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Principles of drug discovery

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Page 1: Principles of drug discovery
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SUBMITTED TO

JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD,

In the partial fulfillment of M.Pharm.I year, I semester.

CMR GROUP OF INSTITOTIONS ·

(Approved by AICTE and affiliated to JNTU Hyderabad.)

Kandlakoya(V), Medchal(M), Hyderabad-501401

Under the guidance of, Prepared By,

Dr T.Vedhavathi, V.kavya lakshmi,

Mpharm; PhD, Mpharm IST yr pharmacology,

CMR collage of pharmacy. Reg. No. 10T21S0105.

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C.M.R COLLEGE OF PHARMACY

(Approved by AICTE & PCI)

(Affiliated to JNTU)

. Kandlakoya, Medchal

CERTIFICATE

This is to verify that this is a bonafied record of the seminar entitled “Principles of

drug discovery” presented by V.kavya lakshmi (10T21S0105), during the

academic year 2010-2011 for partial fulfillment in degree of Masters of Pharmacy

of Jawaharlal Nehru Technological University, Hyderabad.

PRINCIPAL: INTERNAL GUIDE:

Rajeswar dutt Dr T.Vedavathi

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DECLARATION

I here by declare that the seminar work entitled

“Principles of drug discovery”submitted to the {JNTUH} is a

record work of seminar, under the guidance of Dr.Vedhavathi

professor of C.M.R college of pharmacy.

V.kavya lakshmi,

Regno:10T21S0105

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IntroductionTime course involved in drug discoveryHistorical back groundApproachesRational approaches in drug discoveryPreclinical studies

Clinical studiesNovel approaches

INDEX

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Principles of drug discovery

Drug discovery is the process by which drugs are discovered and/or designed.

In the past most drugs have been discovered either by identifying the active ingredient

from traditional remedies or by serendipitous discovery. A new approach has been to understand

how diseases and infections are controlled at the molecular and physiological level and to target

specific entities based on this knowledge.

The process of drug discovery involves the identification of substances, synthesis,

characterization, screening and assays for therapeutic efficacy. Once a compound has shown its

value in these tests, it will begin the process of drug development prior to clinical trails

Despite advances in technology and understanding of biological systems, drug discovery

is still a lengthy, "expensive, difficult, and inefficient process" with low rate of new therapeutic

discovery. Currently, the research and development cost of each new molecular entity (NME) is

approximately US$1.8 billion.

Information on the human genome, its sequence and what it encodes has been hailed as a

potential windfall for drug discovery, promising to virtually eliminate the bottleneck in

therapeutic targets that has been one limiting factor on the rate of therapeutic

discovery. However, data indicates that "new targets" as opposed to "established targets" are

more prone to drug discovery project failure in general. This data collaborates some thinking

underlying a pharmaceutical industry trend beginning at the turn of the twenty-first century and

continuing today which finds more risk aversion in target selection among multi-national

pharmaceutical companies.

Time course involved in drug discovery

The time course in a drug discovery involves at least ten years and more than ten years.

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The processes of new drug discovery and development are long, complicated and

dependent upon the expertise of a wide variety of scientific, technical and managerial

groups.

Here are the different stages of drug discovery and development.

There are four stages in the drug discovery. They are

Synthesis or isolation of the compound which involves different techniques like

extraction methods, chromatographic techniques for isolation and different methods of

synthesis. This section in drug discovery takes 1-2 yrs

Preclinical studies: These are done before testing on humans

So these are called as animal studies. They includes different topics of study like

Screening

Evaluation

Pharmacokinetics

Toxicity testing

All these process takes place 2-4yrs.

Then apply for grant of permission for clinical trail from concerned associations.

This takes 0.5-1yr.

After the approval Pharmaceutical formulation, standardization of

chemicals/biological/immunological assays of new drug applications are estimated.

Clinical studies

Phase IV or post market surveillance is the time involving step, which cannot be

predicted.

As the drug may be success with out any adverse effects or it is rejected or send back for

further optimization.

Success rate in getting from an initial compound to an approved and commercially

available product is very low.

< 2% of new compounds investigated may show suitable biological activity

Modification of an existing drug can yield as little as 1% suitable compounds

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< 10% of these compounds result in successful human clinical trials and reaches the

market place

Schematic representation of drug discovery process

Generally the "target" is the naturally existing cellular or molecular structure involved in

the pathology of interest that the drug-in-development is meant to act on. However, the

distinction between a "new" and "established" target can be made without a full understanding of

just what a "target" is. This distinction is typically made by pharmaceutical companies engaged

in discovery and development of therapeutics.

"Established targets" are those for which there is a good scientific understanding,

supported by a lengthy publication history, of both how the target functions in normal

physiology and how it is involved in human pathology. This does not imply that the mechanism

of action of drugs that are thought to act through particular established targets is fully

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understood. Rather, "established" relates directly to the amount of background information

available on a target, in particular functional information. The more such information is

available, the less investment is (generally) required to develop a therapeutic directed against the

target. The process of gathering such functional information is called "target validation’ in

pharmaceutical industry parlance. Established targets also include those that the pharmaceutical

industry had experience mounting drug discovery campaigns against in the past; such a history

provides information on the chemical feasibility for developing a small molecular

therapeutics, against the target and can provide licensing opportunities and freedom-to-operate

indicators with respect to small-molecule therapeutic candidates.

In general, "new targets" are all those targets that are not "established targets" but which

have been the subjects of drug discovery campaigns. These typically include newly

discovered proteins, or proteins whose function has now become clear as a result of basic

scientific research.

The majority of targets currently selected for drug discovery efforts are proteins. Two

classes predominate: G-protein-coupled receptors (or GPCRs) and protein kinases.

HISTORICAL BACKGROUND

The idea that effect of drug in human body are mediated by specific interactions of the

drug molecule with biological macromolecules, (proteins or nucleic acids in most cases) led

scientists to the conclusion that individual chemicals are required for the biological activity of

the drug. This made for the beginning of the modern era in pharmacology, as pure chemicals,

instead of crude extracts, became the standard drugs. Examples of drug compounds isolated from

crude preparations are morphine as the active agent in opium, and digoxin (a heart stimulant)

originating from Digitalis lanata. Organic chemistry also led to the synthesis of many of the co-

chemicals isolated from biological sources.

