CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

539 Maturation Inhibitor Mechanistic Studies - Differential Inhibition of Gag Polymorphs Zeyu Lin 1 ; Joseph Cantone 1 ;Tricia Protack 1 ; Dieter Drexler 1 ; Beata Nowicka-Sans 1 ;YuanTian 3 ; Zheng Liu 1 ; Mark Krystal 1 ; Alicia Regueiro-Ren 1 ; Ira B. Dicker 1 1 Bristol-Myers Squibb Co, Wallingford, CT, US; 2 Bristol-Myers Squibb Co, Wallingford, CT, US; 3 Bristol-Myers Squibb Co, Princeton, NJ, US; 4 Bristol-Myers Squibb Co, Wallingford, CT, US Background: HIV-1 maturation inhibitors (MIs) are a class of inhibitors that may be effective in the treatment of HIV-1. MIs disrupt the final step in the HIV-1 protease-mediated cleavage of the HIV-1 Gag polyprotein, between capsid (CA) and spacer peptide 1 (SP1), which is responsible for a major conformational rearrangement of viral proteins within the virion, leading to the production of infectious virus. An MI, bevirimat, previously terminated from development as a result of inadequate coverage of polymorphic Gag variants present in the general HIV-1 population, was used as a model compound. Understanding the mechanism of action of MIs in greater detail may be of value to help guide the development of MIs with improved genotypic coverage. Methods: In this study, we developed a novel LC/MS assay using assembled Gag virus-like particles (VLPs) to quantitatively characterize the kinetics of CA/SP1 cleavage of a family of Gag variant genotypes. These variant VLPs contained site-directed mutations that alter susceptibility of HIV-1 to bevirimat. This method was also used to study inhibition by bevirimat and a second MI in this system. Secondarily, we used a radioligand binding assay to measure the kinetics of dissociation of bevirimat and this second MI to the same Gag variant VLPs. Results: The LC/MS cleavage assay allows for the simultaneous quantization of both cleavage steps leading to the production of free SP1 (SP1/nucleocapsid and CA/SP1) while monitoring the effects of MIs on these processes. The innate rates of cleavage at CA/SP1 roughly correlate inversely with the ability of bevirimat to inhibit these HIV-1 polymorphic viruses in antiviral assays; genotypes with more rapid CA/SP1 cleavage profiles are less sensitive to bevirimat. The MI kinetic dissociation data indicate that improvements to polymorphic antiviral activity arise from increases in MI dissociation half-lives toward those polymorphs. Conclusions: Together, the innate polymorph cleavage rates at CA/SP1 and the MI-specific kinetic dissociation data suggest a model for MI inhibition of HIV-1 protease mediated CA/SP1 cleavage that can be used to quantify MI antiviral behavior as a function of both MI and Gag polymorphs. 540 Late-Stage Integrase-LEDGF Inhibitors Mode of Action and Acquisition of Resistance Richard Benarous 1 ; Erwann Le Rouzic 1 ; Nikki van Bel 2 ;YmeVan derVelden 2 ; Damien Bonnard 1 ; Atze Das 2 ; Celine Amadori 1 ; Alessia Zamborlini 4 ; Stephane Emiliani 3 ; Ben Berkhout 2 1 BiodimMutabilis, Romainville, France; 2 Academic Medical centre, Amsterdam, Netherlands; 3 Inserm CNRS, Paris, France; 4 U944 UMR7212, Inserm CNRS, Paris, France Background: We and others described a new class of HIV-1 integrase (IN) allosteric inhibitors that bind to the LEDGF binding pocket of IN and inhibit IN-LEDGF interaction. Mutabilis developed very active compounds of this class. Designed to interfere with IN-LEDGF interaction during integration, the major impact of these inhibitors was found on virus maturation, causing a reverse transcription defect in target cells independent of LEDGF and linked to compound-induced IN oligomerization. We wanted to determine whether viral RNA packaging was affected and study IN mutants resistant to these inhibitors and interaction of IN with other cofactors such as Transportin (TNPO3) and VHL- binding protein 1 (VBP1). Methods: Virus RNA was isolated, submitted to northern blot and t-RNA primer extension, interaction of IN with cofactors, or IN oligomerization, were studied by HTRF. Resistance mutations were identified by serial passages with increasing concentration of inhibitors during NL4-3 infection of MT4 cells. Results: Wild-type level of HIV-1 genomic RNA