Brian A. Johns

In Vitro Antiretroviral Properties of S/GSK1349572, a Next-Generation HIV Integrase Inhibitor

ABSTRACT S/GSK1349572 is a next-generation HIV integrase (IN) inhibitor designed to deliver potent antiviral activity with a low-milligram once-daily dose requiring no pharmacokinetic (PK) booster. In addition, S/GSK1349572 demonstrates activity against clinically relevant IN mutant viruses and has potential for a high genetic barrier to resistance. S/GSK1349572 is a two-metal-binding HIV integrase strand transfer inhibitor whose mechanism of action was established through in vitro integrase enzyme assays, resistance passage experiments, activity against viral strains resistant to other classes of anti-HIV agents, and mechanistic cellular assays. In a variety of cellular antiviral assays, S/GSK1349572 inhibited HIV replication with low-nanomolar or subnanomolar potency and with a selectivity index of 9,400. The protein-adjusted half-maximal effective concentration (PA-EC50) extrapolated to 100% human serum was 38 nM. When virus was passaged in the presence of S/GSK1349572, highly resistant mutants were not selected, but mutations that effected a low fold change (FC) in the EC50 (up to 4.1 fold) were identified in the vicinity of the integrase active site. S/GSK1349572 demonstrated activity against site-directed molecular clones containing the raltegravir-resistant signature mutations Y143R, Q148K, N155H, and G140S/Q148H (FCs, 1.4, 1.1, 1.2, and 2.6, respectively), while these mutants led to a high FC in the EC50 of raltegravir (11- to >130-fold). Either additive or synergistic effects were observed when S/GSK1349572 was tested in combination with representative approved antiretroviral agents; no antagonistic effects were seen. These findings demonstrate that S/GSK1349572 would be classified as a next-generation drug in the integrase inhibitor class, with a resistance profile markedly different from that of first-generation integrase inhibitors.

Dolutegravir (S/GSK1349572) Exhibits Significantly Slower Dissociation than Raltegravir and Elvitegravir from Wild-Type and Integrase Inhibitor-Resistant HIV-1 Integrase-DNA Complexes

ABSTRACT The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10−6 s−1 and a dissociative half-life (t1/2) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t1/2, at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Selection of diverse and clinically relevant integrase inhibitor-resistant human immunodeficiency virus type 1 mutants

Resistance passage studies were conducted with five INIs (integrase inhibitors) that have been tested in clinical trials to date: a new naphthyridinone-type INI S/GSK-364735, raltegravir, elvitegravir, L-870,810 and S-1360. In establishing the passage system and starting from concentrations several fold above the EC(50) value, resistance mutations against S-1360 and related diketoacid-type compounds could be isolated from infected MT-2 cell cultures from day 14 to 28. Q148R and F121Y were the two main pathways of resistance to S/GSK-364735. Q148R/K and N155H, which were found in patients failing raltegravir treatment in Phase IIb studies, were observed during passage with raltegravir with this method. The fold resistance of 40 mutant molecular clones versus wild type virus was compared with these five INIs. The overall resistance pattern of S/GSK-364735 was similar to that of raltegravir and other INIs. However, different fold resistances of particular mutations were noted among different INIs, reflecting a potential to develop INIs with distinctly different resistant profiles.

Carbamoyl pyridone HIV-1 integrase inhibitors 3. A diastereomeric approach to chiral nonracemic tricyclic ring systems and the discovery of dolutegravir (S/GSK1349572) and (S/GSK1265744).

We report herein the discovery of the human immunodeficiency virus type-1 (HIV-1) integrase inhibitors dolutegravir (S/GSK1349572) (3) and S/GSK1265744 (4). These drugs stem from a series of carbamoyl pyridone analogues designed using a two-metal chelation model of the integrase catalytic active site. Structure-activity studies evolved a tricyclic series of carbamoyl pyridines that demonstrated properties indicative of once-daily dosing and superior potency against resistant viral strains. An inherent hemiaminal ring fusion stereocenter within the tricyclic carbamoyl pyridone scaffold led to a critical substrate controlled diastereoselective synthetic strategy whereby chiral information from small readily available amino alcohols was employed to control relative and absolute stereochemistry of the final drug candidates. Modest to extremely high levels of stereochemical control were observed depending on ring size and position of the stereocenter. This approach resulted in the discovery of 3 and 4, which are currently in clinical development.

