1

1-INTRODUCTION

1.1. Tuberculosis

Tuberculosis is a contagious disease1 caused by a bacterial infection of the lungs,

which can also spread to other parts of the body, such as the brain, kidneys, and bones.

Tuberculosis, also known as TB, is caused by the bacterium Mycobacterium tuberculosis.

Tuberculosis (TB) is a bacterium that usually causes disease in the lung. Many people

become symptom-free carriers of the TB bacteria. Although common and deadly in the

third world, tuberculosis was almost non-existent in the developed world, but has been

making a recent resurgence. Certain drug-resistant strains are emerging and people with

immune suppression such as AIDS.

It is estimated that 1.7 million people died of 2 in 2009. There were an estimated

9.4 million new cases of tuberculosis in 2009 of which the majority were in Asia and

Africa. It is thought that the rates of new tuberculosis infections and deaths per capita

have probably been falling globally for several years now. However, the total number of

new tuberculosis cases is still slowly rising due to population growth poor health.

Person can have active or inactive tuberculosis2. Active tuberculosis or TB

disease means the bacteria are active in the body and the immune system is unable to stop

them from causing illness. People with active tuberculosis in their lungs can pass the

bacteria on to anyone they come into close contact with. When a person with active

tuberculosis coughs, sneezes or spits, people nearby may breathe in the tuberculosis

bacteria and become infected. Left untreated, each person with active tuberculosis will

infect on average between 10 and 15 people every year. Between 1990 and 2005,

2

tuberculosis incidence in 2007, Africa accounted for an estimated 78% of tuberculosis

cases among HIV-positive people worldwide.

The largest number of tuberculosis cases occurs in Asia, which in 2009 accounted

for an estimated 56% of the global total. However the estimated incidence per capita in

sub-Saharan Africa is around twice that of South-East Asia. The countries of Eastern

Europe are also facing a serious epidemic; there were over 150,000 new cases in Russia

alone in 2009.

Tuberculosis is not only a problem in low and middle income countries. For

example, there were 11,545 new cases reported in the U.S.A. in 2009.4 In the UK, TB

has been dubbed 'the disease that has never went away', with 9,040 new cases of

tuberculosis reported in the UK in 2009.5 Although the UK's national rate is very low in

comparison with most of the world, London has become one of the world's tuberculosis

hotspots. In parts of London, tuberculosis rates are ten times the national rate - higher

than in some countries of the former Soviet Union. About 10 per cent of people with TB

in London are likely to be co-infected with HIV6.

Of the people who worldwide died of tuberculosis in 2008, it is estimated that

400,000 were infected with HIV. Tuberculosis is the leading cause of death among HIV

infected people in Africa. When someone is infected with tuberculosis the likelihood of

them becoming sick with the disease is increased many times if they are also HIV

positive "The different cultures of the TB and HIV communities raise many challenges in

achieving an effective and productive partnership... TB services are geared towards

chronic-care services with simple and standardized technical procedures, while

3

HIV/AIDS services are clinically oriented and tend to be more individual-patient-

oriented4.

People with latent tuberculosis are increasingly becoming infected with HIV, and many

more are developing active TB because HIV is weakening their immune system. People

who are co-infected with both HIV and latent TB have an up to 800 times greater risk of

developing active tuberculosis disease and becoming infectious compared to people not

infected with HIV1.

Tuberculosis (TB) and HIV Positive People5

Because tuberculosis can spread through the air, the increase in active tuberculosis

among people infected with both tuberculosis and HIV results in:

More transmission of the tuberculosis bacteria

More transmission of the tuberculosis bacteria

More people with latent tuberculosis

More TB disease in the whole population

2There are several important associations between the epidemics of HIV and tuberculosis:

Tuberculosis is harder to diagnose in HIV positive people

Tuberculosis progresses faster in HIV-infected people

Tuberculosis in HIV positive people is more likely to be fatal if undiagnosed or

left untreated

Tuberculosis occurs earlier in the course of HIV infection than other opportunistic

infections

4

Tuberculosis is the only major AIDS-related opportunistic infection that poses a

risk to HIV-negative people.

