CASE STUDY

IN SILICO PREDICTION OF
RESISTANCE TO DRUGS
NEED FOR IN SILICO METHOD

 Resistance is the major problem in case of

diseases like AIDS, TB

 Lot of time is needed to study resistance and

then discover drug accordingly

 In silico method can shorten this time

HIV-1 PROTEASE ENZYME

 Structure:
 Is a homodimeric protein consisting 2 identical
polypeptides of 99 amino acids
 Catalytic active site is a triad D25(25’)-T26(26’)-
G27(27’) which is located in a hydrophobic loop
(cavity) stabilized by hydrophobic interactions and
hydrogen bonding
 Active site is covered by a flap which participates
in binding of inhibitors and substrates
 Structure of
HIV-1 Protease

Binding to
enzyme
HIV-1 PROTEASE ENZYME(conti.)

 Function:

 Necessary for processing precursor polypeptide

into a functional protein essential for viral
assembly and maturation for infectious virus

 Hence one of the major targets for treatment of

AIDS
DRUGS STUDIED

 FDA approved protease inhibitors include:

indinavir, ritonavir, saquinavir, nelfinavir,
saquinavir, amprenavir, lopinavir, atazanavir,
tipranavir, fosamprenavir and darunavir
 Case study -1: Ritonavir
 Case study -2: Lopinavir, Amprenavir,
Saquinavir, Atazanavir.
BINDING OF INHIBITORS

 Binding of inhibitor introduces substantial

conformational changes to the enzyme

 Flap rotation occurs to reveal the active site

residue

 Most of the inhibitors bind in extended

conformation to the active site residue
HOW RESISTANCE DEVELOPS

 Due to mutation in enzyme structure

 mostly develops when antiretroviral therapy

fails to be suppressive

 Since virus replicates under pressure of drug

mutations are mostly compensatory- binding
affinity to drug is minimal but replication
capacity is retained
CASE STUDIES

 Molecular Dynamic and Free Energy Studies

of Primary Resistance Mutations in HIV-1
Protease-Ritonavir Complexes
 Molecular Dynamics and Free Energy Studies
on the Wild-Type and Mutated HIV-1 Protease
Complexed with Four Approved Drugs:
Mechanism of Binding and Drug Resistance
CASE STUDY-1

 Molecular Dynamic and Free Energy Studies

of Primary Resistance Mutations in HIV-1
Protease-Ritonavir Complexes
Selection of mutants
 Experimental thermodynamic studies of the
binding of the drug to the wild-type and to
the V82F/I84V HIV-1 protease indicated that
the V82F/I84V mutant shows a decrease of
700-fold of the binding affinity to ritonavir.
 Also the V82F/I84V mutant reduced dimer
stability relative to the autolysis-resistant
mutant at pH 7.0
 In these mutations affinity was decreased but
activity was retained
Method

preparation of starting structure

molecular dynamic simulations

calculation of inding free energy

Method (conti.)

1) Preparation of starting structures

Wild type HIV-1 PR-RTV complex form PDB

LeAP of amber 7 to add all missing atoms and

H of enzyme

comparative models of V82F, I84V, V84F/I84V

were constructed using wt. model as
template
Method (conti.)

University of Houston Brownian Dynamics

Program – ionization states of all ionisable
residues was assigned

partial geometric optimization (Hartree-Fock

level- Gaussian 98 program)

Each complex immersed in 10Å radius of

TIP3P water model
Method (conti.)

2) MD simulations:
Energy minimization and molecular dynamic
simulation (SANDER module of AMBER 7.0
with cornel force field) (NPT ensemble→
SHAKE algorithm→RMSD)

MD trajectories every 0.1 ps

Method (conti.)

3) Calculation of binding free energy (∆Gbind)

∆Gbind = Gcomplex - (Greceptor + Gligand)
Gbind = EMM - TS + Gsolv
EMM = Einternal + Eelec + EvdW
Gsolv = Gelec,solv + Gnonpolar,solv
Result and discussion

 Structural features of HIV-1 protease:

 Structure of flap is changed and hence no
interaction with inhibitor
 No change in structure of catalytic triad
 Conformational Flexibility of Ritonavir:
 Ritonavir-HIV-1 Protease Binding Energy:
 A net loss of ∆Gbind in the mutant complexes with
respect to the wt. corresponds to a decrease in
scathe inhibition potency
Hydrogen bonding (d1-d6) between HIV-1 PR and ritonavir and the percent
occurrence of the corresponding hydrogen bonds calculated from the MD
trajectories
CASE STUDY-2

 Molecular Dynamics and Free Energy Studies

on the Wild-Type and Mutated HIV-1 Protease
Complexed with Four Approved Drugs:
Mechanism of Binding and Drug Resistance
Mutations studied

 M46I, V82A, I84V, and L76V

 M46I, V82A, and I84V mutations – different

levels of resistance to Pis

 L76V mutation is associated with resistance

to lopinavir and darunavir
Method

 Required structures of drug and wt HIV-1 PR

were taken from PDB
 Mutant structure was generated from wt
 Same procedure was applied as in case
study-1
 Z-score data were obtained experimentally
and were compared with data obtained with
in silico method
Result and discussion

e
Result and discussion(conti.)
Result and discussion(conti.)

 On comparison of table-1 and table-2 it is

evident that method adopted here has
correlated well with experimental data.
 Where there is decrease in binding due to
mutation, binding free energies are shown
unfavourable
 Where there is increase in binding, binding
free energies are shown favourable
CONCLUSION

 Useful in highly active anti-retroviral therapy

(HAART) for AIDS
 Elucidation of molecular recognition of the
currently approved PIs in the presence of
known mutations responsible to confer
resistance is critical for the development of
superior inhibitors
REFERENCES
 J. Chem. Inf. Model. 2009, 49, 1751–1761 Molecular
Dynamics and Free Energy Studies on the Wild-Type
and Mutated HIV-1 Protease Complexed with Four
Approved Drugs: Mechanism of Binding and Drug
Resistance
 J. Chem. Inf. Model. 2006, 46, 2085-2092 Molecular
Dynamic and Free Energy Studies of Primary
Resistance Mutations in HIV-1 Protease-Ritonavir
Complexes
 Google-wikipedia
SHIKHA PANWAR
M PHARM

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