemical and physical properties of the drug and its
target site. In Hansch analysis these properties are described by the parameters which
correlate the biological activity. The most commonly used physiochemical parameters
foe Hansch analysis are log p, π, σ and steric parameters as practically all the parameters
used in Hansch analysis are “linear free energy approach” or “extra thermodynamic
approach”.

If the hydrophobic values are limited to a small range then the equation will be linear as
follows.

log (1/c) = k1 log p + k2 σ + k3 E3 + k4

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 Where

k1, k2 and k3 are constant obtained by least square procedure, c is the molar
concentration that produce certain biological action.

The molecules which are too hydrophilic or too lipophilic will not be able to cross the
lipophilic or hydrophilic barriers respectively. Therefore the p value are spread over a
large range, then the equation will be parabolic and given as

log (1/c) = -k (logp)2 + k2logp + k3σ+ k4Es + k5

The constant k1 - k5 are obtained by least square method. Not all the parameters are
necessarily significant in a QSAR model for biological activity. To derive an extra
thermodynamic equation following rules are formulated by Hansch:

i. Selection of independent variables. A wide range of different parameter like

log p, π, σ, MR, steric parameters etc should be tried. The parameters selected for the
best equation should be essentials independent i.e., the intercorrelation coefficient should
be larger than 0.6-0.7.

ii. All the reasonable parameters must be validated by appropriate statistical

procedure i.e., either by stepwise regression analysis or cross validation. The best
equation is normally one with lower standard deviation and higher F value.

iii. If all the equations are equal then one should accept the simplest one.

iv. Number of terms or variables should be atleast 5 or 6 data point per variable
to avoid chance correlations.

v. It is important to have a model which is consistent with known physical-

organic and bio-medical chemistry of the process under consideration.

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 Applications of Hansch analysis

Hansch equation may be used to predict the activity of an yet unsynthesized

analogue. This enables the medicinal chemist to make a synthesis of analogue which is
worthy. However this prediction should only be regarded as valid, if they are made
within the range of parameter values used to establish the Hansch equation. Hansch
analysis may also be used to give an indication of the importance of the influence of
parameters on the mechanism by which a drug acts.

Example

The adrenergic blocking activity of series of analogue of β-Halo aryl amine was
observed. It was found that only π and σ values only related to the activity and not the
steric factor, from the following Hansch equation

Log1/c = 1.78π – 0.12σ + 1.674.

The smaller the value of coefficient of σ relative to that of π in the above equation
shows that electronic effect do not play an important role in the action of drug.

The accuracy of Hansch equation depends on

i. The number of analogues (n) used. The greater the number, the higher the
probability of obtaining an accurate Hansch equation.

ii. The accuracy of biological data used in the derivation of the equation.

iii. The choice of parameters.

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USES OF COMPUTER GRAPHICS IN COMPUTER-ASSISTED DRUG DESIGN

INTRODUCTION

Computers are essential tool in modern mechanical chemistry and are

important in both drug discovery and development. The development of this powerful
desktop enabled the chemist to predict the structure and the value of the properties of
known, unknown, stable and unstable molecular species using mathematical equation.
Solving this equation gives required data. Graphical package convert the data for the
structure of a chemical species into a variety of visual formats. Consequently, in
medicinal chemistry, it is now possible to visualize the three dimensional shape of both
the ligands and their target sites. In addition, sophisticated computational chemistry
packages also allow the medicinal chemists to evaluate the interaction between a
compound and its target site before synthesizing that compound. This means that,
medicinal chemists need only synthesize and test the compounds that considerably
increase the potency that is, it increase the chance of discovering a potent drug. It also
significantly reduces the cost of development.

MOLECULAR MODELING

Molecular modeling is a general term that covers a wide range of molecular

graphics and computational chemistry techniques used to build, display, manipulate,
simulate and analyze molecular structure and to calculate properties of these structures.
Molecular modeling is used in several different researches and therefore the term does
not have a rigid definition. To a chemical physicist, molecular modeling imply
performing a high quality quantum mechanical calculation using a super computer on the
structure to a medicinal chemists, molecular modeling mean displaying and modifying a
candidate drug molecule on the desktop computer. Molecular modeling techniques can
be divided into molecular graphics and computation chemistry.