TB Organism

TB is caused by repeated exposure to airborne droplets contaminated with

M. tuberculosis, a rod-shaped bacterium. The TB bacterium also is known as the tubercle

bacillus. (A small fraction of cases are caused by related bacteria, M. africanum and M.

bovis.) M. tuberculosis, like other mycobacteria, has an unusual cell wall, a waxy coat

comprised of fatty molecules whose structure and function are not well known. This cell

wall appears to allow M. tuberculosis to survive in its preferred environment: inside

immune cells called macrophages, which ordinarily degrade pathogens with enzymes.

The coat of M. tuberculosis also renders it impermeable to many common drugs.

Biologists call M. tuberculosis and other mycobacteria "acid fast" bacteria

because their fatty cell walls prevent the cells from being decolorized by acid solutions

after staining during diagnostic tests. Several factors make M. tuberculosis a difficult

organism to study in the laboratory, hampering TB research. The bacteria multiply very

slowly, only once every 24 hours, and take a month to form a colony. By comparison,

other bacteria such as E. coli form colonies within eight hours. TB bacilli tend to form

clumps, which makes working with them and counting them difficult. Most daunting, M.

tuberculosis, a dangerous, airborne organism, can be studied only in laboratories that

have specialized safety equipment

5

Treatments for Tuberculosis

Antibiotic combination therapy - using more than one antibiotic reduces the risk of

resistance.

Isoniazid (INH)/rifampicin combination with Pyrazinamide and Ethambutol

Isoniazid

Isoniazid (Laniazid, Nydrazid), also known as isonicotinylhydrazine (INH), is an

organic compound which is the first-line antituberculosis medication in prevention and

treatment. It was first discovered in, and later it was found to be effective against

tuberculosis. Isoniazid is never used on its own to treat active tuberculosis because

resistance quickly develops. Isoniazid also has an antidepressant effect, and it was one of

the first antidepressants discovered.

Isoniazid is a prodrug and must be activated by a bacterial catalase-peroxidase enzyme

that in M. tuberculosis is called KatG5. KatG couples the isonicotinic acyl with NADH to

form isonicotinic acyl-NADH complex. This complex binds tightly to the enoyl-acyl

carrier protein reductase known as InhA, thereby blocking the natural enoyl-AcpM

substrate and the action of fatty acid synthase. This process inhibits the synthesis of

mycolic acid, required for the mycobacterial cell wall. A range of radicals are produced

by KatG activation of Isoniazid, including nitric oxide, 6

which has also been shown to be

N

CONHNH2

Isoniazid Isoniazid

6

important in the action of another antimycobacterial prodrug7. Isoniazid is bactericidal to

rapidly-dividing mycobacteria but is bacteriostatic if the mycobacterium is slow-growing.

Isoniazid inhibits the P450 system8.

Pyrazinamide

N

N CONH2

a Pyrazinamide

Pyrazinamide: A synthetic pyrazinoic acid amide derivative, bactericidal

tuberculostatic Pyrazinamide is particularly active against slowly multiplying

intracellular bacilli (unaffected by other drugs) by an unknown mechanism of action. Its

bactericidal action is dependent upon the presence of bacterial pyrazinamidase, which

removes the amide group to produce active pyrazinoic acid. Pyrazinamide is an important

component of multidrug therapy for tuberculosis. Pyrazinamide: antibacterial derived

from nicotinic acid, used as a tuberculostatic.

7

Ethambutol13

CH2.NH.CH.CH

2.OH

CH2.NH.CH.CH

2.OH

CH2.CH

3

CH2.CH

3

.2 HCL

Ethambutol Hydrochloride

Ethambutol is an antitubercular agent that inhibits the transfer of mycolic acids

into the cell wall of the tubercle bacillus. It may also inhibit the synthesis of spermidine

in mycobacteria. The action is usually bactericidal, and the drug can penetrate human cell

membranes to exert its lethal effect. Ethambutol: antitubercular agent that inhibits the

transfer of mycolic acids into the cell wall of the tubercle bacillus; it may also inhibit the

synthesis of spermidine in mycobacteria. Ethambutol: An antitubercular agent that

inhibits the transfer of mycolic acids into the cell wall of the tubercle bacillus. It may also

inhibit the synthesis of spermidine in mycobacteria. The action is usually bactericidal,

and the drug can penetrate human cell membranes to exert its lethal effect.

