Novel Acylguanidine-Based Inhibitor of HIV-1

Abstract

The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24(Gag) production was unaffected, but virion release (measured as extracellular p24(Gag)) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity.

Importance: New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.

FIG 1

Chemical structures of acylguanidine compounds, namely, 5-(N,N-hexamethylene)amiloride (HMA) (A), N-(5-(1-methyl-1H-pyrazol-4-yl)naphthalene-2-carbonyl)guanidine (BIT-225) (B), 1-(2-(azepan-1-yl)nicotinoyl)guanidine (SM111) (C), and C-(6-azepan-1-yl-pyridin-3-yl)-methylamine (SM113) (D).

FIG 2

SM111 has minimal toxicity in comparison to that of reported acylguanidine compounds. (A to C) Effects of SM111 (blue), SM113 (green), HMA (purple), and BIT-225 (red) on viability of uninfected CEM-GXR cells (A) and cellular ATP in uninfected (B) and infected (C) CEM-GXR cells after 6 days. (D to F) Effects of compounds on viability of uninfected PBMC (D) and cellular ATP in uninfected (E) and infected (F) PBMC after 6 days. For panels A, B, D, and E, data are representative of three independent experiments performed in triplicate. For panels C and F, histograms and error bars represent means ± standard deviations (SD) calculated from three independent experiments (C) and using cells from three different donors (F), performed in triplicate.

FIG 3

SM111 inhibits in vitro replication of HIV-1_NL4.3. (A) CEM-GXR cells were infected with HIV-1_NL4.3 for 24 h (MOI = 0.003), washed, and cultured in the absence or presence of SM111 or SM113 at the indicated concentrations. Cellular GFP, a marker of viral infection, was monitored on days 2 to 6 by flow cytometry. (B) Viral replicative capacity (vRC) of HIV-1_NL4.3 in CEM-GXR cells in the presence of indicated SM111 concentrations, normalized to viral spread in the absence of compound. (C) Replication of HIV-1_NL4.3 in CEM-GXR cells infected at MOIs of 0.03, 0.01, and 0.003 in the absence or presence of 100 μM SM111. (D) Schematic of experimental plan for results shown in panel E, indicating the times of addition and removal of 100 μM SM111 in CEM-GXR cells relative to infection with HIV-1_NL4.3 (MOI = 0.003) at time zero. (E) vRCs of HIV-1_NL4.3 in the absence or presence of 100 μM SM111, annotated (i to v) as shown in panel D. (F) PBMC were infected with HIV-1_NL4.3 for 24 h (MOI = 0.003), washed, and cultured in the absence or presence of 50 μM SM111 or SM113. Supernatant levels of p24^Gag were monitored by ELISA on days 3 and 6 postinfection. For panels A, C, and F, data are representative of three independent experiments performed in triplicate (from three different donors in panel F). For panels B and E, histograms and error bars represent means ± SD calculated from three independent experiments performed in triplicate.

FIG 4

SM111 inhibits in vitro replication of antiretroviral-resistant HIV-1 strains in CEM-GXR cells. (A) Properties of drug-resistant viruses, including primary mutations and representative antiretroviral drugs. (B) Replication of HIV-1_NL4.3 in the absence or presence of 100 nM antiretroviral drugs or 100 μM SM111. (C) Replication of HIV-1_PI-RS in the absence or presence of 100 nM indinavir or 100 μM SM111. (D) Replication of HIV-1_NRTI-RS in the absence or presence of 100 nM azidothymidine or 100 μM SM111. (E) Replication of HIV-1_NNRTI-RS in the absence or presence 100 nM efavirenz or 100 μM SM111. (F) Replication of HIV-1_INI-RS in the absence or presence 100 nM raltegravir or 100 μM SM111. For panels B to F, error bars represent means ± SD. Data are representative of two independent experiments performed in triplicate.

