CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

The efficacy of DTG was quantified against a panel of seven viruses containing one or two InSTI resistance mutations. In contrast with other drug classes but consistent with first- generation InSTIs, mutations had less effect on the slope of the DTG dose-response curve than on the IC 50 . This result was true even for viruses containing the DTG-specific R263K resistance mutation. Conclusions: A quantitative in vitro efficacy model that correlates with clinical efficacy for all drug classes except first-generation InSTIs is consistent with the clinical efficacy of the second-generation InSTI DTG. In combination with previously published results, this model may be used to predict new DTG-containing regimens with high clinical efficacy. 463 HIV-1 Transcription Inhibition by New p300 Inhibitors Kotaro Shirakawa 1 ; Melanie Ott 2 ; EricVerdin 2 1 Kyoto Univ, Kyoto, Japan; 2 Gladstone Inst of Virology and Immunology, San Francisco, CA, USA Background: Understanding the mechanism of HIV-1 transcription is key for developing a new class of antiviral drugs. HIV-1 Tat is an essential viral protein that transactivates HIV-1 transcription by binding to the TAR region of HIV-1 mRNA. p300-mediated acetylation of Tat is required for transactivation of the HIV-1 long terminal repeat (LTR). Full activation of NF-κB also requires p300-mediated acetylation. Since salicylate (SAL) is known to inhibit NF-κB and HIV-1 transcription, we hypothesized that SAL might inhibit p300 acetyltransferase activity. Methods: First, we tested SAL in in vitro HAT assays and checked histone acetylation profile in HEK293T cells upon SAL treatment. Next, we did a secondary screen of FDA-approved chemical compounds that contain structures similar to SAL. Finally, we tested whether SAL and related FDA approved drugs inhibits HIV transcription and infection using HIV-1 LTR = 996μM), which is an old anti- inflammatory drug. We tested these drugs using HIV-1 LTR reporter assays. Co-transfection of both Tat and p300 synergistically activated the HIV-1 promoter, however, both SAL and DFN inhibited synergistic activation of HIV-1 transcription at lower IC 50 than in vitro HAT inhibition (IC 50 = 2.2 mM for SAL and 80 μM for diflunisal). SAL and DFN inhibited HIV-1infection at IC 50 1.09 mM for SAL and 154 μM for DFN measured by single round infection assays using vsv-g pseudotyped luciferase reporter HIV-1 and activated CD4 positive T-cells. Conclusions: These results revealed novel epigenetic therapeutic targets for SAL, CBP and p300, and the precise mechanism how SAL and DFN inhibit HIV-1 transcription. These drugs could be used to suppress HIV-1 transcription and to treat other inflammatory pathologies including diabetes, cancer and neurodegenerative disorder. 464 Maturation Inhibitor BMS-955176: Activity Against PI-Resistant Clinical Isolates Neelanjana Ray 1 ;Tianbo Li 2 ;Tricia Protack 2 ; Zeyu Lin 2 ; Petronella M. van Ham 3 ; Carey Hwang 1 ; Mark Krystal 2 ; Monique Nijhuis 3 ; Max Lataillade 2 ; Ira B. Dicker 2 1 Bristol-Myers Squibb, Princeton, NJ, USA; 2 Bristol-Myers Squibb, Wallingford, CT, USA; 3 Univ Med Cntr Utrecht, Utrecht, Netherlands Background: BMS-955176 is a second-generation HIV-1 maturation inhibitor (MI). HIV-1 isolates resistant to approved protease inhibitors (PI) harbor substitutions in both protease (Pr) (1 o ) and the substrate of Pr, Gag (2 o ). Such isolates reportedly have a higher frequency of Gag substitutions which could potentially exhibit cross-resistance to MIs. To assess the susceptibility of BMS-955176 to PI-resistant (PI R ) viruses with Gag substitutions, we evaluated the virologic profile of a cohort of pre- and post-treatment (PT) clinical isolates from subjects who experienced PI therapy failure. Methods: Longitudinal isolates (n=21) from 15 PI-treated subjects had a median (range) of 6 (2.3-11.7) years on PI therapy. All PT samples had major PI resistance-associated mutations (RAMs) in Pr and 17/21 had 2 o changes in Gag associated with PI resistance (at positions 128,431,436,437,449,452,453). Phenotypic susceptibilities (fold-change-IC 50 , FC) were determined using either the PhenoSense Gag/Pr (PS) assay (Monogram Biosciences) or BMS gag/Pr pseudotype single (SC) and multiple cycle (MC) antiviral assays. Fold changes from baseline (FCFB = FC post/FC pre) < 3 were considered to be within the no-effect level based on current assay reproducibility data in control samples. Seven non- longitudinal highly PI R viruses containing multiple major and minor PI RAMs were also evaluated for BMS-955176 susceptibility. Results: 19/21 PT samples from 15 PI-treated subjects had FCFBs within the no-effect level. The median (range) FCFB was 0.83 (0.05-27.4) [PS assay (11 subjects)] and 1.5 (1.0-2.2) [SC assay (4 subjects)]. The two PT samples with PS FCFB >3 were retested using the SC and MC assays. One of these two samples, with PS FCFB=27.4, was within the no-effect level of the SC assay with FCFB median (range) of 2.2 (1.9-2.5), while the other sample showed variable changes in susceptibility with SC FCFB median (range) =4.2 (2.3-6.1). Neither sample showed meaningful susceptibility changes in the MC assay (FCFB = 2.1 and 1.5, respectively). 