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1. Molecular graphics (Computer graphic displays)

Molecular graphics is the core of a modeling system, providing for the

visualization of molecular structure and its properties. In molecular modeling, the data
produced are converted into visual image on the computer screen by graphic packages.
These images may be displayed in a variety of styles like fill, CPK (Corey-Pauling-
Koltum), stick, ball and stick, mesh and ribbon and colour scheme with visual aids.
Ribbon presentation is used for larger molecules like nucleic acid and protein.

Visualization of molecular properties is an extremely important aspect of

molecular modeling. The properties might be calculated using a computational chemistry
program and visualized as 3D contours along with the associated structure. The most
common computational methods are based on either molecular or quantum mechanics.
Both these approaches produce equation for the total energy of the structure. In this
equation the position of the atom in the structures are represented by either Cartesian or
polar co-ordinates. Once the energy equation is established, the computer computes a set
of co-ordinates which corresponds to minimum total energy value for the system. This
set of co-ordinate is converted into the required visual display by the graphic packages.
The program usually indicates the three dimensional nature of the molecule and it can be
viewed from different angles and allows the structure to be fitted to its target site. In
addition, it is also possible by molecular dynamics, to show how the shape of structure
might vary with time by visualizing the natural vibration of the molecule.

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2. Molecular mechanics

Molecular mechanics is the more popular of the methods used to obtain molecular
models as it is simple to use and requires considerably less computing time to produce a
model. In this technique the energy of structure is calculated. The equation used in
molecular mechanics follow the laws of classical physics and applies them to molecular
nuclei without consideration of the electrons. The molecular mechanics method is based
on the assumption that the position of the nuclei of the atom forming the structure is
determined by the force of attraction and repulsion operating in that structure. It assumes
that the total potential energy [Etotal ] of a molecule is given by the sum of all the energies
of the attractive and repulsive forces between the atoms in the structure. Molecules are
treated as a series of sphere (the atoms) connected by spring (the bond) using this model:
Etotal is expressed mathematically by equation known as force fields given by:

E total = Σ Estretching + Σ Ebend + Σ Etorsion + Σ Evdw + Σ Ecoulombic

Estretching

Estretching is the bond stretching energy. The value of the Estretching bond energy for pair
of atoms joined by a single bond can be estimated by considering the bond to be a
mechanical spring that obeys Hooke’s law. If r is the stretched length of the bond and r0
is the ideal bond length, then

Estretching = ½ K (r- r0)2

26
 Where K is the force constant in other word a measure of the strength of the bond.

If a molecule consist of three atoms, (a-b-c), then

Estretching = Ea-b + Eb-c

= ½ K(a-b) [r(a-b)- r0(a-b)]2 + ½ K(b-c) [r(b-c)- r0(b-c)]2

Ebend

Ebend is bond energy due to the changes in bond angle and estimated as

Ebend = ½ (K0(θ-θ0)2

Where θ0 is the ideal bond length i.e., the minimum energy position of the 3 atoms.

Etorsion

Etorsion is the bond energy due to changes in the conformation of the bond and given
by

Etorsion = 1/2 Kø (1+cos (m (ø+ø offset))

27
Where Kø is the energy barrier to the rotation about the torsion about the torsion

angleø, m is the periodicity of the rotation and øoffset is the ideal torsion angle relative to
staggered arrangement of two atoms.

Evdw

Evdw is the total energy contribution due to the Vander Waal’s force and it is
calculated from the Lennard-Jone6-12 potential equation.

Evdw = ε[(rmin)12 – 2(rmin)6]

r r

The (rmin)6 term in this equation represents attractive force, while (rmin)12 term represents

r r

the short range of repulsive forces between the atoms. The rmin is the distance between

two atoms i and j when the energy at a minimum ε and r is the actual distance between
the atoms.

Ecoulombic

Ecoulombic is the electrostatic attractive and repulsive forces operating in the

molecule between the atoms carrying a partial or full charge.