8

Rifampicin14

CH N NN

NH

O

O

OH

H3C

CH3

O

O

CH3

HO

CH3CO

OH

CH3O

CH3

CH3

CH3 CH

3

O

O

CH3

O

Rifampicin

A semisynthetic antibiotic produced from Streptomyces mediterranei. It has a

broad antibacterial spectrum, including activity against several forms of Mycobacterium.

In susceptible organisms it inhibits DNA-dependent RNA polymerase activity by forming

a stable complex with the enzyme. It thus suppresses the initiation of RNA synthesis.

Rifampin is bactericidal, and acts on both intracellular and extracellular organisms14

.

In earlies, Vaccine against tuberculosis called as BCG3, but the vaccine is now very old

(it was first used in the 1920s), and tests have found it to be very variable in its ability to

protect people from infection in modern settings. The BCG can also cause false-positive

readings on the tuberculin skin test. If given to HIV positive adults or children with very

9

weak immune systems, the BCG can occasionally cause disseminated BCG disease,

which is often fatal.

A drug called isoniazid (INH) can be used as a preventative therapy for those who are at

high risk of becoming infected with tuberculosis or for those who have inactive TB.

People who have inactive tuberculosis but are not yet sick can take a course of isoniazid

for several months to stop them developing active tuberculosis. The WHO recommends

that HIV positive people who have latent tuberculosis (but definitely not active TB)

should be offered isoniazid preventive therapy as needed.

Tuberculosis Treatment and HIV

It is vitally important for people with HIV to have treatment if they have active

tuberculosis. This will cure them of TB and prevent transmission to others. Even in

settings where antiretroviral drugs are unavailable or inaccessible, it is crucial that the

health system is able to offer HIV positive people the simple drugs needed for DOTS.

For some people it can be difficult to take drugs for both tuberculosis and HIV at the

same time. Some anti-HIV drugs can also interact with some tuberculosis drugs making

the treatment more difficult. It is important that the tuberculosis treatment is taken

regularly and exactly as the health care provider has advised. If the drugs are not taken

regularly, the bacteria can become resistant to the drugs and this can be dangerous. As

one of the first opportunistic infections to appear in HIV-infected people, tuberculosis

may be one of the earlier signs of HIV infection. Addressing tuberculosis offers the

opportunity for early HIV intervention. Although treatment of tuberculosis can improve

10

the quality of life of HIV positive people and prolong their life, it cannot stop them from

dying of AIDS. This is why access to antiretroviral treatment is also vitally important.

Around the world7

attempts are being made to improve collaboration between

tuberculosis and HIV programmes. It is being proposed that everyone diagnosed with

tuberculosis should be tested for HIV and vice-versa, and that treatment programmes

should share facilities and expertise. However, achieving such collaboration is not

straightforward: "The different cultures of the TB and HIV communities raise many

challenges in achieving an effective and productive partnership... TB services are geared

towards chronic-care services with simple and standardized technical procedures, while

HIV/AIDS services are clinically oriented and tend to be more individual-patient-

oriented3.

Multi-drug resistant TB (MDR-TB) and extreme drug resistant 8-10

When a strain of tuberculosis bacteria is resistant to two or more 'first-line'

antibiotic drugs it is called multi-drug resistant TB or MDR-TB. When it is resistant to

three or more 'second-line' antibiotics as well, it is classed as extreme drug resistant

tuberculosis, or XDR-TB. Drug resistance usually arises when tuberculosis patients do

not or cannot take their medicine as prescribed and drug-resistant mutations of the

bacteria are allowed to replicate. People can also catch MDR and XDR-TB from others.

MDR-TB is a serious problem and is very difficult to treat. In normal treatment

(sometimes referred to as 'first-line' treatment) for tuberculosis, patients take the drugs

isoniazid and rifampicin (the most effective tuberculosis drug available) plus other drugs

for around six to eight months. If a person is resistant to isoniazid and rifampicin

11

However, they are said to have MDR-TB, and will need to change to a regime

containing newer and often less widely-available 'second-line' drugs. Treatment with

second-line drugs can take a very long time, and is usually far more expensive than

standard DOTS therapy because most of the drugs are still under patent.

