Computer Assisted Drug Design (HIV-1 NNRTIs)

Amol A. Kulkarni

Acquired Immuno Deficiency Syndrome (AIDS) is a systemic and fatal disorder produced by Human Immuno deficiency Virus (HIV), which is retrovirus of the lentivirus family originally reffered to HILV-III or Lav.

By leading to the destuction &/or functional impairement of cells of immune system, notably CD4+ T cells.HIV progressively destroys the body’s ability to fight infection leads to severe depletion of CD4+ T-lymphcytes resulting in opportunistic infection,neurologic and neoplastic disease and ultimately death. Identification of molecular events critical to virus replication has enabled the solution of several strategies for potential chemotherapeutic intervention.

Etiology of AIDS:-

AIDS is caused by Human Immuno Deficiency virus (HIV).It is a retrovirus.The retrovirus known to contain gag, pol and env genes as basic component of a replicating genome.HIV has as its major structural components a core of genomic RNA,a group specific antigen(gag) proteins which play a role both in the structure of the core and assembly of viron,a lipid bilayer and outside envelope glycoprotein.

Fig:-  Structural Features Of Retrovirus

HIV is retrovirus contains ribonucleoprotein core particle surrounded by an outer protein shell. Overall diameter of this spherical structure is around 110 nm. Immune electon microscopy has shown that club shaped inner core particle of HIV-1 consist of capsid(CA) &nucleocapsid (NC) proteins,which are associated with two copies of stranded HIV genome RNA.Within core molecules reverse trancriptase & integrase are present.This ribonucleoprotiens assembly is surrounded by icosahederal outer protein shell. Finally, this outer protein shell is surrounded by lipid bilayer envelope studded with knob like protrusions consisting of outer surface glycoprotein gp 120 in association with the hyrophobic membrane spanning gp 41.

Infectious Cycle Of HIV:-

Since all the steps in the cycle are required for generation of new infectious virons,intervention can be conceived 3 steps involved in the infectious cycle of virus.

1.Invasion

    a. Attachment

    b. Fusion

2. Maturation

   a. Reverse transcription

   b. Integration

3. Release

   a. Budding

   b. Exit from cell

HIV usually initiates infection of a new cell by attaching to the extracellular domain of the CD4 receptor.A specific complex formed primarily between the so called domain of the Cd4 receptor and gp120 surface glycoprotein mediates the attachment.Fusion of viral and cellular membranes is mediated by the transmembrane glycoprotein gp41.

The second stage in the infection of human cells involves making DNA transcripts of viral transcripts using viral reverse transcriptase.Following reverse transcription of the viral DNA genome into a double stranded DNA,Integrase catalyses integration of the latter into the host chromosome through co-ordinated reaction of processing and joining.

Many of the viral proteins are derived via proteolytic processing by virally coded protease.It appears that much of the proteolytic processing that’s leads to production of infectious particles occurs within particularlly formed and budding immature virions.

The final stage of replication is the elimination of virus particles from the host cells.This step is characterised by assembly,budding and release process.

Possible Target Sites for Anti-Hiv Drugs:-

Examples of Drugs Developed As Inhibitors to Act at Different Target Sites:-

Structure of Reverse Transcriptase Enzyme:-   

Reverse transcriptase of human immunodeficiency Virus type1 [HIV-1 RT] plays a key role in the duplication of the human immunodeficiency virus [HIV] by  transcripting the single  stranded RNA into double stranded DNA. As a consequence  RT is an  important target in  the  treatment of Acquired Immuno Deficiency Syndrome ( AIDS) .

Reverse Transcriptase is a protein dimer consisting of 66-kDa [p66] & 51-kDa [p51]   sub units. The p66 & p51 subunits are composed of subdomains, which are named   thumb, palm, fingers & connection. The p66 subunit also contains the RNAs H subdomain.

