Gregory R. Reyes

HIV-1 escape from a small molecule, CCR5-specific entry inhibitor does not involve CXCR4 use.

To study HIV-1 escape from a coreceptor antagonist, the R5 primary isolate CC1/85 was passaged in peripheral blood mononuclear cells with increasing concentrations of the CCR5-specific small molecule inhibitor, AD101. By 19 passages, an escape mutant emerged with a >20,000-fold resistance to AD101. This virus was cross-resistant to a related inhibitor, SCH-C, and partially resistant to RANTES but still sensitive to CCR5-specific mAbs. The resistant phenotype was stable; the mutant virus retained AD101 resistance during nine additional passages of culture in the absence of inhibitor. Replication of the escape mutant in peripheral blood mononuclear cells completely depended on CCR5 expression and did not occur in cells from CCR5-Δ32 homozygous individuals. The escape mutant was unable to use CXCR4 or any other tested coreceptor to enter transfected cells. Acquisition of CXCR4 use is not the dominant in vitro escape pathway for a small molecule CCR5 entry inhibitor. Instead, HIV-1 acquires the ability to use CCR5 despite the inhibitor, first by requiring lower levels of CCR5 for entry and then probably by using the drug-bound form of the receptor.

SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo

We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime–piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activity in vitro against primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50–60% and a serum half-life of 5–6 h. On the basis of its novel mechanism of action, potent antiviral activity, and in vivo pharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.

Discovery and characterization of vicriviroc (SCH 417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1.

ABSTRACT Inhibiting human immunodeficiency virus type 1 (HIV-1) infection by blocking the host cell coreceptors CCR5 and CXCR4 is an emerging strategy for antiretroviral therapy. Currently, several novel coreceptor inhibitors are being developed in the clinic, and early results have proven promising. In this report, we describe a novel CCR5 antagonist, vicriviroc (formerly SCH-D or SCH 417690), with improved antiviral activity and pharmacokinetic properties compared to those of SCH-C, a previously described CCR5 antagonist. Like SCH-C, vicriviroc binds specifically to the CCR5 receptor and prevents infection of target cells by CCR5-tropic HIV-1 isolates. In antiviral assays, vicriviroc showed potent, broad-spectrum activity against genetically diverse and drug-resistant HIV-1 isolates and was consistently more active than SCH-C in inhibiting viral replication. This compound demonstrated synergistic anti-HIV activity in combination with drugs from all other classes of approved antiretrovirals. Competition binding assays revealed that vicriviroc binds with higher affinity to CCR5 than SCH-C. Functional assays, including inhibition of calcium flux, guanosine 5′-[35S]triphosphate exchange, and chemotaxis, confirmed that vicriviroc acts as a receptor antagonist by inhibiting signaling of CCR5 by chemokines. Finally, vicriviroc demonstrated diminished affinity for the human ether a-go-go related gene transcript ion channel compared to SCH-C, suggesting a reduced potential for cardiac effects. Vicriviroc represents a promising new candidate for the treatment of HIV-1 infection.

The nonstructural NS5A protein of hepatitis C virus: an expanding, multifunctional role in enhancing hepatitis C virus pathogenesis.

The hepatitis C virus (HCV) NS5A gene product is a phosphorylated 56- to 58-kD nonstructural protein that displays a multitude of activities related to enhancement of viral pathogenesis. Although associated with other viral encoded proteins as part of the viral replicase complex positioned on the cytoplasmic side of the endoplasmic reticulum, a role for NS5A in viral replication has not been defined. Post-translational modifications of NS5A include phosphorylation and potential proteolytic processing to smaller molecular weight forms able to translocate to the nucleus. Both the identification of a putative interferon (IFN) sensitivity-determining region within NS5A, as well as the direct interaction with and inhibition of the IFN-induced double-stranded RNA-dependent protein kinase (PKR) by NS5A remain controversial. Truncated versions of NS5A can act as transcriptional activators, while other recently characterized interactions of NS5A with cellular proteins indicate its pleiotropic role in HCV-host interactions. NS5A itself has no direct effect on IFN-alpha signaling or activation, but other abundant interactions with members of the cellular signaling apparatus, transcription activation machinery and cell cycle-regulatory kinases have been described (e.g. growth factor receptor-bound protein 2, p53, p21/waf and cyclins). Many of these interactions block the apoptotic cellular response to persistent HCV infection. More recently, another altogether different mechanism attenuating the IFN-alpha response was reported based on induction of interleukin (IL)-8. IL-8, in model systems, potentiates viral replication and mutes the nonspecific intracellular IFN antiviral response. Evidence supporting a complex multimechanistic role of NS5A in promoting viral persistence, pathogenesis and, indirectly, viral-related hepatocarcinogenesis indicates its key role in HCV pathobiology.

