This presentation gives us an information regarding the protease enzyme and its development ,development of agents using molecular modelling techniques

2. CONTENTS
• INTRODUCTION ABOUT PROTEASE ENZYME
• CLASSIFICATION OF PROTEASE ENZYME
• M.O.A OF PROTEASE ENZYME
• CLASSIFICATION OF PI
• MOLECULAR MODELLING
• HIV PROTEASE ENZYME STRUCTURE & FUNCTION
• NEWER PI INHIBITORS
• APPLICATIONS
• REFERENCE

3. PROTEASE
•A protease is an enzyme that catalyzes proteolysis, the breakdown of proteins into
smaller polypeptides or single amino acids by cleaving the peptide bonds within
proteins by hydrolysis of water molecule.

4. CLASSIFICATION OF PROTEASE
On the basis of pH –
Acidic protease, Neutral protease and Alkaline protease.
On the basis of Peptide bond specificity :
Endo- peptidases & Exo- peptidases.
On the basis of Functional group at active site –
Serine protease, Cysteine protease, Aspartic protease and Metallo-proteases.

6. MODE OF ACTION OF PROTEASE ENZYME

7. CLASSIFICATION OF
PROTEASE INHIBITOR

10. a) SAQUINAVIR (FIRST PROTEASE INHIBITOR)
•Saquinavir is the first FDA-approved HIV-1 PI in 1995 .
•First-generation PIs were designed based on Noncleavable hydroxyethylene or
hydroxyethylamine-based isosteres.
•These PIs typically possessed multiple amide/peptide-like bonds and have been
initially developed to extensively interact with all subsites of the protease

11. SQV COMPLEX WITH ENZYME

12. b) RITONAVIR
•It is an peptidomimetic HIV protease inhibitor, was marketed in 1996.
•It was designed to fit the C2-symmetry in the binding site of the protease.
•It is a strong inhibitor of the cytochrome p450 enzyme mediated metabolism and it is only
used in a combination therapy with other protease inhibitors .

13. c) NELFINAVIR
•It is the first protease inhibitor that was not Peptidomimetic.
• It contains a novel 2-methyl-3-hydroxybenzamide group, whereas its carboxyl
terminal contains the same DIQ (DECAHYDROISOQUINOLINE )group as
Saquinavir.
•Nelfinavir was marketed in 1997 and was the first protease inhibitor to be
indicated for pediatric AIDS.

15. MOLECULAR MODELLING
“ Molecular Modeling “ is to visualize three-dimensional
structures and to simulate , predict and analyze the
properties and behavior of the molecules and to organize
many compounds and their properties into database and
to perform virtual drug screening via 3D database
screening for novel drug compounds.

16. HIV PROTEASE STRUCTURE AND FUNCTION:

17.  The HIV-1 protease (HIV-1 PR) is a virus-specific aspartic protease responsible for
processing the polyproteins of gag and gag-pol during virion maturation and for the
proliferation of the retrovirus.
 It is an important target for the development of anti-AIDS drugs.
 The HIV-1 PR can recognize Phe-Pro or Tyr-Pro sequence as the retrovirus-specific
cleavage site.
 The HIV-1 PR is a homodimer with C2 symmetry.
 The Homodimeric protein consists of two identical 99 amino acid subunits, each of
which has one catalytic aspartic acid Asp25, 25′.
 Amino acid sequences of mature HIV-1-Protease
PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFI
KVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF

18.  The C2-symmetric active site is located at the dimer interface and each subunit
contributes one catalytic aspartic acid in the tripeptide sequence, Asp25, 25′ -Thr26, 26′ -
Gly27, 27′.
 The protease subunit fold contains
“An compact structure of β-strands and a short α-helix near the C-terminal”
 The antiparallel β-strands, form a flexible “flap” region that is thought to fold down over
the active site during catalysis ( in order to bind with substrate and exclude water).
 The flap region regulates substrate entry into the active site by its conformation, which
can be open, semiopen or closed.
 The intermolecular interactions stabilize the two subunits.
 The four-stranded antiparallel β-sheets form the largest part of the interface which consists
of residues 1–5 in N-terminal and residues 95–99 in C-terminal of the protease monomers.