Different Types of Reverse Transcriptase Inhibitors:-

Following are the classes of Reverse Transcriptase Inhibitors

Crystal Structures of Reverse Transcriptase with Nucleoside reverse Transcriptase inhibitors complexes:-

Nevirapine:

Nevirapine is the non-nucleoside reverse transcriptase inhibitor. The complex structure of Nevirapine in the allosteric site of Reverse Transcriptase enzyme. The binding site of RT complexed to the NNRTI nevirapine. The amino acid residues composing the non-nucleoside binding site are mainly hydrophobic. They are pro95,Leu100,Lys101, Lys103,Val106,Val108, Val179,Tyr181,Tyr188,Gly190,phe227,Trp229,Leu234,His235,Gly190,phe227,Trp229,Leu234,His235 and pro236 of the p66 palm subdomain and Glu138 of p51 finger subdomain.There are 3 waters at the entrance of the binding pocket forming hydrogen bonds with nevirapine, Lyslol and glu138.

Advantages of NNRTI over NRTI:-

1.NNRTIs are having unique mode of action.

2.NNRTIs lack the toxic effects associated with NRTIS such as oedema ulceration, nailpigmentation, macrolytic anaemia, leucopenia, myopathy and meningoencephalitis. Prolonged use of NRTIS leads to painful peripheral neuropathy, pancreatitis and hepatic failure.

3. NNRTIS bind non-covalently and non-competitively at allosteric site, which is present only in the HIV-1 RT.

4.NNRTIs not suffer from resistance development as that of NRTIs.

Disadvantages of Non-nucleoside Reverse Transcriptase inhibitors:-

1. NNRTIs only restricted to the HIV-1 in activity.

Computer Assisted Drug Design:-

Molecular modeling started about 25 years ago with the presentation and real time rotation of a molecule in front of a computer screen. Within short time, it developed to a highly valuable tool in drug design, especially supporting the medicinal chemist to establish and evaluate working hypothesis on structure activity relationships. A very first computerised approach to generate active molecules de novo was the programmed CAVE AT which replaces a peptide loop by a (rigid) scaffold that is capable to accommodate the relevant amino acid side chains in exactly the same 3D orientation as peptide lead. In this manner a peptidomimetic is created in one step, the conversion of peptidic integrin ligands to benzodiazepines might be considered a successful application of this concept.

Goodfords computer Programme (GRID):-

Inspects the surface of a protein especially its binding site with different chemical probes to search for hot spots where a certain functionality of a ligand should favorably interact. The most impressive application of structure based and computer aided drug design resulted from the application of GRID to the viral enzyme neuroaminidase.

An alternative to GRID is the Programme Isostar which extracts a statistics of non-bonded intermolecular interactions from cambridge crystallographic database. Superstar is an extension of isostar, contour maps are generated from the individual positions of the interacting groups.  Then a first computer programme Dock was developed by Kuntz for geometric docking of ligands into a binding site.

Further progress resulted from the programme LUDI which defined interaction sites and used a scoring function to evaluate the docking results. Programme for a flexible docking of ligands into a rigid binding site are e.g. DOCK 4.0,GOLD, Flexx and public domain programme autodock. Flexx modifications FlexE and Flex-Pharm allow a flexible ligand docking into an ensemble of different binding site conformations and definition of Pharmacophore constraints.

Ligand-Protein Binding Studies Using Docking:-

The docking process involves the prediction of ligand conformation and orientation within a targeted binding site.

Aims of docking:-

1. Accurate structural modeling

2. Correct prediction of activity.

Theoretical aspect of docking:-

For an enzyme and inhibitor docking aims at correct prediction of the structure of the complex (E+I) =(EI) under equilibrium conditions---

the figure illustrates the binding of inhibitor Dmp323 to HIV protease.

When considering the term (EI), the following factors are important:-

1. Steric

2. Electrostatic

3. Hydrogen Bonding

4. Inhibitor Strain@

5. Enzyme Strain

When considering the equilibrium the following factors are important

1. Desolvation

2. Rational entropy

3. Translational entropy

Following are the steps involve in Ligand-Protein binding studies using CADD:-

1. Molecular modeling of Ligand:

1. First step involved in CADD is drawing of the two dimensional (2D) structure of the ligand.
2. Then the conversion of 2D structure to three dimensional (3D) structure of ligand
3. 3D structure of ligand only get bind to the enzyme or receptor

Example:

2D structure of nevirapine          3D structure of nevirapine

2. Energy Minimization:

Energy minimization studies are carried out for ligand. Ligand can exist in different conformations. The conformation, which has lower energy, will be the most stable conformation of the ligand. There are two types of energies.

a) Electrostatic Potential Energy:

It isElectrostatic Potential Energy a pair wise summation of coulombic interactions as described in equilibrium.