SCREENING & DESIGNING

The process of finding a new drug against a chosen target for a particular disease usually

involves high-throughput screening (HTS), wherein large libraries of chemicals are tested for

their ability to modify the target. For example, if the target is a novel GPCR (G protein coupled

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receptors) compounds will be screened for their ability to inhibit or stimulate that receptor. If the

target is a protein kinase, the chemicals will be tested for their ability to inhibit that kinase.

Another important function of HTS is to show how selective the compounds are for the

chosen target. The ideal is to find a molecule which will interfere with only the chosen target, but

not other, related targets. To this end, other screening runs will be made to see whether the "hits"

against the chosen target will interfere with other related targets - this is the process of cross-

screening. Cross-screening is important, because the more unrelated targets are compound hits.

This leads to off-target toxicity with that compound once it reaches the market.

There are two types of screening

Random screening

Non random screening

Random Screening

In the absence of known drugs and other compounds with desired activity, a random

screening is a valuable approach. Random screening involves no intellectualization; all

compounds are tested in the bioassay without regard to their structures. Prior to 1935 (the

discovery of sulfa drugs) this was essentially the only approach; today this method is still an

important approach to discover drugs or leads, particularly because it is now possible to screen

such huge numbers of compounds rapidly with HTSs. This is the lead discovery method of

choice when nothing is known about the receptor target.

The two major classes of materials screened are synthetic chemicals and natural

(Microbial, plant and marine) products. An example of a random screen of synthetic and natural

compounds was the “war on cancer” declared by Congress and the National Cancer Institute in

the early 1970s. Any new compound submitted was screened in a mouse tumor bioassay. Few

new anticancer drugs resulted from that screen, but many known anticancer drugs also did not

show activity in the screen used, so a new set of screens was devised that gave more consistent

results. In the 1940s and 1950s, a random screen of soil samples by various pharmaceutical

companies in search of new antibiotics was undertaken. However, in this case, not only were

numerous leads uncovered, but two important antibiotics, streptomycin and the tetracyclines

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were found. Screening of microbial broths, particular strains of Streptomyces was a common

random screen methodology prior to 1980.

Nonrandom (or Targeted or Focused) Screening

Nonrandom screening also called targeted or focused screening and is a more narrow

approach than the random screening. In this case, compounds having a vague resemblance to

weakly active compounds uncovered in a random screen, or compounds containing different

functional groups than leads, may be tested selectively. By the late 1970s, the National Cancer

Institute’s random screen was modified to a nonrandom screen because of budgetary and

manpower restrictions. Also, the single tumor screen was changed to a variety of tumor screens

because it was realized that cancer is not just a single disease.

It is very unlikely that a perfect drug candidate will emerge from these early screening

runs. It is more often observed that several compounds are found to have some degree of activity,

and if these compounds share common chemical features, one or more pharmacophores can then

be developed

While HTS is a commonly used method for novel drug discovery, it is not the only

method. It is often possible to start from a molecule which already has some of the desired

properties. Such a molecule might be extracted from a natural product or even be a drug on the

market which could be improved upon (so-called "me too" drugs). Other methods, such as virtual

high throughput screening, where screening is done using computer-generated models and

attempting to "dock" virtual libraries to a target are also often used.

Another important method for drug discovery is drug design, whereby the biological and

physical properties of the target are studied and a prediction is made of the sorts of chemicals

that might fit into an active site. One example is fragment-based lead discoveries (FBLD). Novel

pharmacophores can emerge very rapidly from these exercises.

Once a lead compound series has been established with sufficient target potency and

selectivity and favorable drug-like properties, one or two compounds will then be proposed

for drug development. The best of these is generally called the lead compound, while the other

will be designated as the "backup".

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Approaches

Nature of source for drug discovery

Despite the rise of combinatorial chemistry as an integral part of lead discovery process,

natural products still play a major role as starting material for drug discovery.  A report was

published in 2007,[7] covering years 1981-2006 details the contribution of biologically occurring

chemicals in drug development. According to this report, of the 974 small molecule new

chemical entities, 63% were natural derived or semi synthetic derivatives of natural products. For

certain therapy areas, such as antimicrobials, anti neoplastics, antihypertensive and anti-

inflammatory drugs and the numbers were higher. Natural products may be useful as a source of

novel chemical structures for modern techniques of development of antibacterial therapies.

Despite the implied potential, only a fraction of Earth’s living species has been tested for

bioactivity.

Plant-derived

Prior to Paracelsus, the vast majority of traditionally used crude drugs in Western

medicine were plant-derived extracts. This has resulted in a pool of information about the

potential of plant species as an important source of starting material for drug discovery. A

different set of metabolites is sometimes produced in the different anatomical parts of the plant

(e.g. root, leaves and flower), and botanical knowledge is crucial also for the correct

identification of bioactive plant materials.

Microbial metabolites

Microbes compete for living space and nutrients. To survive in these conditions, many

microbes have developed abilities to prevent competing species from proliferating. Microbes are

the main source of antimicrobial drugs. Streptomyces species have been a source of antibiotics.

The classical example of an antibiotic discovered as a defense mechanism against another

microbe is the discovery of penicillin in bacterial cultures contaminated by Penicillium fungi in

1928.

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Marine invertebrates

Marine invertebrates are the potential sources for new agents.[9]. Arabinose nucleosides

discovered from marine invertebrates in 1950s, demonstrating for the first time that sugar

moieties other than ribose and deoxyribose can yield bioactive nucleoside structures. However, it

was 2004 when the first marine-derived drug was approved. The cone snail toxinziconotide, also

known as Prialt, was approved by the Food and Drug Administration to treat severe neuropathic

pain. Several other marine-derived agents are now in clinical trials for indications such as cancer,

anti-inflammatory use and pain. One class of these agents is bryostatin-like compounds, under

investigation as anti-cancer therapy.

Chemical diversity of drug sources

As above mentioned, combinatorial chemistry was a key technology enabling the

efficient generation of large screening libraries for the needs of high-throughput screening.

