Islatravir Selects for HIV-1 Variants in MT4-GFP Cells That Profoundly
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Islatravir Selects for HIV-1 Tracy Diamond1; Winnie Ngo1; Min Xu2; Shih Lin Goh2; Silveria Rodriguez2; Variants in MT4-GFP Cells That Ming-Tain Lai1; Ernest Asante-Appiah1; Profoundly Reduce Replicative Jay Grobler1 Capacity in Peripheral Blood 1Infectious Disease and Vaccines, 2Quantitative Biosciences, Merck & Co., Inc., Mononuclear Cells Kenilworth, NJ, USA Background Activity Against A114S Variants Differentiates ISL A114S-Containing Viruses With Reduced Susceptibility to (an NRTTI) From NRTIs ISL Have Profound Replicative Capacity Defects in PBMCs Islatravir (ISL, MK-8591), a First-in-Class Nucleoside Reverse Transcriptase Translocation Inhibitor (NRTTI) Figure 2A. Potency (IC50) of ISL and NRTIs Against Figure 4A. Replicative Capacity of Viruses in MT4-GFP Cells With Multiple Mechanisms of Action RT Variants in PBMCs OH N O 10000 10000 N NH2 **** N 10000 HO N 8000 Translocation Inhibition F 1000 ) Due to the 4’-ethynyl group WT 6000 WT g/mL (nM) vRNA Delayed Chain Termination 50 A114S 100 (p M184I Due to the 4’-ethynyl and 3’-hydroxyl groups IC M184V vRNA p24 4000 M184V 10 A114S/M184V vDNA A114S/M184V M41L/A114S/M184V Translocation inhibition prevents the opening 2000 • vDNA 1 of the nucleotide binding site • Additional nucleotides cannot bind or be • ISL incorporation changes the vDNA structure 0.1 0 incorporated into the viral DNA • If translocation occurs and a nucleotide is added, the • Viral replication is inhibited structural change prevents further nucleotide incorporation ISLTDF AZT3TC FTC 012345678910 • Viral replication is inhibited Days Post-infection ISL is in clinical development for the • As such, ISL is not in the reverse transcriptase (RT) active site, *IC50 >42000nM treatment and prevention of HIV-1 infection. and it is no longer susceptible to resistance-conferring mutations • ISL displays similar or greater potency against A114S, • All viruses replicated to some extent in MT4-GFP cells M184V, and A114S/M184V compared to TDF, AZT, 3TC, as monitored by GFP (data not shown) and p24 Rationale and FTC against WT The study was conducted to extend our understanding of resistance pathways for ISL in HIV-1 subtypes Figure 2B. Fold Change in IC50 From WT for ISL and NRTIs Figure 4B. Replicative Capacity of Viruses in PBMCs Against RT Variants in PBMCs 5000 100 * * * * Methods Decreased Susceptibility 4000 Perform viral resistance selection studies with HIV-1 subtype 10 ) • vs WT A114S 50 3000 WT A, B, and C wild-type viruses M184V g/mL (p M184I 1 – WT virus was passaged with MT4-GFP [subtype B (R8)] or A114S/M184V M184V p24 2000 MT4-GFP/CCR5 [subtype A (92W026) or C (93MW959)] cells A114S/M184V biweekly in the presence of increasing concentration of ISL 0.1 1000 M41L/A114S/M184V Fold-Change in IC Increased Susceptibility – There were 8 replicates each for subtype A, B, and C virus with 10% FBS and 10% NHS, respectively 0.01 0 ISLTDF AZT3TC FTC 012345678910 – Cultures were monitored for viral breakthrough by measuring Days Post-infection the number of GFP-positive cells at each passage, and the *Fold Change >Max RT region of the viral RNA was periodically genotyped by • In contrast to ISL, A114S maintained or increased population sequencing slope susceptibility to the NRTIs HIV-1 Variant R0 (10 ) • Phenotype selected variants in MT4-GFP cells and PBMCs WT 2.30 M184I 1.94 – Provirus clones were generated by site-directed mutagenesis, and virus was generated by transfection M184V 1.91 A114S Has Differential Effects on Susceptibility of ISL A114S/M184V 1.08 – Potency (IC50) of ISL was assessed in a multiple-cycle viral and NRTIs to Thymidine Analog Mutations (TAM) M41L/A114S/M184V 1.48 kinetic assay in MT4-GFP cells in 