Effects of raltegravir or elvitegravir resistance signature mutations on the barrier to dolutegravir resistance in vitro.

The recently approved HIV-1 integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) (S/GSK1349572) has overall advantageous activity when tested in vitro against HIV-1 with raltegravir (RAL) and elvitegravir (EVG) resistance signature mutations. We conducted an in vitro resistance selection study using wild-type HIV-1 and mutants with the E92Q, Y143C, Y143R, Q148H, Q148K, Q148R, and N155H substitutions to assess the DTG in vitro barrier to resistance. No viral replication was observed at concentrations of ≥ 32 nM DTG, whereas viral replication was observed at 160 nM RAL or EVG in the mutants. In the Q148H, Q148K, or Q148R mutants, G140S/Q148H, E138K/Q148K, E138K/Q148R, and G140S/Q148R secondary mutations were identified with each INSTI and showed high resistance to RAL or EVG but limited resistance to DTG. E138K and G140S, as secondary substitutions to Q148H, Q148K, or Q148R, were associated with partial recovery in viral infectivity and/or INSTI resistance. In the E92Q, Y143C, Y143R, and N155H mutants, no secondary substitutions were associated with DTG. These in vitro results suggest that DTG has a high barrier to the development of resistance in the presence of RAL or EVG signature mutations other than Q148. One explanation for this high barrier to resistance is that no additional secondary substitution of E92Q, Y143C, Y143R, or N155H simultaneously increased the fold change in 50% effective concentration (EC50) to DTG and infectivity. Although increased DTG resistance via the Q148 pathway and secondary substitutions occurs at low concentrations, a higher starting concentration may reduce or eliminate the development of DTG resistance in this pathway in vitro.

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.

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.

Combination of non-natural D-amino acid derivatives and allophenylnorstatine-dimethylthioproline scaffold in HIV protease inhibitors have high efficacy in mutant HIV.

Several non-natural D-amino acid derivatives were introduced as P2/P3 residues in allophenylnorstatine-containing (Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) HIV protease inhibitors. The synthetic analogues exhibited potent inhibitory activity against HIV-1 protease enzyme and HIV-1 replication in MT-4 cells. Structure-activity relationships revealed that D-cysteine or serine derivatives contributed to highly potent anti-HIV activities. Interestingly, anti-HIV activity of all the D-amino acid-introduced inhibitors was remarkably enhanced in their anti-HIV activities against a Nelfinavir-resistant clone, which has a D30N mutation in the protease, over that of the wild-type strain. HIV inhibitory activity of several analogues was moderately affected by an inclusion of alpha1-acid glycoprotein in the test medium.

The naphthyridinone GSK364735 is a novel, potent human immunodeficiency virus type 1 integrase inhibitor and antiretroviral.

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.

Synthesis and antiviral property of allophenylnorstatine-based HIV protease inhibitors incorporating D-cysteine derivatives as P2/P3 moieties.

We designed several HIV protease inhibitors with various d-cysteine derivatives as P(2)/P(3) moieties based on the structure of clinical drug candidate, KNI-764. Herein, we report their synthesis, HIV protease inhibitory activity, HIV IIIB cell inhibitory activity, cellular toxicity, and inhibitory activity against drug-resistant HIV strains. KNI-1931 showed distinct selectivity against HIV proteases and high potency against drug-resistant strains, surpassing those of Ritonavir and Nelfinavir.

Augmented antitumor activity of a secondary lymphoid-tissue chemokine (SLC)-interleukin (IL) 2 fusion protein in mouse.

BACKGROUND: To enhance the antitumor efficacy of IL2 gene therapy, combinations of several other genes, such as p53, a tumor suppressor gene, or lymphotactin, a C-chemokine, and the IL2 gene are attempted, and synergistic effects are observed. We report here on the enhanced antitumor activity of a fusion protein (mSLC-IL2) comprised of a newly identified member of the CC-chemokine family, mouse SLC (mSLC), and mouse IL2 (mIL2). METHODS: We constructed mSLC-IL2 by connecting the N-terminus of mIL-2 to the C-terminus of mSLC using a two-amino-acid linker. The resultant fusion protein retained both mIL2 activity, as measured in a standard proliferation assay using a mouse IL-2 dependent cell line, and chemokine activity, as measured in a chemotaxis assay using a preB cell line expressing mSLC-specific receptor, CCR7. The gene encoding mSLC-IL2 was retrovirally transduced into fibroblast CL.7 cells, derived from Balb/c mice. RESULTS: Intradermal transplantation of fibroblasts expressing mSLC-IL2 into syngenic mice induced a dense accumulation of CD4(+) and CD8(+) cells at the sites of transplantation. Moreover, when CT-26 cells, derived from colon adenocarcinoma cells, were co-transplanted with mSLC-IL2-transduced fibroblasts, the CT-26 cell exhibited significantly lower tumorigenicity than CT-26 cells co-transplanted with mIL2-transduced fibroblasts. CONCLUSIONS: These findings, obtained from both in vitro and in vivo data, suggest that the gene encoding mSLC-IL2 may be a good candidate for inclusion as part of an anticancer gene therapy protocol.