XDR-TB is even more serious. If someone has XDR-TB, it means they are not

only resistant to isoniazid and rifampicin, but to three or more of the six available second-

line drugs too. This can make it virtually impossible to formulate an effective treatment

regime for them. Many people with XDR-TB will die before it is even realised that they

have the extreme resistant strain.

In 2006, 53 people in the province of KwaZulu Natal in South Africa were

identified as having XDR-TB. Of these people, 52 died within 25 days of tuberculosis

being diagnosed. The majority were HIV positive. Although HIV infection does not of

itself increase the chance of drug resistance occurring, both MDR-TB and XDR-TB are

very serious threats to HIV positive people, whose weakened immune systems render

them unlikely to fight off tuberculosis naturally.

WHO Statistical Report11

The WHO's leading infectious disease experts estimate there were 400,000 new

cases of MDR-TB worldwide in 2009. About half of these are among new tuberculosis

patients, and the other half are among patients who have previously received treatment.

There is also new evidence that drug resistant strains are becoming more resistant and

unresponsive to current treatments. More than three quarters of MDR-TB cases are now

12

"super strains", resistant to at least three of the four main drugs used to cure tuberculosis

in first-line treatment. The number of these that could also be classed as extreme drug

resistant strains is however unknown.

For many years, tuberculosis remained relatively overlooked on the global scale,

but the importance of addressing MDR and XDR-TB, and other problematic issues, is

now being recognised internationally. In 2006, the Stop TB Partnership launched "The

Global Plan to Stop TB",8 an initiative that aims to halve the death rates and prevalence

of tuberculosis worldwide by 2015. If successful, the plan will save over 14 million lives,

and will pave the way for the ultimate goal of eradicating tuberculosis by 2050. Much

more needs to be done to achieve this aim, both by directly combating tuberculosis, and

by addressing the HIV epidemic that is fuelling.

Framework for Managing Multiple Drug-Resistant

Management of DR-TB is organized around five components like the DOTS strategy,

because the underlying principles are the same, namely:

Sustained government commitment.

Accurate, timely diagnosis through quality assured culture and drug susceptibility

testing.

Appropriate treatment utilizing second-line drugs under strict supervision.

Uninterrupted supply of quality assured second-line drugs and Standardized

recording and reporting system.

13

1.2 Chemistry of Quinazoline

N

N

Quinazoline

FORMULA C8H6N2

MOL WT. 130.15

SYNONYMS 5,6-Benzopyrimidine; Penmiazine; 1,3-Benzodiazine;

Benzo[alpha]pyrimidine; Chinazolin; Quinazolina;

PHYSICAL PROPERTIES15

PHYSICAL STATE light yellow crystals

MELTING POINT 48 - 49 °C

BOILING POINT 243 °C

SOLUBILITY IN WATER Soluble

FLASH POINT 106 °C

STABILITY Stable under ordinary conditions

14

Chemistry of Quinazolinones

Quinazoline 16

is a fused bicycle compound earlier known as benzo-1 3-diazine

was first prepared in the laboratory by Gabriel1 in 1903, although one of its derivatives

was known much earlier17

.The name quinazoline (German : Chinazolin) was first

proposed for this compound by Weddige18

, on observing that this was isomeric with the

compounds cinnoline17

and quinoxaline18

. Paal and Bush19

suggested the numbering of

quinazoline ring system, which is currently used. The other less commonly used names

for this ring system are ‗phenmiazine‘ and 5, 6-benzopyrimidine. However, the name

quinazoline is now universally accepted.

Of the many derivatives of quinazoline system known so far, keto-quinazolines

also called as quinazolinones, are the most important compounds. Depending upon the

position of the keto or oxo group, these compounds may be classified into two types: 2-

(1H) quinazolinones (or) 1,2-dihydro-2-oxo quinazolines 19

and 4(3H)-quinazolines or

3,4-dihydro-oxoquinazolines 20

. These systems 19&20

exhibit lactam-lactim tautomerism

and undergo hydroxy group replacement reactions. 2-Cyano-4(3H)-quinazolinone was

the first quinazolinone derivative to be synthesized20

.