was packaged in defective virions in dimeric state, tRNAlys3 primer for reverse transcription was properly placed and could be extended. Reverse transcriptase from defective virions was fully active. Several mutations in IN resulted in variable resistance to this class of inhibitors. The most detrimental mutation was T174I with fold change (FC) in EC50 antiretroviral activity over 200. Other mutations were selected, A128T and N222K were the most frequent ones, with much lower FC values around 4-5. These mutants were impaired for interaction with LEDGF to various extents but conserved wt strand transfer activity and IN-IN subunit interaction.. Ternary complexes could be formed between IN LEDGF and TNPO3, and between IN VBP1 and TNPO3. IN was the link between LEDGF and TNPO3. In the IN-VBP1-TNPO3 complex, each protein could interact with its two other partners. Conclusions: Inhibition of reverse transcription promoted by IN-LEDGF inhibitors in target cells likely reflects the mislocalization of the components in the aberrant virus particle. Most of the resistance mutations were in the LEDGF-binding pocket, affecting the affinity of the inhibitors for IN. N222K, in the C-ter domain of IN is a notable exception. The genetic barrier to resistance to this first generation of IN-LEDGF allosteric inhibitors is similar to that observed for Nevirapine. We are developing a second generation of inhibitors with improved resistance profile. 541 BMS-986001: A Promising Candidate for HIV-2 Treatment Robert A. Smith 1 ; Dana Raugi 1 ; Kate Parker 1 ; Mariah Oakes 1 ; Papa Salif Sow 2 ; Selly Ba 2 ; Moussa Seydi 2 ; Geoffrey S. Gottlieb 1 Background: New antiretrovirals (ARV) are urgently needed for the treatment of HIV-2 infection. BMS-986001 is an investigational NRTI that potently suppresses HIV-1 replication in culture and maintains full or partial activity against NRTI-resistant HIV-1 mutants. To examine the potential utility of the drug in HIV-2–infected individuals, we compared the susceptibilities of wild-type HIV-1 and HIV-2 isolates to BMS-986001 in culture and assessed its activity against HIV-2 variants containing clinically-relevant drug resistance mutations in RT. Methods: Wild-type HIV-1 and HIV-2 strains were obtained from the NIH AIDS Reagent Program. Site-directed mutants of HIV-2 and a full-length recombinant clone containing the RT sequence from an ARV-experienced HIV-2 patient (encoding K65R, N69S, V111I, Q151M and M184V) were constructed in the HIV-2 pROD9 background. HeLa-CD4–based indicator cells (MAGIC-5A) were used to quantify drug sensitivity in a single cycle of viral replication. Results: BMS-986001 inhibited HIV-1 isolates NL4-3 (group M/subtype B), LAI (M/B), 92UG029 (M/A) and MVP5180-91 (group O) with EC 50 values ranging from 0.47 to 0.91 m M (mean ± SD = 0.62 ± 0.21 m M). Similar EC 50 values were observed for the parental compound stavudine (d4T) against NL4-3 and ROD9 (1.32 ± 0.33 and 1.29 ± 0.47 m M, respectively). In contrast, all seven of the group A HIV-2 isolates tested in our assays were more sensitive to BMS-986001, with EC 50 s ~10-fold lower than that of HIV-1 (mean = 0.065 ± 0.091 m M, range = 0.030 to 0.081 m M; p = 0.004, Mann-Whitney test). Increased BMS-986001 sensitivity was also observed for a prototypic HIV-2 group B strain (EHO; EC 50 = 0.042 ± 0.009 m M). HIV-2 ROD9 RT mutants K65R and Q151M were equal to or slightly more sensitive to the drug than wild-type HIV-2 ROD9 , and mutants M184V and K65R+M184V showed sensitivities comparable to wild-type HIV-1 NL4-3 . Higher levels of BMS-986001 resistance (>30-fold) were observed for HIV-2 mutants Q151M+M184V, K65R+Q151M+M184V, and the patient-derived RT clone. Conclusions: BMS-986001 inhibits wild-type HIV-2 replication with EC 50 s in the nanomolar range and retains substantial activity against NRTI-resistant HIV-2 mutants. To our knowledge, this is the first and only report of an antiretroviral compound that, when tested against a diverse panel of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than HIV-1. On behalf of the University ofWashington-Dakar HIV-2 Study Group 1 University of Washington, Seattle, WA, US; 2 CHNU de Fann, Dakar, Senegal

Poster Abstracts

350

CROI 2015