10% or 100% NHS Linear regression was performed during the phase with the largest increase in p24 for each Figure 3A. Potency (IC ) of ISL and NRTIs Against Variants – Viral potency in PBMCs (10% NHS) was determined using virus 50 virus to determine the R0 value for each virus. variants expressing GFP Encoding TAMs in MT4-GFP Cells (10% NHS) • No replication was observed for A114S/M184V in PBMCs 10000 • Assess replicative capacity of variants containing A114S (R0 = 1.08) in MT4-GFP cells and PBMCs 1000 • M41L/A114S/M184V had decreased replicative capacity – Replicative capacity was determined by monitoring the number in PBMCs (~36% of WT) of GFP-positive MT4-GFP cells over time and/or the production 100 WT (nM) of p24 protein in infected cultures 50 4 TAMs IC 10 4 TAMs + A114S Results 1 Conclusions • In resistance selection studies, M184I and M184V • Resistance was always associated with M184I or M184V 0.1 across subtypes A, B, and C ISLTDF AZT 3TCFTC were the most common substitutions to emerge across multiple HIV subtypes • No other substitutions were observed in >2 of 48 selection experiments • ISL remained comparable or more potent than the NRTIs • With the exception of A114S, other substitutions – Substitutions observed in 2 experiments – V90I against 4 TAMs observed during these studies had minimal effects – Substitutions observed in 1 experiment – E36K, E36D, M41L, on viral susceptibility alone or in combination with L74I, V75A, A114S, A158T, C162Y, K166R, G196R, H221Y, A400T M184 substitutions Figure 3B. Fold Change in IC50 from WT for ISL and NRTIs • A114S augmented resistance conferred by M184V Figure 1. Effect of Single-codon Substitutions on the Against Variants Encoding TAMs in MT4-GFP Cells but had minimal effect (2-fold) on its own Susceptibility of HIV-1 Subtype B to ISL in MT4-GFP Cells (10% NHS) (100% NHS) 14 • Virus containing A114S in combination with M184V did not replicate in PBMCs 8 12 7 • The differential impact of A114S on ISL vs NRTIs WT vs WT 10 6 50 vs is consistent with its distinct mechanism of action 50 4 TAMs 5 8 IC 4 TAMs + A114S in 4 6 3 4 References 2 Fold Change in IC 1. Rhee SY, et al. Nucleic Acids Res. 2003;31(1):298-303. Fold Change 2 1 2. Grobler J, et al. Conference on Retroviruses and Opportunistic Infections 2019. 0 0 3. Rudd DJ, et al. Conference on Retroviruses and Opportunistic Infections 2020. WT M41L L74I V90I A114SA158T C162Y M184I M184VA400T ISLTDF AZT3TC FTC Abbreviations ISL, islatravir; FBS, fetal bovine serum; GFP, green fluorescent protein; • M184I and M184V were the only single substitutions that • A combination of 4 TAMs had a modest impact (<4-fold) NHS, normal human serum; PBMC, peripheral blood mononuclear cells; conferred >2-fold shift in potency on ISL potency WT, wild-type; AZT, zidovudine; TDF, tenofovir disoproxil fumarate; 3TC, lamivudine; FTC, emtricitabine; R0, replicative ratio; 4 TAMs, D67N/K70R/T215F/K219Q • Only A114S (observed in 1 selection experiment at passage • A combination of 4 TAMs conferred >4-fold potency 38) in combination with M184V augmented resistance reductions to the tested NRTIs Copies of this presentation obtained through conferred by M184V by >2-fold QR (Quick Response) codes are for personal • A114S mitigated resistance to NRTIs caused by 4 TAMs use only and may not be reproduced without • A114S is exceedingly rare in clinical isolates but did not impact susceptibility to ISL permission of the authors. – 8 out of 139,609 people had A114S detected (Stanford HIV Drug Resistance Database)1 https://bit.ly/3lMyghv Presented at HIV Drug Therapy-Glasgow 2020; October 5-8, 2020. Copyright © 2020 Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. All rights reserved. diamontr_210431-0001-HIV-DTG_ePoster_ISL_v1.08 09/23/2020 • Output Size: 11.7” x 6.5” Scale: 100%.