However, now, after two decades of combinatorial chemistry, it has been pointed out that despite

the increased efficiency in chemical synthesis, no increase in lead or drug candidates have been

reached. This has led to analysis of chemical characteristics of combinatorial chemistry products,

compared to existing drugs and/or natural products. The synthetic, combinatorial library

compounds seem to cover only a limited and quite uniform chemical space, whereas existing

drugs and particularly natural products, exhibit much greater chemical diversity, distributing

more evenly to the chemical space.The most prominent differences between natural products and

compounds in combinatorial chemistry libraries is the number of chiral centers (much higher in

natural compounds), structure rigidity (higher in natural compounds) and number of aromatic

moieties (higher in combinatorial chemistry libraries). Other chemical differences between these

two groups include the nature of heteroatoms (O and N enriched in natural products, and S and

halogen atoms more often present in synthetic compounds), as well as level of non-aromatic

unsaturation (higher in natural products). As both structure rigidity and chirality are both well-

established factors in medicinal chemistry known to enhance compounds specificity and efficacy

as a drug, it has been suggested that natural products compare favorable to today's combinatorial

chemistry libraries as potential lead molecules.

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Rational approach

Drug discovery hit to lead

Early drug discovery involves several phases from target identification to preclinical

development. The identification of small molecule modulators of protein function and the

process of transforming these into high-content lead series are key activities in modern drug

discovery. The Hit-to-Lead phase is usually the follow-up of high-throughput screening (HTS). It

includes the following steps:

Hit confirmation

The Hit confirmation phase will be performed during several weeks as follows:

Re-testing: compounds that were found active against the selected target are re-tested using

the same assay conditions used during the HTS.

Dose response curve generation: several compound concentrations are tested using the same

assay, an IC50 or EC50 value is then generated. Methods are being developed that may allow

the reuse of the compound that generated the hit in the initial HTS step. These molecules are

removed from beads and transferred to a microarray for quantitative assessment of binding

affinities in a "seamless" approach that could allow for the investigation of more hits and

larger libraries

Orthogonal testing: Confirmed hits are assayed using a different assay which is usually

closer to the target physiological condition or using a different technology.

Secondary screening: Confirmed hits are tested in a functional assay or in a cellular

environment. Membrane permeability is usually a critical parameter.

Chemical amenability: Medicinal chemists will evaluate compounds according to their

synthesis feasibility and other parameters such as up-scaling or costs

Intellectual property evaluation: Hit compound structures are quickly checked in specialized

databases to define patentability

Biophysical testing: Nuclear magnetic resonance (NMR), Isothermal Titration Calorimetry,

dynamic light scattering, surface Plasmon resonance  dual polarization interferometry, micro

scale thermophoresis(MST) are commonly used to assess whether the compound binds

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effectively to the target, the stoichiometry of binding, any associated conformational change

and to identify promiscuous inhibitors.

Hit ranking and clustering: Confirmed hit compounds are then ranked according to the

various hit confirmation experiments.

Hit expansion

Following hit confirmation, several compound clusters will be chosen according to their

characteristics in the previously defined tests. An Ideal compound cluster will:

have compound members that exhibit a high affinity towards the target (less than 1 µM)

Moderate molecular weight and lipophilicity (usually measured as cLogP). Affinity,

molecular weight and lipophilicity can be linked in single parameter such as ligand

efficiency  and lipophilic efficiency to assess drug likeness

Show chemical tractability

Be free of Intellectual property

Interfere neither with the P450  enzymes nor with the P-glycoprotein’s

Not bind to human serum albumin

Be soluble in water (above 100 µM)

Be stable

Have a good drug likeness

Exhibit cell membrane permeability

Show significant biological activity in a cellular assay

Not exhibit cytotoxicity

Not be metabolized rapidly

Show selectivity versus other related targets

The project team will usually select between three and six compound series to be further

explored. The next step will allow testing analogous compounds to define Quantitative structural

activity relationship (QSAR). Analogs can be quickly selected from an internal library or

purchased from commercially available sources. Medicinal chemists will also start synthesizing

related compounds using different methods such as combinatorial chemistry, high-throughput

chemistry or more classical organic chemistry synthesis.

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Lead optimizations

The objective of this drug discovery phase is to synthesize lead compounds, new analogs

with improved potency, reduced off-target activities, and physiochemical/metabolic properties

suggestive of reasonable in vivo pharmacokinetics. This optimization is accomplished through

chemical modification of the hit structure, with modifications chosen by employing structure-

activity analysis (SAR) as well as structure-based design if structural information about the

target is available.

Drug designing based on receptor and ligand structure

Structural elucidation

The elucidation of the chemical structure is critical to avoid the re-discovery of a

chemical agent that is already known for its structure and chemical activity. Mass spectrometry,

often used to determine structure is a method in which individual compounds are identified based

on their mass/charge ratio, after ionization. Chemical compounds exist in nature as mixtures, so

the combination of liquid chromatography and mass spectrometry (LC-MS) is often used to

separate the individual chemicals. Databases of mass spectra’s for known compounds are

available. Nuclear magnetic resonance spectroscopy is another important technique for

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determining chemical structures of natural products. NMR yields information about individual

hydrogen and carbon atoms in the structure, allowing detailed reconstruction of the molecule’s

architecture.

Molecular modeling

Structural Modifications to Increase Potency and the Therapeutic Index

1. Homologation

2. Chain Branching

3. Ring-Chain Transformations

4. Bioisosterism

5. Combinatorial Chemistry

a Combnitorial synthesis

b Split Synthesis: Peptide Libraries

c. Encoding Combinatorial Libraries

d. Nonpeptide Libraries

6. SAR by NMR/SAR by MS

7. Peptidomimetic

8. CADD

Homologation

A homologous series is a group of compounds that differ by a constant unit, generally a

CH2 group.

For many series of compounds, lengthening of a saturated carbon side chain from one

(methyl) to five to nine atoms (pentyl to nonyl) produces an increase in pharmacological

effects. Lengthening results in a sudden decrease in potency

This phenomenon corresponds to increased lipophilicity of the molecule to permit

penetration into cell membranes until its lowered water solubility becomes problematic in

its transport through aqueous media.

