Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers, part I: core region.

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.

Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.

HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.

Mutations in multiple domains of Gag drive the emergence of in vitro resistance to the phosphonate-containing HIV-1 protease inhibitor GS-8374.

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4(+) T cells (50% effective concentration [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC(50)s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

The two-component hybrid kinase regulator CaNIK1 of Candida albicans.

Using degenerate primers of highly conserved regions of two-component response regulators for PCR amplification, a two-component response regulator was cloned from Candida albicans that is homologous to nik-1+ of Neurospora crassa. This two-component hybrid kinase, CaNIK1, also shows features of bacterial two-component response regulators, including a putative unorthodox second histidine kinase motif at the carboxy-terminal end. CaNIK1 was expressed at low levels in both the white and opaque switch phenotypes and in the bud and hyphal growth forms of C. albicans strain WO-1, but in both developmental programmes, the level of transcript was modulated (levels were higher in opaque cells and in hyphae). Partial deletion of both CaNIK1 alleles, by which the histidine autokinase- and ATP-binding domains were removed, did not inhibit either high-frequency phenotypic switching or the bud-hypha transition in high salt concentrations, but in both cases the efficiency of the developmental process was reduced.