Fig 1.
NRTTIs inhibit reverse transcription through multiple unique mechanisms, including inhibition of translocation.
DCT, delayed chain termination; ICT, immediate chain termination; NRTI, nucleos(t)ide reverse transcriptase inhibitor; NRTTI, nucleoside reverse transcriptase translocation inhibitor; RT, reverse transcriptase; vDNA, viral DNA, vRNA, viral RNA.
Fig 2.
Medicinal chemistry strategy and overview of expansive SAR campaign.
Modifications evaluated changes to the periphery of the ribose core, including 4′, 2′, and nucleobase positions, as well as replacement of the ribose core itself. SAR, structure–activity relationship.
Fig 3.
In vitro persistence assay to assess maintenance of effective compound levels after washout.
Cells were incubated with test compound for 24 h, allowing time for uptake and phosphorylation, followed by washout. Infection was performed immediately after washout (A) or 48 h later (B). Plates were analyzed 24 h post-infection for the number of GFP–positive cells. IC50 values were determined, and a persistence ratio was calculated for each compound. GFP, green fluorescent protein; hpi, hours post-infection; IC50, half maximal inhibitory concentration.
Fig 4.
Evaluation of novel nucleobase substitutions.
Potency, cytotoxicity, and persistence ratios in MT4-GFP cells and/or PBMCs for ISL and 16 compounds with nucleobase substitutions. CC50, concentration of test compound required to reduce cell viability by 50%; CTG, CellTiter-Glo; GFP, green fluorescent protein; IC50, half maximal inhibitory effect; ISL, islatravir; NC, not calculated (i.e., persistence ratios could not be calculated due to qualifiers in IC50A [infection performed immediately after washout] and/or IC50B [infection performed 48 h after washout]); PBMC, peripheral blood mononuclear cell.
Fig 5.
Evaluation of carbaribose modifications.
Potency, cytotoxicity, and persistence ratios in MT4-GFP cells and/or PBMCs for four compounds with carbaribose modifications. CH2, methylene; CC50, concentration of test compound required to reduce cell viability by 50%; CTG, CellTiter-Glo; GFP, green fluorescent protein; IC50, half maximal inhibitory effect; NC, not calculated (i.e., persistence ratios could not be calculated due to qualifiers in IC50A [infection performed immediately after washout] and/or IC50B [infection performed 48 h after washout]); PBMC, peripheral blood mononuclear cell.
Table 1.
Antiviral activity of MK-8527 and ISL in multiple assays and cell types.
Fig 6.
Activity of MK-8527 against HIV-1 subtypes.
Antiviral activity of MK-8527 was evaluated across 11 HIV-1 subtypes with the PhenoSense assay using vectors encoding protease and RT sequences derived from HIV-1 in human plasma for WT subtypes A (n = 3), A1 (n = 5), AE (n = 5), AG (n = 6), B (n = 5), BF (n = 5), C (n = 5), D (n = 4), F1 (n = 5), and G (n = 2). Geometric mean fold change from CNDO control is shown; error bars denote standard deviation. The underlying data for Fig 6 can be found in the S1 Data. RT, reverse transcriptase; WT, wild-type.
Fig 7.
Iron footprinting assay [24] to evaluate the position of RT on the primer and template.
An iron footprinting assay (A) was performed in the presence of (B) 5 µM ddATP; (C) 1 µM ISL-TP; or (D) µM MK-8527-TP; and escalating concentrations of dTTP. The original uncropped blots can be found in S1 Raw Images. DCT, delayed chain termination; ddATP, dideoxyadenosine triphosphate; dTTP, deoxythymidine triphosphate; ICT, immediate chain termination; ISL, islatravir; MP, monophosphate; RT, reverse transcriptase; TP, triphosphate.
Fig 8.
HIV-RT primer extension assay [23] to evaluate the chain termination mechanism of MK-8527-TP compared with ISL-TP and ddATP (an NRTI).
(A) Primer and template used along with representative gel with DCT and ICT sites marked. Dose-dependent chain termination patterns of (B) ddATP, (C) ISL-TP, and (D) MK-8527-TP are shown. The original uncropped blots can be found in S2 Raw Images. DCT, delayed chain termination; ddATP, dideoxyadenosine triphosphate; ICT, immediate chain termination; ISL-TP, islatravir-triphosphate; NRTI; nucleos(t)ide reverse transcriptase inhibitor, RT, reverse transcriptase; TP, triphosphate.
Fig 9.
Crystal structure of MK-8527-TP bound in the N-site of a HIV-RT/DNA complex.
RT, reverse transcriptase; TP, triphosphate.
Table 2.
Pharmacokinetics of MK-8527 in plasma from rats and monkeys.
Fig 10.
Concentration-vs-time profiles of MK-8527 in plasma and MK-8527-TP in PBMCs following oral administration to rhesus monkeys at 50 mg/kg.
The underlying data for Fig 10 can be found in the S2 Data. PBMC, peripheral blood mononuclear cells; TP, triphosphate.
Fig 11.
Reagents and conditions: (a) acetyl chloride, methanol (MeOH); (b) p-toluoyl chloride, pyridine; (c) HCl, diethyl ether; (d) 2,6-dichloro-7-deazapurine, NaH, acetonitrile (MeCN); (e) ammonia in isopropanol, sodium methoxide (NaOMe) in methanol (MeOH); (f) TBSCl, imidazole, N,N-dimethylformamide; (g) trifluoroacetic acid (TFA), tetrahydrofuran (THF), H2O; (h) IBX, MeCN, dimethyl sulfoxide (DMSO); (i) 1. formaldehyde, NaOH, 2. NaBH4, ethanol; (j) IBX, MeCN; (k) Bestmann−Ohira reagent, K2CO3; (l) TBAF, THF.