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In the case of aliphatic amines, another problem is micelle formation, which begins at

about C12.

Chain branching

This effectively removes the compound from potential interaction with the appropriate

receptors.

Then chain branching flowers the potency of a compound because a branched alkyl chain

is less lipophilic than the corresponding straight alkyl chain as a result of larger molar

volumes and shapes of branched compounds.

This phenomenon is exemplified by the lower potency of the compounds having isoalkyl

chains

For example, phenethylamine (PhCH2CH2NH2) is an excellent substrate for monoamine

oxidase.

Ring-Chain Transformations

Another modification that can be made is the transformation of alkyl substituent’s into

cyclic analogs, which often does not affect potency greatly.

However, a ring-chain transformation could have an important pharmacokinetic effect,

such as to increase lipophilicity or decrease metabolism, which could make the drug more

effective in vivo. Also by connecting substituents into a ring, pharmacodynamic

properties could be enhanced by constraining the groups into a particularly favorable

conformation. Of course, it also could constrain the molecule into an unfavorable

conformation, and potency could drop different activities can result from a ring-chain

transformation as well. For example, if the dimethylamino group of the tranquilizer

chlorpromazine is substituted by a methyl piperazine ring (X = Cl, R = CH2CH2CH2N

NCH3; prochlorperazine), antiemetic (prevents nausea)

Bioisosterism

Bioisosteres are substituents or groups that have chemical or physical similarities, and

which produce broadly similar biological properties.

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Bioisosterism is an important lead modification approach that has been shown to be

useful to attenuate toxicity or to modify the activity of a lead, and may have a significant

role in the alteration of pharmacokinetics of a lead.

There are classical isosteres and nonclassical isosteress

Nonclassical bioisosteres do not have the same number of atoms and do not fit the steric

and electronic rules of the classical isosteres, but do produce similar biological activity

change: size, shape, electronic distribution, lipid solubility, water solubility, pKa,

chemical reactivites and hydrogen bonding. Because a drug must get to the site of action,

then interact with it bioisosteric modifications made to a molecule may have one or more

of the effects.

Combinatorial Chemistry

General Aspects

Combinatorial chemistry involves the synthesis or biosynthesis of chemical libraries (a

family of compounds having a certain base chemical structure) of molecules with in a

short period of time for the purpose of biological screening, particularly for lead

discovery or lead modification.

Typically, these chemical libraries are prepared in a systematic and repetitive way by

covalent assembly of building blocks (various reactant molecules that build up parts of

the overall structure)

To give a diverse array of molecules with a common scaffold (the parent structure in the

family of compounds).

Combinatorial synthesis

Split synthesis

Encoding combinatorial library

Nonpeptidal synthesis

Combinatorial synthesis

The advantage of this methodology is that it is carried out on a solid (polymeric) support,

so that isolation and purification of the product of each reaction can be performed by

simple filtration and washing with a variety of solvents of the polymeric support to which

the building blocks have been attached.

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Because of the insolubility of the polymer, everything not attached to the polymer is

removed, which allows the use of excess reagents to drive the synthetic reactions.

The disadvantages of this methodology are the difficulty in scaling up the reactions and

the sluggishness of reactions.

An alternative strategy (covalent scavenger technology) is to carry out the reactions in

solution with excess reagent, which is then scavenged with a polymeric-supported

scavenger after the reaction is completed

In this approach, filtration removes the excess reagent attached to the scavenger polymer,

leaving the product in solution.

Another approach is to use polymer-supported reagents with solution reactions.

To avoid problems of heterogeneous polymer reactions, soluble polyethylene glycol

polymers can be used

Split synthesis (Peptide Libraries)

The initial approach, known as a split synthesis (also called mix and split, split and pool,

or the divide, couple, recombine method), is the most common general lead discovery

approach for making large libraries (104–106 compounds) that are assayed as library

mixtures.

The result of a split synthesis is a collection of polymer beads, each containing one

library member, i.e., one bead, one compound.

The library contains every possible combination of every building block.

The serious limitations are that it is applicable only to the synthesis of sequenceable

oligomers and each bead carries only about 100–500 pmol of product, which makes

structure determination difficult or impossible.

For simple compounds mass spectrometric methods may be used but this is not

applicable if the library contains many thousands or millions of members that may not be

pure or are isomeric with other library members. In that case, encoding methods need to

be utilized.

For example: how the split synthesis approach would be applied to a small (27-member)

library of all possible tripeptides of three amino acids.

This method can be extrapolated to any size library.

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A homogeneous mixture of all of the tripeptides of His, Val, and Ser could be

synthesized on a Merrifield resin

Note that a Merrifield synthesis starts at the ‘C’ terminus and builds to the ‘N’ terminus.

The homogenization step is very important to ensure that each tube contains the same

mixture of resin-bound compounds.

The process shown in Scheme was carried out, but starting with 20 separate tubes

containing methyl benzhydryl amine (MBHA) polystyrene as the resin. (This resin

produces peptide amides when peptides are cleaved from it.)

A combinatorial library of penta=peptides containing the 20 standard amino acids was

constructed on the MBHA resin, homogenized, then separated into 20 tubes.

To each of the 20 different tubes was added a different N-acetylamino acid, so that in

each tube there was a combinatorial library of all possible resin-linked N-acetyl hexa

peptides having the same N terminus.

Each tube contained all of the N-acetyl hexa peptides starting with a different N-terminal

amino acid.

An aliquot from each of the 20 tubes is removed and assayed.

The most potent aliquot indicates which amino acid is best.

Then this process is repeated, except in the next interaction a combinatorial library of

MBHA-bound tetrapeptides is made, is split into 20 tubes, a different amino acid is

coupled in each tube at the next-to-N-terminal position, then each tube is N-terminal

capped with N-acetyl Arg, because that was shown in the previous assay

Encoding Combinatorial Libraries

A more rapid approach would be to test the entire library at once and identify the active

component of the library directly.

As mentioned above, with large libraries of complex molecules it is not readily possible

to determine the structure of the active component.