Brief Account of reactivity of 4(3H)-Quinazolinones

Reactions associated with tautomeric nature of the quinazolinones are often quite

complex and generally unpredictable. The recorded chemical investigation on the subject

is voluminous. The amide linkages in quinazolinones should not be looked on as

15

predominantly the keto or the enol form but as true keto-enol tautomers, showing reaction

characteristic of both the forms.

Quinazolinones are always high melting crystalline solids, insoluble in water and

in most organic solvents but soluble in aqueous alkali. They are generally insoluble in

dilute acids but are sometimes soluble in concentrated acids. Simple 4(3H)-

quinazolinones, although insoluble in dilute acids, are soluble in 6N hydrochloric acid.

4(3H)-quinazolinones form stable monohydrochlorides, chloroplatinate, chloroaurates

and picrates6 and their metal salts of silver, mercury, zinc, copper, sodium and

potassium21

.

Stability of the ring system

The ring system in quinazolinone is exceedingly stable in oxidation, reduction, hydrolysis

reactions and other treatment designed to break the ring. There is no report of degradation

of quinazolinone by simple chemical oxidation.

Aromatization

When a simple and 2-substituted- 4(3H) - quinazolinone is heated with an equivalent

amount of phosphorous pentachloride in phosphorous oxychloride, the corresponding 4-

chloroquinazoline20

is obtained. If a methyl group is present at 3-position, prohibiting the

usual tautomerism, the methyl group is lost during the chlorination22

.

Alkylation

The position of alkylation of quinazolinones is similar to all the aromatic nitrogen

heterocyclic systems in which a hydroxyl group is found ortho or para to the nitrogen

position. Such compounds exist in tautomeric mixture 21

, the two structures being inter-

16

convertible by the shift of one proton and one pair of electrons. In alkaline solution the

ions of such compounds exist as resonance hybrids of the two major forms differing only

by the position of two pairs of electrons, as shown. Thus in alkylation of such hydroxyl

derivatives of pyridine, pyrimidine and similar heterocycles, the entering group may

become attached to either the nitrogen atom, thus giving for instance, an N-alkyl-pyridine

or to the oxygen atom, giving an alkoxy pyridine. Alkylating agent9 and the conditions of

alkylation but not the hetercyclic nucleus, were the factors determining the course of

alkylation.

Nitration

4(3H)-Quinazolinone on boiling with nitric acid undergoes substitution to give 6-nitro-4

(3H)-quinazolinone 22.

On further nitration it has been observed that the second nitro

group enters the 8-position to give 6, 8-dinitro derivatives 23

2-Substituted-4(3H)-

quinazolinones were also found to behave similarly, under such conditions 24-27

.

Reduction

2, 3-Dihydro-3-methyl- 4(1H)-quinazolinone (10) could be obtained on reduction of 3-

methyl-4(3H)-quinazolinone with Lithium Aluminium Hydride (LiAIH4) in benzene28

.

Reactivity of the 2-methyl group

The methyl group in 2-position of 4(3H)-quinazolinone system was found to be quite

reactive since it is linked to an azomethine carbon and condenses with aldehydes to give

the styryl compounds 25,29-30

. These studies, interestingly, revealed that quite a few of

such quinazolinone derivatives possess a wide variety of pharmacological activities.

17

The summary of methods of preparation of 4(3H)-Quinazolinones

Most of the methods employed for the synthesis of 4(3H)-quinazolinones make use of

anthranilic acid or one of their functional derivatives as the starting materials. Based on

this factor, the general methods of synthesis are:

Condensation of Anthranilic acid with acid amides

When anthranilic acid is heated in an open container with excess of formamide at 120°C,

water is expelled and a nearly quantitative (90%) conversion to 4(3H)-quinazolinones 26,

31 is achieved.