Ecoulombic = qi qj

28
 Drij

Where

qi and qj are the point charges on atoms i and j.

rij is the distance between the charges and

D is the dielectric constant of the medium surrounding the charges.

The values of the parameters r, r0, k . . . . etc used in the expression for the energy term in
the above equation is either obtained/calculated from experimental observations. The
experimental values are derived from variety of spectroscopic techniques.
Thermodynamic data measurement and crystal structure measurement for inter atomic
distances.

The best fit parameters are obtained by looking with known parameter values
and stored in the data base of the molecular modeling computer program.

Creating a molecular model using molecular mechanics

Molecular modeling can be created by any of these methods.

• Commercial force field computer program

• Assembling model

Commercial force field computer program

Commercial packages usually have several different force fields within the same
package and it is necessary to pick the most appropriate one for the structure being
modeled.

29
Assembling model

Molecular models are created by assembling a model from structural fragments

held in the database of the molecular modeling program. Initially, these fragments are
put together in a reasonably sensible manner to give a structure that does not allow for
steric hindrance. It is necessary to check that, the computer has selected atoms for the
structure whose configuration corresponds to the type of bond required in structure. For
example, if the atom in the structure is double bonded, then the computer has selected a
form of atom that is double bonded. These checks are carried out by matching a code for
the atoms on the screen against the code given in the manual for the program and
replacing atom where necessary.

An outline of the steps involved using INSIGHT II to produce a stick model of the
structure of paracetmol.

STEP 1

The selection of the structure fragments from the database of the INSIGHT II program.
The molecule with the relevant functional group and/or structure is selected.

The INSIGHT II models of these structures.

STEP 2

The fragments are linked together. Fragments are joined to each other by removing
hydrogen atoms at the points at which the fragments are to be linked. The bonding state
of each atom is checked and if necessary adjusted.

30
STEP 3

A representation of the change in the value of Etotal demonstrating how the

computation could stop at a local(x) rather than the true (global) minimum value. The
use of molecular dynamics gives the structure kinetic energy which allows it to
overcome energy barriers, such as Y, to reach the global minimum energy structure of
the molecule.

Once the structure is created energy minimization should be carried out. This is
because the construction process may have resulted in unfavourable bond lengths, bond
angle or torsion angle. The energy minimization process is carried out by a molecular
mechanics program, calculates the energy of the starting molecule, then varies the bond
lengths, bond angle and torsion angle to create a new structure in whatever software
program used. The program will interpret the most stable structure and will stop at that
stage when the force field reaches the nearest local minimum energy value. This final
structure may be around the screen and expanded or reduced in size. It can also be
rotated about the x and y axis to view different elevation of the model.

The molecular mechanic method requires less computing time than the quantum
mechanical approach and may be used for large molecules containing more than a
thousand atoms. Energy calculation has a range of application in molecular modeling.

• They can be used in the conformational analysis to evaluate the relative

stability of different conformers and to predict the equilibrium geometry of a structure.

31
• They can also be used to evaluate the energy of two or more interacting
molecules, such as when docking a substrate the enzyme active site.

It is not useful for computing properties such as electron density. The accuracy of
the structure obtained will depend on the quality and appropriateness of the parameters
used in the force field. Molecular mechanical calculations are normally based on isolated
structures at zero Kelvin and not normally take into account the effect of the
environment on the structure.

3. Molecular dynamics

Molecular mechanics calculations are made at zero Kelvin, that is on structure

that are frozen in time and so do not show the natural motion in the structure. Molecular
dynamics programs allow the modular to show the dynamic nature of the molecule by
stimulating the natural motion of the atom in a structure.

Starting with the molecular mechanics energy description of the structure as

described above, the force acting as the atom can be evaluated. Since the masses of the
atom are known, Newton’s second law of motion (force=mass*acceleration) may be
used to compute the acceleration and thus the velocities of the atoms. The acceleration
and velocities may be used to calculate new position for the atom over a short time step
thus moving each atom to a new position in the space. The velocities of the atoms are
related directly to the temperature at which the stimulation is run. Higher temperature
stimulations are used to search conformational shape, since more energy is available to
climb and cross barriers. These variations are displayed on the monitor in as a moving
picture. The appearance of this picture will depend on the force field selected for the
structure and the time interval and temperature used for the integration of the Newtonian
equation. Molecular dynamics can be used to find minimal energy structure and
conformational analysis.