In that case, encoding methods are needed. This is similar to the way in which proteins

are often sequenced in biology.

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the protein is not sequenced, but the gene that encodes the protein is Although the

structure of the actual compound may not be directly elucidated, certain tag molecules

that encode the structure may be determined.

One important approach that involves the attachment of unique arrays of readily

analyzable, chemically inert, small molecule tags to each bead in a split synthesis

Ideal encoding tags must survive organic synthesis conditions, not interfere with

screening assays, be readily decoded without ambiguity, encode large numbers of

compounds the test compound and the encoding tag must be able to be packed into a

very small volume.

In the Still method, groups of tags are attached to a bead at each combinatorial step in a

split synthesis.

The tags create a record of the building blocks used in that step. At the end of the

synthesis the tags are removed and analyzed, which decodes the structure of the

compound attached to that bead.

one or more readily cleavable tag molecules (TagsX) are attached to about 1% of the

Polymer bead sites (about 1 pmol/bead), and these encode building block 1 (BB1).

Non peptidal synthesis

Peptides do not make very useful drugs, especially if an orally active drug is sought. The

same techniques described above for the synthesis of peptide libraries could be utilized to

prepare nonpeptide libraries;

However, there is an important difference between the chemistry with peptides versus

nonpeptides, namely, reactivity.

In a typical peptide coupling reaction the carbodimide-activated N-protected amino acids

are all about the same in reactivity with the different amino acids in the growing peptide

chain. Because of that, the split synthesis method works well.

However, with nonamino acid reagents, such as different acid chlorides, the structure of

the acid chloride will affect the rates of reaction with different nucleophiles.

That could lead to mixtures in which some of the components have reacted and others have not.

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For each reaction the conditions have to be worked out to be sure complete reaction has

occurred.

Over the years it has been recognized that when large numbers of nonpeptide analogs are

screened simultaneously, many false negatives (an active compound that does not

produce a hit, i.e., a compound that shows a predetermined level of activity in the assay)

and false Positives (an inactive compound that gives a hit) are observed.

A false positive may arise from an impurity in the sample tested or as a result of a

complex between more than one compound.

False positives are a waste of time, but false negatives mean that potential drugs (or at

least lead compounds) are being overlooked.

It is typical for pharmaceutical companies to carry out single entity screens to avoid these

problems. Because of this, individual compounds, rather than mixtures, are synthesized.

Nonetheless, synthesis on a solid support allows the synthesis of large numbers of

individual compounds rapidly and robotically.

The reactions are carried out individually in separate micro tubes containing the

polymeric support.

Because the library of compounds (in the range of 50–104 compounds in amounts of 1–

50 mg) is synthesized in parallel without combining any of the tubes.S

One strategy that can be used for potentially more effective libraries is to select

privileged structures as the scaffold.

Another strategy is to design a scaffold based on an important molecular recognition

motif in the target receptor.

The libraries should incorporate different sets of (commercially available) building

blocks to provide a large number of diverse structures, and they should contain as much

functionality as possible as recognition elements.

Molecular diversity, however, is difficult to determine;

Dixon and Villar have found that a protein can bind a set of structurally diverse

molecules with similar potent binding affinities, but analogs closely related to these

compounds can exhibit very weak binding.

Parallel synthesis can generate many more compounds than can be synthesized

traditionally, and the cost per compound is much lower.

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The main differences among the various combinatorial approaches are the solid support

used, the methods for assembling the building blocks, the state (immobilized or in solution)

and numbers (a fraction of the total library or individual entities)

there different types of combinatorial synthesis.

SAR by NMR/SAR by MS

Fesik and coworkers at Abbott Laboratories developed a NMR-based approach to screen

libraries of small organic molecules and to identify and optimize high-affinity ligands

(compounds that bind to receptors) for proteins.

This approach, termed SAR by NMR, was initially used to discover compounds with

nanomolar affinities (highly potent for the immunosuppressant FK506 binding protein by

tethering two molecules with micromolar affinities (low potency).

The first step of the process involves screening a library of small compounds, 10 at a

time, by observation of the amide 15N-chemical shift in the heteronuclear single quantum

coherence (HSQC) NMR spectrum.

Once a lead is identified, a library of analogs is screened to identify compounds with

optimal binding at that site.

Then a second library of compounds is screened to find a compound that binds at a

nearby site, and again this compound is optimized by screening a library of related

compounds.

Based on the NMR spectrum of the ternary complex of the protein and the two bound

ligands, the location and orientation of these ligands are determined, and compounds are

synthesized in which the two ligands are covalently attached

Although each individual ligand may be a relatively weak binder, when the two are

attached, the binding affinity increases dramatically.

This is because the free energy of binding becomes the sum of three free energies: the

two ligands and the linker; the binding affinity is the multiplier of the three binding

affinities.

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There is a gain of about a factor of 100 in binding affinity by freezing out one bond

rotation. Therefore, it is not necessary to optimize the lead much, because ligands with

micromolar or even mill molar affinities can attain nanomolar affinities

When linked. An example of this is the identification of the first potent inhibitor of the

enzyme stromelysin, a matrix metalloprotease (a family of zinc-containing hydrolytic

enzymes responsible for degradation of extracellular matrix components such as collagen

and proteoglycans

In normal tissue remodeling and in many disease states such as arthritis, osteoporosis,

and cancer) as a potential antitumor agent

The method relies on the development of structure-activity relationships by mass

spectrometry (SAR by MS) and utilizes information derived from the binding of known

inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic

effort. Non covalent complexes of known inhibitors with a target protein are analyzed;

these inhibitors are deconstructed into sets of fragments which compete for common or

overlapping binding sites on the target protein. The binding of each fragment set can be

studied independently. With the use of competition studies, novel members of each

fragment set are identified from compound libraries that bind to the same site on the

target protein. A novel inhibitor of the target protein was then constructed by chemically

linking a combination of members of each fragment set in a manner guided by the

proximity and orientation of the fragments derived from the known inhibitors. In the case

of stromelysin, a novel inhibitor composed of favorably linked fragments was observed

to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately

formed higher order complexes with stoichiometries of 2:1 or greater. These linked

molecules were subsequently assessed for their ability to block stromelysin function in a

chromogenic substrate assay.

