3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

IN VITRO SELECTION AND CHARACTERIZATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) VARIANTS WITH DECREASED SENSITIVITY TO HYDROXYETHYLUREA ISOSTERE CONTAINING PROTEASE INHIBITORS

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:4 (abstract no. 3)

Karen E. Potts¹, Mary L. Smidt¹, William C. Stallings Jr.¹, Michael Clare², Deenan Pillay³, Douglas D. Richman³ and Martin L. Bryant^1
1G.D. Searle, St. Louis, Missouri, USA; ²G.D. Searle, Skokie, Illinois, USA; ³University of California San Diego, La Jolla, California, USA.

Protease inhibitors SC-52151 and SC-55389 (G.D. Searle), which contain a unique hydroxyethylurea isostere, were used to select drug resistant HIV-1 variants in vitro. Clinical and laboratory HIV-1 strains were passaged in T cell lines or peripheral blood mononuclear cells (PBMCs) in the presence of increasing drug concentrations. Resistant variants consistently exhibited IC₅₀ values at least 10-fold higher than control virus passaged for an identical period, but in the absence of inhibitor. Viral DNA was amplified by PCR and the nucleotide sequence of the protease gene was analyzed from multiple subclones. In one laboratory HIV strain with reduced sensitivity to SC-52151, both amino acid changes at positions 48 and 82 were observed relative to the untreated controls. In a second laboratory HIV strain and a clinical strain grown in the presence of SC-55389A and SC-52151, respectively, only one amino acid change at position 88 was consistently observed in all the variants selected. The Asn residue at 88 lies within a structurally conserved helical domain, present in both monomeric and dimeric aspartic proteinases. The corresponding carboxy terminal sequence Gly-Arg-Asp/Asn (residues 86-88) is unique to retroviral aspartic proteinases. Modelling studies based on templates derived from high resolution x-ray structures of prototypical hydroxyethylurea inhibitors bound to recombinant HIV-1 PR suggest that the Asn88 mutations may alter the conformation of the protease at this site. Subtle perturbations of essential H-bon interactions among Asn88, Thr 31, Thr 74 and Asp29 could in turn influence the S2/S2´ inhibitor binding subsites. Other amino acid changes (positions 10, 46, 53 and 71) were found in some, but not all, selected resistant variants. The significance of the observed changes and their ability to confer cross resistance to other asymmetric protease inhibitors is currently under investigation.

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1994-08-02
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