Condensation of acetanilides with urethanes:

A number of attempts have been made to condense a urethane derivative with aniline to

give 4(3H)-quinazolinone, directly. Urethane and acetanilide, heated for 3 hours with

phosphorus pentoxide in toluene, give 2-methyl-4(3H)-quinazolinone 27,32

.

Condensation of N-acylanthranilic acids with primary amines:

4(3H)-Quinazolinones may also be synthesized directly from the corresponding N-

acylanthranilic acid by heating with ammonia or substituted amines. Bogert and Steiner35

have prepared 2-methyl-3-alkyl-6-nitro- 4(3H)-quinazolinones 28

from N-acyl-5-

nitroanthranilic acid and a variety of primary amines.

18

Biological importance of 4(3H)-Quinazolinones

The quinazolinone skeleton is a frequently encountered heterocycle in medicinal

chemistry literature with applications including antibacterial34

, analgesic35

, anti-

inflammatory 36, 37

antifungal38

, antimalarial39

, antihypertensive40

, CNS depressant41

,

anticonvulsant42

, antihistaminic & local anaesthetic 43

, antiparkinsonism 44

, and antiviral

and cancer activities 45

. Little number of quinazolinones was reported as potent

chemotherapeutic agents 47-48

in the treatment of tuberculosis. For example 3-aryl-6, 8-

dichloro-2H-1, 3-benzoxazine-2, 4(3H)-diones and 3-arylquinazoline-2, 4(1H, 3H)-

diones46 as antimycobacterial agents, quinazolinone derivatives 49

as antitubercular

agents.

19

1.3 Introduction to Docking

The demand for a rapid search for small molecules that may bind to the targets of

the biological interest is of vital importance in the drug discovery process. One way of

achieving this is the in silico or virtual screening51

((VS) of large compound collections

in identifying a subset of compounds that contain relatively many hits against the target

molecules, compared to the random selection from collection. If a three dimensional

structure or a model of the target [8, 10] is available, the commonly used technique is

structural based virtual screening58

(SBVS). Here it is also called docking program50

.

It is used to place computer generated representations of small molecules into the

target or in a user-defined part thereof, e.g., the active site of an enzyme) in variety of

positions, confirmations and orientations. Each such docking mode is called „pose‟. This

approach aims to predict correctly the structure of the intermolecular complex formed

between the target receptor and the ligand. To correctly dock a molecule or a fragment in

the region of interest60

(e.g., active site) leads two technical challenges.

The first challenge is the pose generation or docking of the ligand in the active

site of the biological target. The second challenge is the evaluation of different poses, or

Scoring. Scoring evaluates the binding energy between the target and the ligand in a

reliable manner and produces a correct correlative rank ordering for a number of

compounds. Docking programs usually do both the programs in tandem: they first sample

the different poses and then a scoring function used to estimate the affinity between the

receptor and the ligand for each pose.

20

Computer methods of drug design are based on a postulate that pharmacologically

active compounds act by interaction with their macro molecule targets, mainly proteins

and nucleic acids. Major factors of such interactions include steric complementary of

interacting surfaces of molecules, electrostatic forces, hydrophobic interactions, and

hydrogen bond formation. These factors are mainly considered during analysis and

prediction of interaction of two molecules.

Definition of molecular docking

Molecular docking can be defined as an optimization problem which

would describe the ―best –fit‖ orientation of a ligand that binds to a particular protein of

interest. However, since both the ligand and the protein are flexible, a ―hand-in-glove ―is

more appropriate than a “lock-and-key”52

. During the course of the processes the ligand

and the protein both adjust their confirmations to get the ‖best-fit‖ and this kind of

conformational adjustments resulting in the overall binding is referred to as “Induced

fit”53

. The aim of the molecular docking is to achieve an optimized confirmation for

both the protein and ligand such that the free energy of the overall system is minimized.