Peptidomimetics

Plants and animals, including human skin, contain a variety of antibiotic peptides.

Endogenous peptides also function as analgesics antihypertensive agents, and antitumor

agents

However, peptides do not make good drug candidates because they are rapidly

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Proteolyzed in the GI tract and serum, and they are poorly bioavailable, rapidly excreted,

and can bind to multiple receptors.

What is needed is a compound that mimics or blocks the biological effect of a peptide by

interacting with its receptor or enzyme, but does not have the undesirable characteristics

of peptides; these are peptidomimetics.

A remarkable resemblance was demonstrated between the N-terminal tyrosine structure

of these opioid peptides and the morphine phenol ring system, which suggested why they

all interacted with these receptors in a similar way.

The design of peptidomimetics can be a lead optimization approach, which uses the

desired peptide as the lead compound and modifies it to minimize (or preferably,

eliminate) the undesirable pharmokinetic properties.

The generation of peptidomimetics is based on the conformational, topochemical, and

electronic properties of the lead peptide when bound toits target receptor or enzyme.The

7 goal is to replace as much of the peptide backbone as possible with nonpeptide

fragments while still maintaining the pharmacophoric groups (usually the amino acid side

chains) of the peptide. This makes the compound more lipophilic, which increases its

bioavailability.

Replacement of the amide bond with alternative groups prevents proteolysis and

promotes metabolic stability.

Initially, conformational flexibility has to be retained to allow the pharmacophoric groups

a better opportunity to find their binding sites, but further lead refinement should favor

the formation of more conformationally restricted analogs that hold appropriate

pharmacophoric groups in the bioactive conformation for binding to the target receptor.

Increased lipophilicity and conformational modification of amino acids can be designed

into the peptidomimetic. These groups may not be recognized by peptidases. For example

conformational restricted analogs of phenylalanine can be incorporated into

peptidomimetic receptor ligands.

Likewise, conformational restriction and lipophilicity can be incorporated into peptides

Another approach involves the design of conformationally restricted analogs that mimic

characteristics of the receptor-bound conformation of the endogenous peptide, such as

turns,α-helices-loops and β-strands This idea can be extended to scaffold

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peptidomimetics in which importantPharmacophoric residues are held in the appropriate

orientation by a rigid template.

Compounds that block the binding of fibrinogen to its receptor (glycoprotein IIb/IIIa)

can prevent platelet aggregation and are of potential value in the treatment of strokes and

heart attacks. Common and important approach for the conversion of a peptide lead into

a peptidomimetic is the use of peptide backbone isosteres .

Peptides in which the amide bonds are replaced with alternative groups are known as

pseudo peptides.

These isosteric replacements remove the peptide linkage (thereby stabilizing the

peptidomimetics to metabolism) and/or make them less polar and more lipophilic.

The hydroxymethylene (also called statine) isostere is one of the early mimetics used in the

design of inhibitors of proteases, particularly of HIV protease.Other variants of azapeptides

Computer-aided design (CAD), also known as computer-aided design and

drafting (CADD) , is the use of computer technology for the process of design and design-

documentation. Computer Aided Drafting describes the process of drafting with a computer.

CADD software, or environments, provides the user with input-tools for the purpose of

streamlining design processes; drafting, documentation, and manufacturing processes. CADD

output is often in the form of electronic files for print or machining operations. The development

of CADD-based software is in direct correlation with the processes it seeks to economize;

industry-based software (construction, manufacturing, etc.) typically uses vector-based (linear)

environments whereas graphic-based software utilizes raster-based (pixelated) environments.

CADD environments often involve more than just shapes. As in the

manual drafting of technical and engineering drawings, the output of CAD must convey

information, such as materials, processes, dimensions, and tolerances, according to application-

specific conventions.CAD may be used to design curves and figures in two-dimensional (2D)

space; or curves, surfaces, and solids in three-dimensional (3D) objects.

Pre-clinical development is a stage of research that begins before clinical trials (testing in

humans) can begin, and during which important feasibility, iterative testing and safety (also

known as Good Laboratory Practice or "GLP") data is collected.

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The main goals of pre-clinical studies (also named preclinical studies and nonclinical

studies) are to determine a product's ultimate safety profile. Products may include new or iterated

or like-kind medical devices, drugs, gene therapy solutions, etc. Each class of product may

undergo different types of preclinical research. For instance, drugs may undergo

pharmacodynamics (PD), pharmacokinetics (PK), ADME, and toxicity testing through animal

testing. This data allows researchers to allometrically estimate a safe starting dose of the drug

for clinical trials in humans. Medical devices that do not have drug attached will not undergo

these additional tests and may go directly to GLP testing for safety of the device and its

components. Some medical devices will also undergo biocompatibility testing which helps to

show whether a component of the device or all components are sustainable in a living model.

Most pre-clinical studies must adhere to Good Laboratory Practices (GLP) in ICH Guidelines to

be acceptable for submission to regulatory agencies such as the Food & Drug Administration in

the United States.

Typically, both in vitro and in vivo tests will be performed. Studies of a

drug's toxicity include which organs are targeted by that drug, as well as if there are any long-

term carcinogenic effects or toxic effects on mammalian reproduction.

The information collected from these studies is vital so that safe human testing can begin.

Typically, in drug development studies animal testing involves two species. The most commonly

used models are murine and canine, although primate and porcine are also used. The choice of

species is based on which will give the best correlation to human trials. Differences in

the gut, enzyme activity, circulatory system, or other considerations make certain models more

appropriate based on the dosage form, site of activity, or noxious metabolites. For example,

canines may not be good models for solid oral dosage forms because the characteristic carnivore

intestine is underdeveloped compared to the omnivores, and gastric emptying rates are increased.

