HIV-1 PROTEASEMike Krzyskowski

Intro, Background• HIV-1 – Human Immunodeficiency Virus 1

• Highly virulent, highly contagious• Left untreated progresses to AIDS

• About 1.6 million deaths worldwide in 2012 (WHO)

• Integral protein in HIV-1 lifecycle

• “chops” polyprotein into

appropriate size

Image: HIV-1 Protease (pdb 3FX5)

Sequence Alignment

• HIV-1 protease, colored by chain

Structure

• Red: Flap region• Orange: Active site triad (Asp-Thr-Gly)• Purple: Helices• Black: AA 93, mutation I93L implicated in drug resistance

Structure: Active Site

KNI-272 Inhibitor

Asp25 (protonated)

Mechanism• Hydrolysis

• H2O molecule held in active site (purple)

Asp25Asp125

KNI-272

Mechanism• Only 1 Asp is protonated

• Asp farther from H2O

1. Negative Asp activates H2O

2. Attacks Carbonyl• Generation of oxyanion tetrahedral intermediate

3. Protonation of scissile N atom• Breakdown to hydrolysis products

Drug Resistance: Not so simple• ANAM-11

• “Wild type”

• A-1• Active site

mutation

• NAM-10• Non-active site

mutations

Image source: Muzzamil et, al. (2003)

Drug Resistance

• Substrates: synthetic proteins that mimic natural cleavage sites

References• Adachi, M.,  Ohhara, T.,  Kurihara, K.,  Tamada, T.,  Honjo, E.,  Okazaki, N.,  Arai, S.,  Shoyama, Y.,  Kimura, K.,  Matsumura, H.,  Sugiyama, S.,  Adachi,

H.,  Takano, K.,  Mori, Y.,  Hidaka, K.,  Kimura, T.,  Hayashi, Y.,  Kiso, Y.,  Kuroki, R. (2009) Structure of HIV-1 Protease in Complex with Potent Inhibitor KNI-272 Determined by High Resolution X-ray Crystallography Proc.Natl.Acad.Sci.USA

• Brik, A and Wong, C. (2003) HIV-1 Protease: Mechanism and Drug Discovery. Org. Bio.Chem. 1: 5-14

• Hidaka, K.,  Kimura, T.,  Abdel-Rahman, H.M.,  Nguyen, J.T.,  McDaniel, K.F.,  Kohlbrenner, W.E.,  Molla, A.,  Adachi, M.,  Tamada, T.,  Kuroki, R.,  Katsuki, N.,  Tanaka, Y.,  Matsumoto, H.,  Wang, J.,  Hayashi, Y.,  Kempf, D.J.,  Kiso, Y. (2009) Crystal Structure of HIV-1 Protease Complexed with KNI-1689. J.Med.Chem. 52: 7604-7617

• Jaskólski, M, Tomasselli, G Sawyer, T K Staples, D G Heinrikson, R L Schneider, J Kent, S B, Wlodawer, A Structure at 2.5-A resolution of chemically synthesized human immunodeficiency virus type 1 protease complexed with a hydroxyethylene-based inhibitor. (1991) 30: 1600-9 

• Kohl, N E Emini, E a Schleif, W a Davis, L J Heimbach, J C Dixon, R a Scolnick, E M Sigal, I S Active human immunodeficiency virus protease is required for viral infectivity. (1988) Proc. Nat. Acad. Sci. 85: 4686-90

• Kovalevsky, A.Y.,  Liu, F.,  Leshchenko, S.,  Ghosh, A.K.,  Louis, J.M.,  Harrison, R.W.,  Weber, I.T. (2006) Ultra-high resolution X-ray crystal structure of HIV-1 protease V32I mutant with TMC114 (darunavir) inhibitor J.Mol.Biol. 363: 161-173

• Muzammil, S Ross, P and Freire, E. A Major Role for a Set of Non-Active Site Mutations in the Development of HIV-1 Protease Drug Resistance (2003) Biochemistry 42:631-638

• Servais, J., Lambert, C., Fontaine, E., Plesse, J., Robert, I., Arendt, V. I. C., … Schmit, J. (2001). Variant human immunodeficiency virus type 1 proteases and response to combination therapy including a protease inhibitor. Antimicrobial Agents …, 45(3), 893–900.

• Strisovsky K, Tessmer U, Langner J, Konvalinka J, Kräusslich HG.. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis. (2000) Pro. Sci 9(9):1631-41.

• K. Suguna, Richard R. Bott†, Eduardo A. Padlan, E. Subramanian‡,, Steven Sheriff,, Gerson H. Cohen, Davies David (1987) Structure and refinement at 1.8 Å resolution of the aspartic proteinase from Rhizopus chinensis J. Mol. Bio. 196(4):877-900

• http://www.who.int/gho/hiv/en/