4. Conformational analysis

32
 Using molecular mechanics (MM2), it is possible to generate a variety or different
conformations by using a molecular dynamics program which ‘heats’ the molecule to
800-900K. Of course, this does not mean that the inside of your computer is about to
melt. It means that the program allows the structure to undergo bond stretching and bond
rotation as if it was being heated. As a result, energy barriers between different
conformations are overcome, allowing the crossing of energy saddles. In the process, the
molecule is ‘heated’ at a high T(900K) for a certain period, then ‘cooled’ to 300K for
another period to give a final structure. The process can be repeated automatically as
many times a wished to give as many different structures as required. Each of these
structures can then be recovered, energy minimized and its steric energy measured. By
carrying out this procedure, it is usually possible to identify distinct conformations, some
of which might be more stable than the initial conformation.

Example

The 2D drawing of butane was imported into Chem3D and energy minimized.
Because of the way molecule was represented, energy minimization stopped at the first
local energy minimum it found, which was the gauche conformation having a steric
energy of 3.038Kcal/mol. The molecular dynamic program was run to generate other
conformations and successfully produced the fully staggered trans conformation which,
after optimization, had a steric energy of 2.175Kcal/mol, showing that the latter was
more stable by about 1Kcal/mol.

33
In fact, this particular problem could be solved more efficiently by the stepwise rotation
of bonds described below. Molecular dynamic is more useful for creating different
conformations of molecule which are not conductive to stepwise bond rotation (cyclic
system), or which would take too long analyse by that process (large molecular).

Example

The twist boat conformation of cyclohexane remains as the twist boat when
energy minimization is carried out. ‘Heating’ the molecule by molecular dynamics in
Chem3D produces a variety of different conformations, including the more stable chair
conformation.

5. Quantum mechanics

Unlike molecular mechanisms the quantum mechanic approach to molecular

modeling does not require the use of parameters similar to those used in molecular
mechanics. It is based on the realization that electrons and all material particles exhibit
wave like properties. This allows the well defined, parameter free, mathematics of wave
motion to be applied to electrons, atomic and molecular structure. The basis of this
calculation is the Schrodinger wave equation, which in its simplest form may be stated as

Hφ = Eφ

34
 In molecular modeling term Eφ represents the total potential and kinetic
energy of all the particles in the structure and H is the Hamiltonium operator acting on
the wave functionφ.

The energy of a structure calculated via quantum mechanics can be used in

conformational searches, in the same way that the molecular mechanics energy is used.
Quantum mechanics calculations can also be used for energy minimization. However,
quantum mechanics calculation typically consume a far greater amount of computer
resource than molecular mechanics calculations and are therefore generally limited to
small molecules, where as molecular mechanics can be applied to structures up to the
size of large proteins. Molecular mechanics and quantum mechanics should thus be
viewed as complementary techniques. For instance, conformational energy calculations
for a peptide are best carried out using molecular mechanics. However, molecular
mechanics is generally ineffective for handling conjugated systems, while quantum
mechanics, in calculating electronic structure, takes account of conjugation automatically
and is therefore recommended for optimizing the structure of a small molecule
containing conjugated systems.

The wave function can be used to calculate a range of chemical properties,

which can be in structure activity studies. These include electrostatic potential, electron
density, dipole moment and the energies and positions of frontier orbital. As with the
analysis of a molecular dynamics calculation, molecular graphics is essential for
visualizing these properties. Quantum mechanics calculations are also used frequently to
derive atom centered partial charges (although the term charge itself does not have a
strict quantum mechanical definition). Charges have a wide range of applications in
modeling and are used in the calculation of electrostatic energies in molecular mechanics
calculations and in computing electrostatic potentials.