FIG 5

SM111 selects for mutations in Vpu and Env. Culture of HIV-1_NL4.3 in CEM-GXR cells in the presence of 100 μM SM111 is shown. Strains A to N represent 14 independent infected cultures. Viral outgrowth was observed in 2 of 14 (14.3%) cultures (strains C and H) by day 9. Mutations identified in each strain by full-genome viral sequencing are annotated.

FIG 6

Validation of SM111 resistance mutations in CEM-GXR cells and PBMC. (A to F) Replication of HIV-1_{VpuW22*/EnvN136Y} (A), HIV-1_VpuI17R (B), HIV-1_ΔVpu (C), HIV-1_VpuW22* (D), and HIV-1_EnvN136Y (E) in CEM-GXR cells in comparison to HIV-1_NL4.3 in the absence and presence of 100 μM SM111. (F) Percent inhibition of each virus strain at day 6 postinfection at various SM111 concentrations. (G to I) Replication of HIV-1_{VpuW22*/EnvN136Y} (G), HIV-1_VpuI17R (H), and HIV-1_ΔVpu (I) in PBMC in comparison to HIV-1_NL4.3 in the absence and presence of 56 μM SM111. Data are representative of three independent experiments performed in triplicate in CEM-GXR cells (A to F) or using PBMC from three different donors in triplicate (G to I). Data for HIV-1_NL4.3 are shown repetitively to facilitate comparisons with replication of mutant strains. Error bars represent means ± SD.

FIG 7

SM111-induced Vpu mutations abrogate CD4 and tetherin downregulation functions. (A) Representative flow cytometry data showing cell surface expression of CD4 (top) or tetherin (bottom) in CEM T cells transfected with a plasmid encoding GFP and wild-type (WT) or mutant Vpu sequences. Numbers in each plot indicate MFIs of CD4 or tetherin staining. Data are representative of three independent experiments. (B and C) CEM-GXR cells were infected with VSV-G-pseudotyped HIV-1_NL4.3 or Vpu/Env mutant viruses as indicated, and cell surface expression of CD4 (B) or tetherin (C) was measured after 48 h by flow cytometry. Data are normalized to CD4 or tetherin expression on uninfected cells (dashed lines). (D) Uninfected CEM-GXR cells were treated with SM111, and cell surface expression of CD4, tetherin, or CXCR4 was measured after 2 days. Data are normalized to CD4, tetherin, or CXCR4 expression on untreated cells (dashed lines). For panels B to D, the means ± SD of results from three independent experiments are shown. *, P < 0.05 (two-tailed Student's t test).

FIG 8

SM111 inhibits particle release, virion infectivity, and Env surface expression. (A) Viral p24^Gag levels were measured by ELISA in supernatants from CEM-GXR cells infected with VSV-G-pseudotyped HIV-1 strains following treatment with the indicated concentrations of SM111 at 48 h. (B) The infectivities of culture supernatants of strains shown in panel A were measured by incubating TZM-bl cells with 0.5 ng p24^Gag. Results are shown as luminescence in arbitrary light units. (C) Env surface expression on CEM-GXR cells was detected by flow cytometry at 48 h postinfection in the absence or presence of SM111 at the indicated concentrations. MFI of infected cells minus background staining (based on uninfected cells) is shown. Data for all panels are representative of three independent experiments performed in triplicate.

FIG 9

SM111 does not inhibit intracellular p24^Gag expression. HEK-293T cells were transfected with wild-type pNL4.3 plasmid, followed by detection of intracellular p24^Gag by flow cytometry and extracellular p24^Gag by ELISA. (A) Representative flow cytometry data showing intracellular p24^Gag expression in the absence or presence of 50 μM or 100 μM SM111. Mean fluorescence intensity (MFI) of p24^Gag staining is indicated. (B) Dose-dependent effects of SM111 on intracellular and extracellular p24^Gag. (C) Cellular ATP levels for untransfected cells treated with various concentrations of SM111. For panels B and C, means ± SD of results from three independent experiments are shown.