ASPARTIC PROTEASE INHIBITORS:

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• ASPARTIC PROTEASE INHIBITORS
• IMPLICATIONS IN AIDS THERAPY
• Chandravanu Dash Mala Rao

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• INTRODUCTION
• RESEARCH ON ANTIVIRAL AGENTS HAS INTENSIFIED
  GREATLY IN RESPONSE TO THE NEED TO TREAT
  INFECTIONS OF HUMAN IMMUNODEFICIENCY VIRUS (HIV),
  THE CAUSATIVE AGNET OD AIDS (ACQUIRED
  IMMUNODEFICIENCY SYNDROME)

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• RECENT TREATMENTS ARE BASED ON COMBINATION
  THERAPIES
• HIGHLY ACTIVE ANTIRETROVIRAL THERAPY (HAART)
  REVERSE TRANSCRIPTASE INHIBITORS ARE ASSOCIATED
  WITH ANTIPROTEASE INHIBITORS.
• SHOWN TO MANIFEST SIGNIFICANT IMPROVEMENTS IN THE
  DISEASE STATE

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• ROLE OF PROTEASE IN HIV REPLICATION
• PROTEASES PLAY AN ESSENTIAL ROLE IN THE
  REPLICATION CYCLE OF HIV
• FOR PROCESSING OF THE GAG-POL PRECURSOR PROTEINS
  
• MATURATION OF INFECTIONS VIRONS

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• COMPARISION OF ASPARTIC HIV PROTEASE
• HIV PROTEASE IS STRUCTURALLY AND MECHANISTICALLY
  RELATED TO MAMMALIAN AND MICROBIAL ASPARTIC
  PROTEASES SUCH AS PEPSIN, CATHEPSIN, RENIN AND
  ENDOTHIOPEPSIN
• CLASSIFICATION OF HIV-1 PROTEASE IN THE ASPARTYL
  FAMILY WAS ALSO PREDICTED FROM ITS PRIMARY
  SEQUENCE.

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• A SEQUENCE ASP-THR-GLY (DTG) WHICH IS HIGHLY
  CONSERVED IN RETROVIRAL PROTEASES IS ALSO
  CONSERVED IN THE ACTIVE SITE OF CELLULAR AND
  FUNGAL ASPARTIC PROTEASES
• MOLECULAR MODELLING STUDIES FURTHER PROPOSED THAT
  THE RETROVIRAL PROTEASES RESEMBLE OTHER ASPARTYL
  PROTEASES FUNCTIONALLY AND STRUCTURALLY

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• INHIBITION OF RETROVIRAL PROTEOLYTIC ACTIVITY IN
  THE PRESENCE OF GENERAL ASPARTYL PROTEINASE
  INHIBITOR PEPSTATIN-A
• PROTEASE INHIBITORS GAINED IMPORTANCE DUE TO
  THEIR ATTRACTIVE ANTIVIRAL PROPERTIES
• HIV-1 PROTEASE INHIBITORS REPRESENT MAJOR
  ADVANTAGES OVER COMPOUNDS SUCH AS AZIDOTHYMIDINE
  (AZT)
• High degree of specificity for the target
• Lack of cellular toxicity
• Metabolic stability
• Good availability

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• SPECIFIC INHIBITORS OF HIV-1 ASPARTIV PROTEASE
  MIGHT BE INTERESTING CHEMICAL LEADS TO DEVELOP
  EFFECTIVE THERAPEUCTIC AGENTS FOR THE TREATMENT
  OF AIDS

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• HIGHLIGHTS OF THE WORK
• Identification of bioactive molecules from
  diverse microbial flora
• Identification of a potent HIV-1 protease
  inhibitor from the extracellular culture filtrate
  of extremophilic Bacillus sp.

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• INHIBITORS OF MICROBIAL ORIGIN
• Novel, and complex molecule
• Economical source for the rapid production
• Cost effective

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PURIFICATION OF ATBI
Extracellular Culture Filtrate
Charcoal Treatment
Membrane Filtration
Lyophilization
Reverse Phase-HPLC
Production of ATBI 250 ?g/litre
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BIOCHEMICAL PROPERTIES OF ATBI
Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu.
Mr 1147 Da Electrospray mass spectrometry
Inhibits HIV-1 protease non-competitively.
IC50 value 18 nM
Ki value 17.8 nM.
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FDA APPROVED PROTEASE INHIBITORS
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• PROPOSED MECHANISM FOR HIV PROTEASE INHIBITION
• Active site cleft of HIV-1 protease possess two
  identical conformational mobile loops known as
  Flaps, which are important for the substrate
  binding and catalysis
• Fluoremetric analysis of HIV-1 Protease-ATBI
  complex.
• Localized conformational chagnes induced in the
  HIV-1 protease due to the interaction of ATBI was
  monitored by Trp fluorescence.
• ATBI binds to the active site of the HIV-1
  protease which results in the loss of flexibility
  of the flaps.

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• FUTURE PROSPECTS.
• The action of ATBI on HIV infection in cell
  cultures.
• ATBI as the lead molecule for designing potent
  inhibitors.
• X-ray crystallographic studies to decipher the
  mechanism of action of the inhibition of HIV-1
  protease.
• J. Biol. Chem. 2001. Dash, C. and Rao, M.
• NewsIndia, Nature Supplemnet
• Indian patent granted. 1998
• US patent filed, 1999.