In Equation N=the number of atoms in molecules A &B respectively q = charge on each atom.

b) Vanders Waals Potential Energy:

For general treatment of non-bonded interactions is often modeled by a Lennard-Jones 12-6 function as in equilibrium

In equation

S = is the well depth of the potential

s = is the collision diameter of respective atoms i & j

Figure shows a representation of Lennard-Jones 12-6 function

The exp (12) term of the equilibrium is responsible for small distance repulsion.

The exp (6) provides an attractive term which approaches zero as the distance between the two atoms increased. Before energy minimization, water molecules must be removed from receptor or enzyme active site before ligand docking is done.

3. Validation of Docking Software Packages

There are number of software packages available for docking in the market. To know the best software package which predicts the closest match to the crystallographic data on protein-ligand complex, the following comparative evaluation is carried out.

1. Take a known standard ligand e.g. Nevirapine.

2. From X-ray crystallography co –ordinates of binding position of Nevirapine and co-ordinates of active site amino acids is already known experimentally.

3. Take a ligand dock it into the active site of different enzyme software packages carry out translation and rotation of ligand as a whole.Then determine final binding or clocking energy and grade them.

4. Best fit of ligand will be compared with the X-raycrystallographic binding co-ordinates.From these comparision we can know the best software package for docking studies.

4. Different Protocols or Approaches:

There are number of approaches exist for docking as follows:-

a. Monte Carlo Approach:-It generates an initial configuration of a ligand in an active site consisting of random conformation, translation & rotation. It scores initial configuration. Then it generates new configuration & score it. It use Metropolis criterion to determine whether the new configuration is retained. (Metropolis criterion- If new solution scores better than the previous one, it is immediately accepted. If the configuration is not new one, a boltzman-based probability function is applied. If the solution passes the probability function test, it is accepted; if not the configuration rejected).

b. Fragment based method:-Fragment based methods can be described as dividing the ligand into separate protons or fragments, docking the fragments & finally linking these fragments together.

c. Distance Geometry:-Many types of structural information can be expressed as intra or intermolecular distances. The distance geometry formalism allows these distance to be assembled & 3-dimentional structures consistent with them to be calculated.

d. Matching approach:-These approach focus on complimentarity. Ligand atom is placed at the ‘best’ position in the site, generating a ligand receptor configuration that may require optimization.

e. Ligand fit approach:- Ligand fit term provide a rapid accurate protocol for docking small molecules ligand into protein active sites for considering shape complimentarity between ligand & protein active sites.

f. Point Complimentarity approach:- These methods are based on evaluating a shape &/or chemical complimentarity between interacting molecules.

g. Blind Docking:-It was introduced for detection of possible binding sites & modes of peptide ligand by scanning the entire surface of protein targets.

h. Inverse Docking:-In this use of a computer method for finding toxicity & side effect protein targets of a small molecule. Knowledge of these targets combined with that of proteomics pharmacokinetic profile can facilitates the assessment of potential toxicities side effect of drug candidate. One of these protocols is selected for docking studies of particular ligand.

5. Binding or Docking Energy Calculation:

After formation of the complex of ligand with protein there will be calculation of binding energy or docking energy. If there is no. of proposed compounds then binding energy will be different for all of them. The grading of these energies is done. After grading we will come to know best-fit ligand complex.

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About Authors:

Amol A. Kulkarni (M.Pharm. Pharmaceutical Chemistry)
Pharmaceutical Chemistry Department, Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences & Research, Pimri, Pune.

Suraj S. Bhosale (M.Pharm.Sem IV. Pharmaceutical Chemistry)
Pharmaceutical Chemistry Department,Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences & Research, Pimri, Pune.

Sheetal B. Nikam (M.Pharm.Sem IV. Pharmaceutical Chemistry)
Pharmaceutical Chemistry Department,  Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences & Research, Pimri, Pune.

Sohan S. Chitlange (M.Pharm. Pharmaceutical Chemistry)
Pharmaceutical Chemistry Department, Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences & Research, Pimri, Pune.

R.K.Nanda

Pharmaceutical Chemistry Department, Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences & Research, Pimri, Pune.