Quantum mechanical methods are suitable for calculating the following

• Molecular orbital energies and coefficients

• Heat of formation for specific conformations

• Partial atomic charges calculated from molecular orbital coefficients

35
• Electrostatic potentials

• Dipole moments

• Transition state geometries and energies

• Bond dissociation energies

HYBRID QM/MM

QM. (quantum-mechanical) methods are very powerful however they are

computationally expensive, while the MM (classical or molecular mechanics) methods
are fast but suffer from several limitations (require extensive parameterization; energy
estimates obtained are not very accurate; cannot be used to simulate reactions where
covalent bonds are broken/formed; and are limited in their abilities for providing
accurate details regarding the chemical environment). A new class of method has
emerged that combines the good points of QM (accuracy) are MM (speed) calculations.
These methods are known as mixed or hybrid quantum-mechanical and molecular
mechanics methods (hybrid QM/MM). The methodology for techniques was introduced
by Warshel and coworkers.

IMPORTANT TECHNIQUES FOR DRUG DESIGN

36
 To obtain the structural information about molecules necessary for mechanistic
design of drugs, a variety of chemical, physical, and theoretical techniques must be used.
Different techniques provide complementary types of information, which together can be
used to determine how molecules interact.

X-RAY CRYSTALLOGRAPHY

X-ray crystallography is often the starting point for gathering information

from mechanistic drug design. This technology has the potential to determine total
structural information about a molecule. Furthermore it provides the critically important
coordinates needed for the handling of data by computer modeling systems12. It is the
only technique at present that will give the complete three-dimensional structure in detail
at high resolution including bond distance, angles, stereochemistry and absolute
configuration. The use of such a powerful technique for drug design was recognized over
a decade ago .

To carry out an X-ray crystallographic analysis, material of very high purity

is needed. This material must be carefully crystallized to yield crystals of a suitably high
quality for study. Small molecules can generally be crystallized using standard chemical
techniques13. Macromolecules such as proteins, however, require specialized techniques
to produce suitable crystals. Even with suitable crystals, the solution of a
macromolecular structure is much more difficult than for a small molecule. The larger
number of atoms in a macromolecule makes it hard to attain the high degree of resolution
needed. Furthermore, the instrumentation required is complex, and the data analysis and
refinement take substantial computer time14. Finally, because X-ray crystallography must
be carried out with molecules in the solid phase, the three-dimensional structure obtained
may differ from the molecule in its biologically active state.

37
Nevertheless, this technology is very important for determining the structure of the drug
(effector), the structure of the drug’s target, and the interaction of the two. It is
reasonable to assume then the future of large molecule crystallography in medical
chemistry may well be of monumental proportions. The reactivity of the receptor
certainty lies in the nature of the environment and position of various amino acid
residues15. When the structured knowledge of the binding capabilities of the active site
residues to specify groups on the agonist or antagonists becomes known, it should lead to
proposals for synthesis of very specific agents with a high probability of biological
action. Combined with what is known about transport of drugs through a Hansch-type

38
analysis, etc., it is feasible that the drugs of the future will be tailor-made in this
fashion16. Certainly, and unfortunately, however, this day is not as close as one would
like. The X-ray technique for large molecules, crystallization techniques, isolation
techniques of biological systems, mechanism studies of active sites and synthetic talents
have not been extremely interwined because of the existing barriers between vastly
different sciences.

39
40
 Since that time, interdisciplinary scientists have broken down a number of the
walls between the different disciplines. Today it is not unusual to see individuals who
can, with their own hands, synthesize organic heavy-atom derivatives, grow crystals, and
solve X-ray structures of the hardest magnitude, clone genes, and talk rationally, in
mechanistic terms, about substrate specificity. However, the best rational design by
modeling from the surface of known receptors determined from X-ray analysis will not
prevent the compound from bypassing the oxidative enzymes in the liver or deter it from
being taken up by fat depots or serum proteins, or keep it out of the urine, or stop it from
having neurotoxicity17. Will we do any better with the rational design of new agents
based on the structural knowledge of the receptor than with older methods? The score as
of this writing is that one drug, Captopril, has made it to the market place, and a few
others appear to be on their way. The hope for the success of any new agents will rest in
the rational design of compounds with sufficient specificity to circumvent or greater
reduce the distribution, toxicity

41
d metabolism problems mentioned above.