Also, rodents can not act as models for antibiotic drugs because the resulting alteration to their

intestinal flora causes significant adverse effects. Depending on a drugs functional groups, it may

be metabolized in similar or different ways between species, which will affect both efficacy and

toxicology. Medical device studies also use this basic premise. Most studies are performed in

larger species such as dogs, pigs and sheep which allow for testing in a similar sized model as

that of a human. In addition, some species are used for similarity in specific organs or organ

Page 31: Principles of drug discovery

system physiology (swine for dermatological and coronary stent studies; goats for mammary

implant studies; dogs for gastric studies)

Based on pre-clinical trials, No Observable Effect Levels (NOEL) on drugs are

established, which are used to determine initial phase 1 clinical trial dosage levels on a

mass API per mass patient basis. Generally a 1/100 uncertainty factor or "safety margin" is

included to account for interspecies (1/10) and inter-individual (1/10) differences.

Animal testing in the research-based pharmaceutical industry has been reduced in recent

years both for ethical and cost reasons. However, most research will still involve animal based

testing for the need of similarity in anatomy and physiology that is required for diverse product

development.

Preclinical Toxicology Testing and IND Application

Preclinical testing analyzes the bioactivity, safety, and efficacy of the formulated drug

product. This testing is critical to a drug’s eventual success and, as such, is scrutinized by many

regulatory entities. During the preclinical stage of the development process, plans for clinical

trials and an Investigative New Drug (IND) application are prepared. Studies taking place during

the preclinical stage should be designed to support the clinical studies that will follow.

Acute Studies: Acute toxic studies look at the effects of one or more doses administered over a

period of up to 24 hours. The goal is to determine toxic dose levels and observe clinical

indications of toxicity. Usually, at least two mammalian species are tested. Data from acute toxic

studies helps determine doses for repeated dose studies in animals and Phase I studies in humans

Repeated Dose Studies: Depending on the duration of the studies, repeated dose studies may be

referred to as subacute, subchronic, or chronic. The specific duration should anticipate the length

of the clinical trial that will be conducted on the new drug. Again, two species are typically

required.

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PRECLINICAL STUDIES & CLINICAL STUDIES

Genetic Toxicity Studies: These studies assess the likelihood that the drug compound is

mutagenic or carcinogenic. Procedures such as the Ames test (conducted in bacteria) detect

genetic changes. DNA damage is assessed in tests using mammalian cells such as the Mouse

Micronucleus Test. The Chromosomal Aberration Test and similar procedures detect damage at

the chromosomal level.

Reproductive Toxicity Studies: Segment I reproductive toxic studies look at the effects of the

drug on fertility. Segment II and III studies detect effects on embryonic and post-natal

development. In general, reproductive toxic studies must be completed before a drug can be

administered to women of child-bearing age.

Carcinogenicity Studies: Carcinogenicity studies are usually needed only for drugs intended for

chronic or recurring conditions. They are time consuming and expensive, and must be planned

for early in the preclinical testing process.

Page 33: Principles of drug discovery

Toxicokinetic Studies:These are typically similar in design to PK/ADME studies except that

they use much higher dose levels. They examine the effects of toxic doses of the drug and help

estimate the clinical margin of safety.

Grant of permission for clinical trails

There are numerous FDA and ICH guidelines that give a wealth of detail on the different

types of preclinical toxicology studies and the appropriate timing for them relative to IND and

NDA or BLAIn India pharmaceuticals are governed by the Drugs & Cosmetics Act and the

Rules framed to implement the provisions in the Act. New chemical entities may not be

administered to human subjects in a clinical trial without permission from the

Drugs Controller General of India (DCGI). Such permission may be obtained by

submitting to the DCGI an application for a clinical trial (CTA). It takes approximately 12 weeks

to obtain permission for a clinical trial for most investigational drugs. The duration may be

longer for drugs with special significance to the healthcare concerns of the country or those that

may be considered controversial since these are liable to be referred to the Indian Council of

Medical Research for comments. Ethic Committee approval is not a necessary precondition for

regulatory permission to conduct a clinical trial provided the applicant submits an undertaking

that the study will not be initiated at individual sites without prior EC approval. If clinical

supplies are to be imported, a "Test-Import License" must be applied for. This is done using the

format provided in Form 12 of the Drugs & Cosmetics Rules. Import and manufacture of clinical

trial supplies is governed by Rules 33 & 34 and provisions contained in Part X-A of the rules

Adverse drug reactions occurring during the course of a clinical trial need to be submitted

to the DCGI within 14 days if these are unexpected or serious and causally related or result in

death. All other serious adverse events need to be submitted along with periodic progress reports.

Compliance with GCP guidelines issued by the CDSCO is recommended although this does not

have statutory status at the present time. A report on the status of the study with details of

enrollment and safety issues needs to be submitted annually and on completion of the study. 

CLINICAL TRAILS

The Clinical studies are grouped according to their objective into three types or phases:

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Phase I Clinical Development (Human Pharmacology) - Thirty days after a biopharmaceutical

company has filed its IND, it may begin a small-scale Phase I clinical trial unless the FDA places

a hold on the study. Phase I studies are used to evaluate pharmacokinetic parameters and

tolerance, generally in healthy volunteers.  These studies include initial single-dose studies, dose

escalation and short-term repeated-dose studies.

Phase II Clinical Development (Therapeutic Exploratory) – Phase II clinical studies are

small-scale trials to evaluate a drug’s preliminary efficacy and side-effect profile in 100 to

250patients.Additional safety and clinical pharmacology studies are also included.

Phase III Clinical Development (Therapeutic Confirmatory) - Phase III studies are large-

scale clinical trials for safety and efficacy in large patient populations. While phase III studies

20-40 max 50 Healthy volunteersSometimess patients are exposed to drug one by one

Number of subjects

Carried out by qualified clinical pharmacologist & trained physicianDose is given in cumulative manner to achive the effective dose

Associated members

P’kinetics & P’dynamicEmphesis of safty and tolerebilityPurpose of study

100-400patients or volunterrsAccording to specific inclusion and exclusion criteriaNumber of subjects

Physicians These are trained as investigatorsAssociated members

To establish therapeutic efficacy of drug ,dosage regimen & ceiling effect in controlled settingsTolerability & P’cokinetics are studided as phase I extension

Purpose of study

Page 35: Principles of drug discovery

are in progress, preparations are made for submitting the Biologics License Application (BLA)

or the New Drug Application (NDA).  BLAs are currently reviewed by the FDA’s Center for

Biologics Evaluation and Research (CBER).  NDAs are reviewed by the Center for Drug

Evaluation and Research (CDER).

