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 p24Gag production was unaffected, but virion release (measured as extracellular p24Gag ) 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. T he development and scale-up of effective combination antiretroviral therapies have significantly reduced human immunodeficiency virus type 1 (HIV-1)-related morbidity and mortality globally (1); however, selection and transmission of drug-resistant HIV-1 strains remain a concern (2, 3). Thus, new antivirals that overcome drug resistance or act via novel mechanisms are needed. Acylguanidines are a broad class of antiviral compounds that target ion channels and other host and viral factors (4-8). Their anti-HIV-1 activity is exemplified by 5-(N,N-hexamethylene) amiloride (HMA; Fig. 1A) (5, 6), as well as N-(5-(1-methyl-1H-pyrazol-4-yl)naphthalene-2-carbonyl)guanidine (BIT-225; Fig. 1B) (7). HMA and BIT-225 inhibit HIV-1 replication in monocyte-derived macrophages at low micromolar concentrations (6, 7). Moreover, treatment of infected cells with these compounds results in reduced budding and release of viral particles without affecting intracellular viral protein production, suggesting that blockade occurs at a late stage of viral replication (5, 7). 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 preventHMA and BIT-225 are both reported to target the HIV-1 accessory protein Vpu, an ϳ81-amino-acid transmembrane protein that is found in HIV-1 and some simian immunodeficiency virus (SIV) lineages and enhances viral replication through multiple mechanisms (...

In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach

“…nearly 8-fold reduction in CD4 median fluorescence intensity [MFI] between GFP high and GFP neg gates in the representative experiment shown in Figure 2A), and no receptor downregulation by empty vector. In line with studies that have assessed Vpu function by cloning the gene directly at the start codon (Chen et al, 2015; Galaski et al, 2016; Mwimanzi et al, 2016), pSel-NL4.3 Vpu displayed modest ( e.g. less than two-fold) CD4 downregulation function (Figure 2A).…”

“…Though autonomous ( i.e. non-Rev/RRE-dependent) in vitro Vpu expression can be achieved by cloning the vpu open reading frame directly at the start codon (Chen et al, 2015; Douglas et al, 2013; Galaski et al, 2016; Mwimanzi et al, 2016; Verma et al, 2013), protein expression is generally not robust. Moreover, the extensive genetic diversity in and upstream of vpu 's 5' coding sequence necessitates the design of primers specific for each HIV-1 isolate (Chen et al, 2015; Douglas et al, 2013; Galaski et al, 2016), or panels of primers specific for each HIV-1 subtype (Verma et al, 2013), which limits scalability.…”

Various plus unique: Viral protein U as a plurifunctional protein for HIV-1 replication

“…96 More importantly, SM111-mediated inhibition of HIV-1 replication was partially overcome by a Vpu I17R substitution. 96 These observations suggest that SM111 targets Vpu and that acylguanidine-containing compounds including SM111 provide promising opportunities to develop new treatments targeting Vpu.…”

The African natural product knipholone anthrone and its analogue anthralin (dithranol) enhance HIV-1 latency reversal

“…We next sought to investigate whether KA and/or anthralin can reactivate HIV proviruses in primary cells directly isolated from HIV-infected, cART-suppressed individuals. However, we first assessed the extent to which LRAs affected viability of isolated CD4+ cells obtained from 3 uninfected donors, as determined using ViaCount cell viability dye (32). LRAs assessed in this assay included 10 µM prostratin, KA, or anthralin, while 100 ng/mL phorbol myristate acetate (PMA) + 0.1 µg/mL ionomycin was applied as a positive control.…”

“…Viability in uninfected PBMCs was measured in the presence of test agents for 24 hours using Guava ViaCount Reagent (Millipore) and flow cytometry as described previously (32).…”