Crystallography is moving in two directions: 1. macro and 2. mini. The solution of

larger and more complex systems will continue to provide drug designers with atomic
details that promote imaginative approaches to drug design18. The most recent and truly
amazing development in data collection indicates that a whole set of protein data may be
acquired in a second or less using Laue photographs. Such short analysis times may
soon provide structural features at near atomic resolutions of the movements involved in

42
native and substrate bound proteins. On the opposite end of the kilodalton scale, detailed
crystallographic analyses of the electron charge distribution in small molecules will
permit the assignment of electrostatic potentials to atoms that could aid in the
understanding of drug receptor interactions and how side chains pack in proteins. The
addition to the understanding of packing, with a better understanding of water
interactions in maintaining secondary and tertiary structure, may solve the protein
folding problem19. If that happens, then the nature of any receptor might be deduced from
the genome and X-ray crystallography will take a back seat to the dynamic
computational and spectral methods of analyses of molecules20. Until that day, however,
crystallography will continue to have a dominant role in rational drug design.

NMR SPECTROSCOPY

The major limitations of X-ray crystallography are the necessity to obtain

good crystals and the fact that three-dimensional data obtained with crystals may not
reflect the molecular structure under biological conditions that involve molecules in
solution21. The best technique for determining structural information on molecules in
solution is nuclear magnetic resonance (NMR) spectroscopy. NMR uses much softer
radiation which can examine molecules in the more mobile liquid phase, so the three-
dimensional information obtained may be more representative of the molecule in its
biological environment22. Another advantage of NMR is its ability to examine small
molecule-macromolecule complexes, such as an enzyme inhibitor in the active site of the
enzyme. Such information can be obtained by X-ray crystallography only after co-
crystallization or crystal “soaking” techniques. In addition, NMR can often be used to
gather structural information more rapidly than X-ray crystallography. Consequently,
NMR has proved to be a valuable tool in pharmaceutical research23. In addition to its
importance as an analytical method to elucidate the primary structures of chemically
synthesized compounds and isolated natural products.

43
44
NMR can provide information on the three-dimensional structures of small molecules in
solution, high-molecular-weight complexes and the details of the enzyme mechanisms
that can be used to aid in drug design. Some of the recent advances in NMR that have
allowed this information to be obtained include the availability of high magnetic fields
improved software, probe design and electronics, more versatile pulse programmers and
perhaps most importantly, the development of two-dimensional NMR techniques24.

45
46
 NMR spectroscopy can provide detailed information on the conformational
properties of small molecules in solution, the structure of large molecular complexes and
enzyme reaction mechanisms. It is expected that future developments in NMR and other
fields will contribute to even further progress in the ability of these new developments
which are expected in the near future include

• The availability of large quantities of enzymes and drug receptors through

improved expression systems and cloning technology.

• The availability of isotopically labeled (13C, 15N, 2H) inhibitors, enzymes and
soluble receptors suitable for NMR studies by chemical synthesis and biosynthetic
means.

• Improvements in NMR techniques, especially those designed for NMR

studies of large systems

• The availability of increased magnetic-field strengths at a low cost due to the

recently demonstrated improvements in superconducting materials.

47
These developments should vastly increase our capability to study the three-dimensional
structures of enzyme-bound ligands, enzyme active sites and soluble drug-receptor
complexes. In addition, improvements in solid-state NMR techniques and NMR imaging
should allow structural studies of drugs bound to membrane-bound receptors and the
physiological effects of drugs to be examined, respectively25. Clearly, the future holds

48
even more exciting prospects for the use of NMR spectroscopy in the rational design of
new pharmaceutical agents.

The disadvantage of NMR is that the data obtained are not as precise or
complete as those from an X-ray structure determination. There is also a limit on the size
of molecule that can be studied with present equipment. Modern high-field NMR
spectrometers have recently been developed that can obtain data on smaller samples and,
by the use of two-dimensional techniques, are able to obtain more precise information
about macromolecules.