The activity of the integrase inhibitor dolutegravir against HIV-1 variants isolated from raltegravir-treated adults.

Background:Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Methods:Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Results:Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87), while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9–19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H, G140S + Q148R, T97A + Y143R, and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Conclusions:Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir.

The use of oxadiazole and triazole substituted naphthyridines as HIV-1 integrase inhibitors. Part 1: Establishing the pharmacophore.

A series of HIV-1 integrase inhibitors containing a novel metal binding motif consisting of the 8-hydroxy-1,6-naphthyridine core and either an oxadiazole or triazole has been identified. The design of the key structural components was based on a two-metal coordination pharmacophore. This report presents initial structure-activity data that shows the new chelation architecture delivers potent inhibition in both enzymatic and antiviral assays.

The Naphthyridinone GSK364735 Is a Novel, Potent Human Immunodeficiency Virus Type 1 Integrase Inhibitor and Antiretroviral

ABSTRACT The naphthyridinone GSK364735 potently inhibited recombinant human immunodeficiency virus type 1 (HIV-1) integrase in a strand transfer assay (mean 50% inhibitory concentration ± standard deviation, 8 ± 2 nM). As expected based on the structure of the drug, it bound competitively with another two-metal binding inhibitor (Kd [binding constant], 6 ± 4 nM). In a number of different cellular assays, GSK364735 inhibited HIV replication with potency at nanomolar concentrations (e.g., in peripheral blood mononuclear cells and MT-4 cells, 50% effective concentrations were 1.2 ± 0.4 and 5 ± 1 nM, respectively), with selectivity indexes of antiviral activity versus in-assay cytotoxicity of at least 2,200. When human serum was added, the antiviral potency decreased (e.g., a 35-fold decrease in the presence of 100% human serum was calculated by extrapolation from the results of the MT-4 cell assay). In cellular assays, GSK364735 blocked viral DNA integration, with a concomitant increase in two-long-terminal-repeat circles. As expected, this integrase inhibitor was equally active against wild-type viruses and mutant viruses resistant to approved drugs targeting either reverse transcriptase or protease. In contrast, some but not all viruses resistant to other integrase inhibitors were resistant to GSK364735. When virus was passaged in the presence of the inhibitor, we identified resistance mutations within the integrase active site that were the same as or similar to mutations arising in response to other two-metal binding inhibitors. Finally, either additive or synergistic effects were observed when GSK364735 was tested in combination with approved antiretrovirals (i.e., no antagonistic effects were seen). Thus, based on all the data, GSK364735 exerted potent antiviral activity through the inhibition of viral DNA integration by interacting at the two-metal binding site within the catalytic center of HIV integrase.

1,3,4-Oxadiazole substituted naphthyridines as HIV-1 integrase inhibitors. Part 2: SAR of the C5 position

The use of a 1,3,4-oxadiazole in combination with an 8-hydroxy-1,6-naphthyridine ring system has been shown to deliver potent enzyme and antiviral activity through inhibition of viral DNA integration. This report presents a detailed structure-activity investigation of the C5 position resulting in low nM potency for several analogs with an excellent therapeutic index.

Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics

Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations.

Carbamoyl pyridone HIV-1 integrase inhibitors. 2. Bi- and tricyclic derivatives result in superior antiviral and pharmacokinetic profiles.

This work is a continuation of our initial discovery of a potent monocyclic carbamoyl pyridone human immunodeficiency virus type-1 (HIV-1) integrase inhibitor that displayed favorable antiviral and pharmacokinetic properties. We report herein a series of bicyclic carbamoyl pyridone analogues to address conformational issues from our initial SAR studies. This modification of the core unit succeeded to deliver low nanomolar potency in standard antiviral assays. An additional hydroxyl substituent on the bicyclic scaffold provides remarkable improvement of antiviral efficacies against clinically relevant resistant viruses. These findings led to additional cyclic tethering of the naked hydroxyl group resulting in tricyclic carbamoyl pyridone inhibitors to address remaining issues and deliver potential clinical candidates. The tricyclic carbamoyl pyridone derivatives described herein served as the immediate leads in molecules to the next generation integrase inhibitor dolutegravir which is currently in late stage clinical evaluation.