2° Gag changes were not associated with a greater median BMS-955176 FCFB. A panel of 7 highly PI R non-longitudinal viruses were all susceptible (FC range 0.16-0.68) to BMS-955176. Conclusions: BMS-955176 maintains antiviral activity against PI R isolates with emergent Pr and/or Gag mutations. This finding supports continued development of the second- generation MI BMS-955176 in treatment-experienced subjects with or without prior PI therapy. 465 Resistance Pathways for Potent and Broadly Active HIV-1 Maturation Inhibitors Emiko Urano 1 ; Sherimay D. Ablan 1 ; Justin A. Kaplan 1 ; Nishani Kuruppu 1 ; David E. Martin 2 ;Theodore J. Nitz 2 ; CarlT.Wild 2 ; Eric O. Freed 1 1 NCI, Frederick, MD, USA; 2 DFH Pharma, Inc, Gaithersburg, MD, USA Background: A betulinic acid-based compound, bevirimat (BVM), the first-in-class HIV-1 maturation inhibitor, acts by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown to be safe and effective in reducing viral loads in HIV-1-infected patients. However, single-amino-acid polymorphisms in the SP1 region of Gag reduced HIV-1 susceptibility to BVM in patients. Methods: We carried out an extensive medicinal chemistry campaign to identify BVM derivatives that demonstrate increased potency against consensus clade B strains of HIV-1 and are active against primary isolates with polymorphisms in SP1. Compound activity was tested in assays that measure CA-SP1 processing and virus replication kinetics. Selection experiments were performed to identify mutations that confer resistance to these novel compounds and a variety of virological, structural, and molecular approaches were applied to elucidate the mechanism of resistance for each mutant. To evaluate the effect of Gag polymorphisms and resistance mutations on the kinetics of Gag processing, pulse-chase radiolabeling assays were performed. Results: We identified a set of BVM derivatives that are more potent than BVM against WT HIV-1 and show robust antiviral activity against SP1 polymorphic strains and clinical isolates. The best of these analogs retain significant activity against BVM-resistant mutants. Selection experiments identified an Ala-to-Val mutation at SP1 residue 1 (SP1-A1V). In addition, we also selected for the mutation CA-P157A, located in the major homology region (MHR) of CA. Remarkably, the P157A mutant was resistant to not only BVM and the second-generation BVM analogs but also to the structurally distinct maturation inhibitor PF-46396. Pulse-chase data demonstrate that CA-SP1 processing kinetics for P157A are similar to those of the WT. Analysis of the HIV-1 database reveals that Ala1 of SP1 and Pro157 of CA are conserved in ~99.95% of available sequences. Conclusions: This study identifies a panel of BVM derivatives that display marked improvements relative to BVM in antiviral potency and breadth of activity. The characterization of resistant mutants provides novel insights into the structure of the maturation inhibitor-binding site and the role of SP1 and the CA MHR in virus assembly and maturation. This study will support ongoing clinical development of this class of inhibitors. reporter assay and single round infection assays. Results: We found that SAL inhibits p300 (IC 50 of 10.2 mM) and other related acetyltransferase CBP (IC 50 = 5.7 mM), but not PCAF and GCN5 in in vitro HAT assays. Kinetic analysis identified that SAL competes with acetyl CoA but not with histones. SAL suppressed the acetylation of histones residues H2A K5/K9 , H2B K12/K15, and H3 K56 in HEK293T cells. IC 50 for SAL-mediated inhibition of H2B K12/K15 acetylation (4.8 mM) was close to drug plasma concentrations measured in humans following oral SAL administration (1-3 mM). A secondary screen of FDA-approved chemical compounds that contain structures similar to SAL identified a more potent p300 inhibitor, diflunisal (DFN, IC 50

Poster Abstracts

179

CROI 2016