Mechanics of docking

To perform a docking, the first requirement is the protein of interest.

usually the structure have been determined using the biophysical technique such as x-ray

crystallaography or less often, NMR spectroscopy. The protein structure and a database

of potential ligands serve as an input to a docking program. The success of a docking

program depends on two components: the search algorithm and the scoring function

Search algorithm

21

The search space in theory consists of all possible orientations

and confirmations of the protein paired with the ligand. However in practice with current

computational resources, it is impossible to exhaustively explore the search space—this

would involve enumerating all possible distortions of each molecule (molecules are

dynamic and exist in an ensemble of conformational states) and all possible rotation and

translational orientations of the ligand relative to the protein at a given level

of granularity. Most docking programs in use account for a flexible ligand, and several

attempt to model a flexible protein receptor. Each "snapshot" of the pair is referred to as

a pose.

A variety of conformational search strategies have been applied to the ligand and to the

receptor. These include:

systematic or stochastic torsional searches about rotatable bonds

molecular dynamics simulations

genetic algorithms to "evolve" new low energy conformations

Ligand flexibility

Conformations of the ligand may be generated in the absence of the receptor and

subsequently docked61

or conformations may be generated on-the-fly in the presence of

the receptor binding cavity62

force field energy evaluation are most often used to select

energetically reasonable conformations63

but knowledge-based methods have also been

used.

22

Receptor flexibility

Computational capacity has increased dramatically over the last decade making possible

the use of more sophisticated and computationally intensive methods in computer-

assisted drug design. However, dealing with receptor flexibility61

in docking

methodologies is still a thorny issue. The main reason behind this difficulty is the large

number of degrees of freedom that have to be considered in this kind of calculations.

However, neglecting it, leads to poor docking results in terms of binding pose

prediction65

. Multiple static structures experimentally determined for the same protein in

different conformations are often used to emulate receptor

flexibility66

Alternatively rotamer libraries of amino acid side chains that surround the

binding cavity may be searched to generate alternate but energetically reasonable protein

conformations 67-68

Scoring function

The scoring function takes a pose as input and returns a number indicating the likelihood

that the pose represents a favorable binding interaction. Most scoring functions are

physics-based molecular mechanics force fields that estimate the energy54

of the pose; a

low (negative) energy indicates a stable system and thus a likely binding interaction. An

alternative approach is to derive a statistical potential for interactions from a large

database of protein-ligand complexes, such as the Protein Data Bank, and evaluate the fit

of the pose according to this inferred potential.

There are a large number of structures from X-ray crystallography for complexes

between proteins and high affinity ligands, but comparatively fewer for low affinity

ligands as the later complexes tend to be less stable and therefore more difficult to

23

crystallize. Scoring functions trained with this data can dock high affinity ligands

correctly, but they will also give plausible docked conformations for ligands that do not

bind. This gives a large number of false hits, i.e., ligands predicted to bind to the protein

that actually doesn‘t when placed together in a test tube.

One way to reduce the number of false positives is to recalculate the energy of the top

scoring poses using (potentially) more accurate but computationally more intensive

techniques such as Generalized Born or Poisson-Boltzmann methods. 56

Applications

A binding interaction between a small molecule ligand and an enzyme protein may result

in activation or inhibition of the enzyme. If the protein is a receptor, ligand binding may

result in agonism or antagonism. Docking is most commonly used in the field of drug

design — most drugs are small organic molecules, and docking may be applied to:

1. Hit identification – docking combined with a scoring function can be used to quickly

screen large databases of potential drugs in silico to identify molecules that are likely to

bind to protein target of interest.

2. Lead optimization – docking can be used to predict in where and in which relative

orientation a ligand binds to a protein (also referred to as the binding mode or pose). This

information may in turn be used to design more potent and selective analogs.

3. Bioremediation – Protein ligand docking can also be used to predict pollutants that can be

degraded by enzymes.69

24

Docking software

The docking program used in our benchmark is GOLD ver. 3.0.1(Genetic Optimization

of Ligand Docking). This program uses the genetic algorithm to perform the

conformational search.

GOLD

The program was developed by Jones et al 70

and uses a genetic algorithm, with the

adopted island model, to generate the ligand confirmers in an active site. Four scoring

functions implemented in this software are force field based Goldscore and piecewise

linear potential (PLP), empirically based chemscore and knowledge-based Astex

statistical potential (ASP) 71

. Various levels of accuracy can be chosen. In our test it was

set to 100%, i.e., around 30,000 genetic algorithms can be performed during docking.