OTHER IMPORTANT CONSIDERATIONS

It has been realized that biological molecules can exist in a variety if

different conformations and depending on the energetics of the molecules and the
environmental conditions, will shift among these conformations. The initial application
of molecular modeling to design drugs generally begins with the use of rigid constructs
for structures and their targets. This concept of molecular behavior is often satisfactory
for answering simple questions, such as whether a drug will fit into the active site of the
target. As the questions about molecular interactions become more complex, however,
the concept of molecules in different dynamic energetic states and configurations
becomes much more important. Sophisticated questions such as what is the most
favorable position for a drug in its target’s active site require more information, based on
additional physical parameters, than simply answering the question, will a molecule fit
into a given space.

The flexibility of molecular conformations, both in single molecules and

in molecules interacting with each other, is an important and challenging concept in drug
design. One of the major potentials of computer-aided drug design is the development of
completely new effector compounds for targets. To date, however, this has been very
difficult. A significant reason is our lack of knowledge about the factors that govern
conformational states and flexibility. These concepts and the problems they attempt to
understand and handle are important, since it is in these areas that breakthroughs are still
needed to realize the real potential of computer-aided drug design in predicting new
chemical structures that will interact with the desired targets.

49
APPLICATIONS

• Computer-aided design and evaluation of Angiotensin-Converting enzyme

inhibitors.

• Role of computer-aided molecular modeling in the design of novel inhibitors

of Renin.

• Inhibitors of Dihydrofolate reductase.

• Approaches to Antiviral drug design.

• Conformation biological activity relationships for receptor-selective,

conformationally constrained opioid peptides.

• Design of conformationally restricted cyclopeptides for the inhibition of

cholate uptake of Heepatocytes

CONCLUSIONS

. The process of drug discovery and development is a long and difficult one,
and the costs of developing are increasing rapidly. Today it takes appropriately 10years
and $100million to bring a new drug to market. Inspite of the tremendous costs involved,
the payoff is also high, both in dollars and in the improvements made in preventing and
controlling human diseases. The emphasis now is not just on finding new ways to treat
human disease, but also on improving the quality of life of people in general. The use of
new computer-based drug design techniques has the ability to accomplish both of these
goals and to improve the efficiency of the process as well, thus reducing costs.

Mechanism-based drug design tackles medical problems directly. It

provides an opportunity to discover entirely new lead compounds not possible using
other techniques for drug development. Thus it offers the potential for treating diseases
that are not currently controllable by existing drugs. Similarly, these new techniques in
drug design can improve the lead optimization process. By understanding the physical
interaction of a drug and its receptor, one has the means to improve the potency and
selectivity of a drug and thereby reduce its undesirable interactions with other
physiological processes in the body. The quality of life of patients receiving these newer

50
drugs, which have greater potency and fewer side effects, is improving. Finally, since the
traditional lead optimization process typically requires the synthesis of hundreds or even
thousands of new compounds, it is a time-Consuming and labor-intensive process. The
use of newer computer-based techniques in combination with techniques in combination
with techniques that have been successful in the past provides a means to greatly reduce
the number of new compounds that must be synthesized and tested and thus speeds up
the process of drug discovery.

Future developments will continue to improve the efficiency of all aspects of

drug discovery. Knowledge about the molecular basis of diseases is rapidly expanding on
all fronts and will continue unabated. Molecular biologists will soon be able to provide
quantities of receptor molecules and enzymes that have not yet been available to drug
researchers. Improvements in X-ray and NMR techniques will yield needed structural
information in shorter times and will give more details of the drug-target complex. With
these new data, will come improvements in computational techniques and their ability to
predict the conformational state of a small compound and its macro-molecular receptor.
In addition, these techniques will be able to depict more clearly the biological molecules
under physiological conditions. Finally, as more and more drug researchers understood
and become familiar with the concepts and methods of mechanistic, computer-aided drug
design, new applications of the integration of these techniques will emerge and will have
a major impact both on basic science and on discovering new drugs fir the future.

51
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