19. Red spheres represent amino acid positions and are indicated only on one
monomer for clarity

20.  The active site contains eight C2-symmetric subsites (S4, S3, S2, S1, S1′, S2′,
S3′, and S4′), which have the binding sites for the P4, P3, P2, P1, P1′, P2′, P3′,
and P4′ residues of an octapeptide substrate numbered from the scissile bond.
 The scissile bond is defined as the bond at which the hydrolysis cleavage
occurs

21. “INDINAVIR”
FIRST DEVELOPED PI-INHIBITOR
USING MOLECULAR MODELLING

22.  The development of Indinavir (MK-639, L-735,524, Crixivan) by Merck was based on a
transition-state mimetic concept , previously utilized in design of renin inhibitors .
 A series of peptidomimetic inhibitors of different lengths, containing a hydroxyethylene
isostere in an S-configuration, was examined, with a number of substitutions of the side
chains.
 Inclusion of (aminomethyl) benzimidazole provided the most potent compounds in that series,
as the imidazole portion appeared to be mimicking a carboxamide, while the phenyl portion
was probably contributing additional hydrophobic binding.

23.  Some of the inhibitors with excellent IC50 ( INHIBITORY CONSTANT)values were
considerably less potent in cell culture, presumably because of their inability to
penetrate the hydrophobic cell membrane.
 The terminal amide increased in size or polarity, the intrinsic potency improved but not
the minimum inhibitory concentration.
 The design of Indinavir was guided by molecular modelling and X-ray crystal
structure of the inhibited enzyme complex
 Indinavir (Crixivan) was orally bioavailable in three animal models and gained FDA
approval at the beginning of 1996

24.  (A) Indinavir and (B) novel hit docking profiles calculated by AutoDock software.
 Both ligands are gray. HIV protease chain A and B
 Amino acids are painted in green and magenta respectively.
 The hydrogen bonds are shown for each of both molecules.

25. COMPARISON OF BINDING
ENERGIES WITH SQV & IDV

26. NEW PI INHIBITORS YET TO BE APPROVED
Three new HIV protease inhibitors are receiving attention
because of recent clinical studies
i) Atazanavir (ATV, reyataz®)
ii) Fosamprenavir (APV, lexiva) are already approved by the US
food and drug administration
iii) Tipranavir is now in phase III clinical trials

27. APPLICATIONS OF PROTEASE ENZYME:
•Industries uses proteases include detergents, leather processing, silver recovery,
medical purposes, food processing, feeds, chemical and waste treatment.
•For industrial application of proteases different temperature, salt concentration,
optimal pH, type of media and incubation period .
•Proteases contribute for use in products that require the enzyme-aided or digestion of
proteins from different sources.
•Proteases that are consumed in the pharmaceutical industry differ from those used in food
and detergent industries.

28. • Adamson, Catherine S. (2012). “Protease-Mediated Maturation of HIV: Inhibitors of Protease and the
MaturationProcess.” Molecular Biology International 2012: 1-13.
•http://scholar.google.com/scholar_url?url=http://downloads.hindawi.com/journals/mbi/2012/604261.pdf&h
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• Ali, Akbar et al. (2010).
• “Molecular Basis for Drug Resistance in HIV-1 Protease.” Viruses 2: 2509-2525.
• http://scholar.google.com/scholar_url?url=http://www.mdpi.com/19994915/2/11/2509/pdf&hl=en&sa=X
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• Cruciani, Mario. (2014). “Virological efficacy of abacavir: systematic review and meta-analysis.” Journal
ofAntimicrobial Chemotherapy 69: 3169-3180.
• Davies, David R. (1990). “The Structure and Function of the Aspartic Proteases.” Annual Review of
Biophysics and Biophysical Chemistry 19: 189-215.
• Eron Jr., Joseph J. (2000). “HIV-1 Protease Inhibitors.” Infectious Diseases Society of America 30: S160-
170.
REFERENCE