• Randomized double blind comparative trails are done

• Indications are finalized & guidelines for therapeutic use are formulated

• Submission of NDA for licensing is done who if satisfied grants permission for

marketing

Phase 4 (post market surveillance) – On approval of new drug, the importer Should

conduct the necessary surveillance and report back to aid the study of ADR’s of the specific

drug. the drug approved may be even rejected in this phase in the case of its toxicity beyond

the therapeutic effect .so the time period involved cannot be even predicted .

Registration of new drugs for marketing in India requires submission of data generated on

Indian patients. A 100-patient non-comparative open-label study on patients treated for the

primary indication is sufficient. In addition to local data, the NDA must include various other

items of information listed in Schedule Y of the Drugs & Cosmetics Rules. 

Data from prospective post-marketing surveillance is usually required to be submitted to the

CDSCO within 2 years of approval of a product. PMS data is considered a prerequisite for

renewal of the import license on expiration of validity 3 years from the date of issue. 

Clinical trials and research conducted on human beings can now be accessed by the general

public too. Hitherto, research institutions and companies obtained permission from the regulatory

Number of subjects

500-3000

Associated members

physicians

Purpose of study

To establish value of drug in relatn to existing oneADR’S on wide scale in which p’cokinetic data may be obtained

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authorities and registration of the trials was voluntary. Now, the Drugs Controller General of

India (DCGI) has asked the Indian Council of Medical Research to ensure that while granting

permission for clinical trials, the applicants are advised to get the trial registered before initiation

of the study. The new rule mandates that trials should be registered before the enrolment of the

first patient. Not just fresh human trials but even ongoing trials must be registered.

Advanced approaches

Bioinformatics :is application of computer technology to the management of biological

information it deals with algorithms databases and information systems, web technologies,

artificial intelligence and soft computing, information and computation theory ,software

engineering, data mining, image processing, modeling and simulation, signal

processing ,discrete mathematics ,control and system theory ,circuit theory and statistics For

generating new knowledge of biology in medicine improving and discovering new model of

computation

Major research areas

Genome annotation, analysis of gene expression, analysis of mutations in cancer,

modeling biological systems, structural bioinformatics like prediction of protein structure,

molecular interaction, docking algorithms

Chemi-informatics:The chemoinformatics concept chemical diversity, depicted as

distribution of compounds in the chemical space based on their physicochemical characteristics,

is often used to describe the difference between the combinatorial chemistry libraries and natural

products

micro array techniques: an array is an orderly arrangement of samples were matching of

known and unknown DNA samples is done based on base pairing rules .an experiment makes

use of common assay systems such as micro plates are standard blotting membranes. the samples

spot sizes(probe) are typically less than 200microns in diameter usually contains thousands of

spots

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A typical micro array ex involves hybridization of mRNA molecule to the DNA template

from which it is originated .Many DNA samples are used to construct an array. The amount of

mRNA bound to each site on the array indicates the expression level of various genes.

Conclusion: One should follow the above laid guide lines in order to produce a safe and

efficient drug

The drug discovery process should follow the ethics with out any compromise in any stages

of the process

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REFERENCES

1. “Textbook of Drug Design and Discovery”. Madsen, Ulf; Krogsgaard-Larsen, Povl; Liljefors,

Tommy (2002).  Washington, DC: Taylor & Francis.povl 235-423

2. “Guidebook on Molecular Modeling in Drug Design.” .Boston:Academic

Press.  . Pharmacophore Perception, Development, and use in Drug Design. La Jolla, Calif:

International University Line. Cohen, N. Claude (1996) Guner, Osman F. (2000) cohen vol II

541-671

3. “Structure-based Drug Discovery” Leach, Andrew R.; Harren Jhoti (2007). . Berlin:

Springer. Wang R,Gao Y,Lai L (2000).LigBuilder: A Multi-Purpose Program for Structure-

Based Drug Design". Journal of Molecular Modeling 6 (7-8): 498–516.

4. "The many roles of computation in drug discovery". Science 303 (5665) Jorgensen WL (March

2004). s1813–8. 

5. “Essentials of medical pharmacology’KD Tripati 6th addition ,process of drug development, p-12-

27.

6. “How Drugs Work”, Oxford University Press, Oxford, 2000Hare, R. The Birth of Penicillin,

Allen & Unwin, London,1970.Beveridge, W. I. B. , p. 255

7. "Natural products as sources of new drugs over the last 25 years".Newman DJ, Cragg GM

(March 2007 J. Nat. Prod. 70 (3): 461–77. 

8. "Structure-based drug design: progress, results and challenges" Verlinde CL, Hol WG..Böhm HJ.

"The development of a simple empirical scoring function to estimate the binding constant for a

protein-ligand complex of known three-dimensional structure". J(June 1994). Comput. Aided Mol

(July 1994).. Structure 2 (7): 577–87

9. “Book of drug discovery “. Fleming, A. Brit. J. Exp. Stone, T.; Darlington, G. Pills, Potions and

Poisons. Pathol. 1929, P gen(3)10, 226

10. “Seeds of Discovery”, W. W. Norton, Abraham, E. P.; Chain, E.; Fletcher, C. M.; sGardner, A.

D.; Heatley, N. G.;Jennings, M. A.; Florey, H. W. New York, 1981.Lancet 1941, 2, 177.

11. http://www.ncbi.nlm.nih.gov/pubmed/14697768

12. "Comparison of consensus scoring strategies for evaluating computational models of protein-

ligand complexes". J Chem Inf Model 46 (1): Oda A, Tsuchida K, Takakura T, Yamaotsu N,

Hirono S (2006). 380–91.

13. “ Structural interaction fingerprint (SIFt): a novel method for analyzing three-dimensional

protein-ligand binding interactions’Deng Z, Chuaqui C, Singh J (January 2004). "". J. Med.

Chem. 47(2): 337–44. 

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