title: A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease.
abstract: Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.
authors: Department of Molecular and Experimental Medicine, The Scripps Research Institute, CA 92037, USA.
authors: Sebastian
authors: S
authors: Breuer
authors: Homero
authors: H
authors: Sepulveda
authors: Yu
authors: Y
authors: Chen
authors: Joseph
authors: J
authors: Trotter
authors: Bruce E
authors: BE
authors: Torbett
mesh_terms: D001665:Binding Sites; D024882:Drug Resistance, Viral / Q000235:genetics*; D057075:Enzyme Assays / Q000295:instrumentation*; D005434:Flow Cytometry / Q000295:instrumentation*; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008863:Microspheres; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D017320:HIV Protease Inhibitors; D011993:Recombinant Fusion Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease