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

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Lack of precision and reproducibility.

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= (n 2 -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 γ 1/4

D

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

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σx= log KBX

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

25

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))

26

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[ (r min) 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

27

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.

28

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.

29

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.

30

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

31

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.

32

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

33

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

34

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

35

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.

36

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

37

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.

38

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

39

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

40

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.

41

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

42

improved software, probe design and electronics, more versatile pulse programmers and

perhaps most importantly, the development of two-dimensional NMR techniques24.

43

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.

44

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

45

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.

46

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

47

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.

48

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