TY - JOUR

T1 - Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis

AU - Zakharova, Maria Yu

AU - Kuznetsova, Alexandra A.

AU - Kaliberda, Elena N.

AU - Dronina, Maria A.

AU - Kolesnikov, Alexander V.

AU - Kozyr, Arina V.

AU - Smirnov, Ivan V.

AU - Rumsh, Lev D.

AU - Fedorova, Olga S.

AU - Knorre, Dmitry G.

AU - Gabibov, Alexander G.

AU - Kuznetsov, Nikita A.

N1 - Publisher Copyright: © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)

PY - 2017/11/1

Y1 - 2017/11/1

N2 - Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage “minimal” kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of “elementary” kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site.

AB - Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage “minimal” kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of “elementary” kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site.

KW - Active site evolution

KW - Enzyme-substrate interaction

KW - FRET

KW - HIV-1 protease

KW - Mechanism of protease action

KW - Multidrug-resistant mutant

KW - Pre-steady state kinetics

KW - Stopped-flow analysis

KW - RECOGNITION

KW - MECHANISM

KW - DRUG-RESISTANCE

KW - CONFORMATION

KW - SITES

KW - MUTATIONS

KW - BINDING

KW - REVEALS

UR - http://www.scopus.com/inward/record.url?scp=85028721975&partnerID=8YFLogxK

U2 - 10.1016/j.biochi.2017.08.014

DO - 10.1016/j.biochi.2017.08.014

M3 - Article

AN - SCOPUS:85028721975

VL - 142

SP - 125

EP - 134

JO - Biochimie

JF - Biochimie

SN - 0300-9084

ER -