Confirmations were scoted using only gold score function. The active site was chosen

based on the native ligand placement in the considered complex. In our evaluation we

used version 3.0.1 of the program, instead of newest program version 4.1. How-ever, in

our opinion, no new features regarding docking using only GoldScore functions were

added, thus results obtained using those two versions of the program should be virtually

identical.

The mechanism for the ligand placement is based on fitting points. The program add

fitting points to hydrogen-bonding groups on both the protein and the ligand, and maps

acceptor points in the lignad on donor points in the protein and vice versa. Additionally,

Gold generates Hydrophobic fitting points in the protein cavity on to which ligand CH

groups are mapped. The genetic algorithm optimizes the flexible ligand dihedrals, ligand

ring geometries, dihedrals of protein OH and NH3+ groups, and the mappings of the

25

fitting points. The docking poses are ranked based on a molecular mechanics like Scoring

function, which include a hydrogen bond term, a 4-8 intermolecular van der Waals term ,

and a 6-12 intermolecular van der Waals term for the internal energy of the ligand. In this

work the binding site was defined as spherical region of 10Å radius centered on the mass

centre structures of crystallographic ligand. The protein and ligand input structures were

prepared. Default GA settings number 472

were used for all calculations, with the

exception that 10 runs were performed. The top 3 docking poses were energy minimized

with macromolecule in both OPLS-AA (Optimized potentials for liquids stimulations-All

atoms) and MMFF forcefeilds and reran ked.

The underling algorithm of GOLD mimics the process of evolution by applying

genetic operators to a collection of putative poses for a given ligand(in GA terms a

population of chromosomes). GOLD chromosomes contain the conformational

information of the flexible parts of the protein (OH of ser, Thr and Tyr as well as Lysine

NH3+) and of the ligand., which is followed by least square (LS) fitting procedure 73

.,

with the objective to maximize the overlap between ligand and receptor features. The

energy of the resulting pose (fitness) consists of three terms: 1. Hydrogen-bonding energy

2. Internal energy of the ligand. 3. Steric interaction energy.

26

The population of chromosomes evolves through sequential application of genetic

operations. Newly generated chromosomes are decoded, the fitness of the corresponding

pose is evaluated, and the chromosome is kept if is fitter than the least fit chromosome in

the pool. After the application of the genetic operations the algorithm terminates the

algorithm terminates, and the poses with the highest scores are sav

Other Docking Programs

A large number of docking programs and search algorithm have been published. One

criterion for classifying the algorithms is the way the ligands are treated during docking.

In some of the algorithms the ligands are built up incrementally, staring form the docked

‗base fragment‘. Programs that follow this approach include Hammerhead 74

, DOCK 75-77

and FlexX78

. In the other programs such as Auto Dock 79-80

, Genetic Optimization for the

Ligand Docking (GOLD) 81

ICM-Dock 82, 83

and QXP84

the ligand is treated in its entirety.

In addition to the ligand flexibility, it may be desirable to keep at least part of the receptor

flexible in order to allow for conformational changes that are necessary to accommodate

the ligand, a phenomenon referred to as ―induced fit‖. Because it is computationally

expensive, few docking programs allow protein flexibility. Notable exceptions are the

latest versions of Auto dock 79

, FlexX 85

, QXP 84

, affinity 86

and the latest version of the

ICM-Dock 82, 83

. The way flexibility handled is differs from program to program.

27

Program ALGORITHM REFERENCES

Auto Dock Lamarckian GA [55,80,79]

DOCK Shape matching (sphere

images)

[75-77]

DOCK (NWU version) Shape matching (sphere

images)

[88-89]

FlexX Incremental construction [78]

FRED Shape matching (Gaussian

functions)

[86,87]

GLIDE Descriptor matching /MC [62,90-92]

GOLD GA [81]

Hammerhead Incremental construction [74]

ICM MC minimization [82,83]

Ligand Fit Shape matching (moments

of inertia)

[93]

QXP MC minimization, tree

searching and pruning

[84]

Slide Descriptor matching [94]

Surflex Dock Surface-based molecular

similarity

[95]