The Differential Sensitivity of Human and Rhesus Macaque CCR5 to Small-Molecule Inhibitors of Human Immunodeficiency Virus Type 1 Entry Is Explained by a Single Amino Acid Difference and Suggests a Mechanism of Action for These Inhibitors

ABSTRACT AD101 and SCH-C are two chemically related small molecules that inhibit the entry of human immunodeficiency virus type 1 (HIV-1) via human CCR5. AD101 also inhibits HIV-1 entry via rhesus macaque CCR5, but SCH-C does not. Among the eight residues that differ between the human and macaque versions of the coreceptor, only one, methionine-198, accounts for the insensitivity of macaque CCR5 to inhibition by SCH-C. Thus, the macaque coreceptor engineered to contain the natural human CCR5 residue (isoleucine) at position 198 is sensitive to HIV-1 entry inhibition by SCH-C, whereas a human CCR5 mutant containing the corresponding macaque residue (methionine) is resistant. Position 198 is in CCR5 transmembrane (TM) helix 5 and is not located within the previously defined binding site for AD101 and SCH-C, which involves residues in TM helices 1, 2, 3, and 7. SCH-C binds to human CCR5 whether residue 198 is isoleucine or methionine, and it also binds to macaque CCR5. However, the binding of a conformation-dependent monoclonal antibody to human CCR5 is inhibited by SCH-C only when residue 198 is isoleucine. These observations, taken together, suggest that the antiviral effects of SCH-C and AD101 involve stabilization, or induction, of a CCR5 conformation that is not compatible with HIV-1 infection. However, SCH-C is unable to exert this effect on CCR5 conformation when residue 198 is methionine. The region of CCR5 near residue 198 has, therefore, an important influence on the conformational state of this receptor.

Entry inhibitors SCH-C, RANTES, and T-20 block HIV type 1 replication in multiple cell types.

The small-molecule CCR5 antagonist SCH-C (SCH 351125) was tested for its ability to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells, immature dendritic cells (DCs), and macrophages. Inhibition of infection of PBMCs by virus associated with mature DC in trans was also studied. For comparison, the peptide-based fusion inhibitor T-20 and the CC-chemokine RANTES were also evaluated. Although some cell type-dependent differences in potency were observed, each of the three entry inhibitors was active against the replication of three different CCR5-using primary isolates in each cell type. CCR5-dependent HIV-1 infectivity, whether DC associated or not, is thus vulnerable to inhibitors that block the virus-cell fusion process by different mechanisms. Together, these results suggest that SCH-C and other entry inhibitors should be evaluated for their clinical potential as inhibitors of HIV-1 replication in several settings, including the prevention of maternal-infant transmission and the prevention of sexual transmission by topical application as a microbicide.

Molecular biology of non-A, non-B hepatitis agents : hepatitis C and hepatitis E viruses

Publisher Summary The discovery of the “Australia antigen” as a marker of hepatitis B infection, and the visualization of the 27-nm hepatitis A viral particle made it possible to develop reliable diagnostic assays for the detection of hepatitis A virus (HAV) and hepatitis B virus (HBV). Nevertheless, the appearance of post-transfusion associated hepatitis in patients negative for markers of either virus led researchers to believe that another new agent(s) existed. Such an agent(s) came to be known as non-A, non-B hepatitis (NANBH). In the past two years, major discoveries in the area of viral hepatitis have led to the identification of two new agents: hepatitis C virus (HCV), which is believed to cause the majority (at least 90%) of parenterally transmitted non-A, non-B hepatitis (PT-NANBH), and hepatitis E virus (HEW), responsible for enterically transmitted non-A, non-B hepatitis (ET-NANBH). This brings the number of known hepatotropic agents to five— namely, hepatitis A, B, C, D, and E viruses. HAV and HEV are spread by the fecal/oral route, whereas HBV, HCV, and HDV are transmitted principally by parenteral routes (e.g., blood and blood-derived products). These viral agents, with the exception of HBV, have RNA genomes. The successful development and application of molecular cloning strategies that had previously been applied to the elucidation of rare or low-abundance genes and gene transcripts have hastened the cloning and characterization of low-titer viruses that have not yet been successfully propagated in vitro. This chapter reviews the developments of the past two years that led to the successful identification of HCV and HEV.

The Molecular Biology of Hepatitis E Virus

There are two characterized forms of fecal-orally transmitted viral hepatitis: A and E (hepatitis A virus [HAV] and hepatitis E virus [HEV] respectively). The first confirmed epidemic of hepatitis E was in New Delhi, India in 1955 (1) and although the epidemiological features (e.g., fecal-oral transmission) suggested HAV as the etiologic agent, by 1980 there was clear evidence that a unique form of fecal-orally transmitted hepatitis was responsible for much of the epidemic hepatitis formerly ascribed to HAV. Indeed, HAV was excluded as the cause of the New Delhi epidemic by retrospective analysis of archived samples for evidence of HAV infection (2). The original observation of a common mode of transmission for hepatitis A and E is now eclipsed by our knowledge of the distinct epidemiology and molecular biology of HAV and HEV. Since the initial molecular cloning reported in 1990 (3) much has been learned about this virus. The unique features and our current understanding of HEV will be reviewed while highlighting issues requiring further investigation.