Benzoxazole and benzothiazole amides as novel pharmacokinetic enhancers of HIV protease inhibitors.

A new class of benzoxazole and benzothiazole amide derivatives exhibiting potent CYP3A4 inhibiting properties was identified. Extensive lead optimization was aimed at improving the CYP3A4 inhibitory properties as well as overall ADME profile of these amide derivatives. This led to the identification of thiazol-5-ylmethyl (2S,3R)-4-(2-(ethyl(methyl)amino)-N-isobutylbenzo[d]oxazole-6-carboxamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (C1) as a lead candidate for this class. This compound together with structurally similar analogues demonstrated excellent 'boosting' properties when tested in dogs. These findings warrant further evaluation of their properties in an effort to identify valuable alternatives to Ritonavir as pharmacokinetic enhancers.

Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis.

The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G-Apobec3G and the Apobec3G-Vif interactions, the mammalian protein-protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G-Apobec3G interaction. All mutations that inhibit Apobec3G-Apobec3G binding also inhibit the Apobec3G-Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G-Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer.

Resistance mutations in human immunodeficiency virus type 1 integrase selected with elvitegravir confer reduced susceptibility to a wide range of integrase inhibitors.

Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.

Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against "nucleotide-competing reverse transcriptase inhibitors".

Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). Although the structure of these compounds differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the polymerase active site. NRTI-associated mutations that are clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1. Here we have studied the underlying biochemical mechanism. RT enzymes containing the isolated mutations M184V and Y115F cause 2-3-fold increases in IC(50) values, while the combination of the two mutations causes a >15-fold increase. K65R can partially counteract these effects. Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of the enzyme to discriminate against the inhibitor. Studies with other strategic mutations at residues Phe-61 and Ala-62, as well as the use of chemically modified templates shed further light on the putative binding site of the inhibitor and ternary complex formation. An abasic site residue at position n, i.e. opposite the 3'-end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect. Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleoside triphosphates (dNTPs). We therefore propose to refer to members of this class of compounds as "nucleotide-competing RT inhibitors" (NcRTIs).

MAPPIT (MAmmalian Protein-Protein Interaction Trap) as a tool to study HIV reverse transcriptase dimerization in intact human cells.

The high mutation rate of Human Immunodeficiency Virus (HIV) leads to the rapid derivation of compound-resistant virus strains and thus necessitates the identification and development of compounds with alternative mode of actions. MAPPIT (MAmmalian Protein-Protein Interaction Trap) is a highly efficient tool to study protein-protein interactions in intact human cells and is applied to study the dimerization process of the HIV reverse transcriptase complex. Highly specific signals for the p66/p51 and p66/p66 interactions could readily be detected. Specificity was established further by introducing mutations in either subunit. Treatment with efavirenz resulted in an increased MAPPIT signal, with an EC50 value of 64nM for the p66/p51 interaction, and allowed detection of the p51/p51 homodimerization, confirming the context-dependent asymmetric contribution of both subunits. These results show that MAPPIT can be used as a novel screening tool for anti-HIV compounds in intact human cells.

Development of robust antiviral assays for profiling compounds against a panel of positive-strand RNA viruses using ATP/luminescence readout.

The development of antiviral assays using an ATP/luminescence-based readout to profile antiviral compounds against the positive-strand RNA viruses: yellow fever virus (YFV), West Nile virus (WNV), Sindbis virus, and Coxsackie B virus, representing three virus families, is described. This assay readout is based upon the bioluminescent measurement of ATP in metabolically active cells. Antiviral efficacy was determined by measuring the ATP level in cells that were protected from the viral cytopathic effect (CPE) by the presence of antiviral compounds. The antiviral assay parameters were optimized and the assays were validated using a panel of different reference compounds to determine the intra- and inter-assay reproducibility. The signal-to-noise ratios for the yellow fever virus and West Nile virus assays were 7.5 and 36, respectively, comparing favorably with a signal-to-noise ratio of only 1.5 in the yellow fever virus neutral red dye uptake assay, an alternative readout for CPE inhibition. For Coxsackie B and Sindbis viruses, the signal-to-noise ratios were 40 and 50, respectively. These assays are robust, high-throughput, reproducible, and give much improved signal-to-noise ratios than those of dye uptake assays.

Binding kinetics of darunavir to human immunodeficiency virus type 1 protease explain the potent antiviral activity and high genetic barrier.

The high incidence of cross-resistance between human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) limits their sequential use. This necessitates the development of PIs with a high genetic barrier and a broad spectrum of activity against PI-resistant HIV, such as tipranavir and darunavir (TMC114). We performed a surface plasmon resonance-based kinetic study to investigate the impact of PI resistance-associated mutations on the protease binding of five PIs used clinically: amprenavir, atazanavir, darunavir, lopinavir, and tipranavir. With wild-type protease, the binding affinity of darunavir was more than 100-fold higher than with the other PIs, due to a very slow dissociation rate. Consequently, the dissociative half-life of darunavir was much higher (>240 h) than that of the other PIs, including darunavir's structural analogue amprenavir. The influence of protease mutations on the binding kinetics was tested with five multidrug-resistant (MDR) proteases derived from clinical isolates harboring 10 to 14 PI resistance-associated mutations with a decreased susceptibility to various PIs. In general, all PIs bound to the MDR proteases with lower binding affinities, caused mainly by a faster dissociation rate. For amprenavir, atazanavir, lopinavir, and tipranavir, the decrease in affinity with MDR proteases resulted in reduced antiviral activity. For darunavir, however, a nearly 1,000-fold decrease in binding affinity did not translate into a weaker antiviral activity; a further decrease in affinity was required for the reduced antiviral effect. These observations provide a mechanistic explanation for darunavir's potent antiviral activity and high genetic barrier to the development of resistance.

Binding kinetics, uptake and intracellular accumulation of F105, an anti-gp120 human IgG1kappa monoclonal antibody, in HIV-1 infected cells.

The use of targeting moieties is a new and exciting field of scientific research for facilitating the specific delivery of therapeutic agents in HIV-infected patients. The interaction of a potential targeting moiety with its ligand is a crucial factor in the evaluation of a targeted approach for chemotherapeutic intervention. Therefore, we have further characterized the interaction between a potential targeting agent, the monoclonal human antibody F105, and its ligand gp120, a glycoprotein expressed on the surface of HIV-1 infected cells. We demonstrate the specificity of binding and entry of F105 to infected cells. F105 was rapidly taken up into the cell and accumulated in the Golgi apparatus. Kinetic analysis of the F105-gp120 interaction revealed an equilibrium dissociation constant (K(D)) of 0.62 nM, compared with the gp120-CD4 interaction where the K(D) was determined at 35 nM. Consequently, F105 displayed a higher gp120 affinity. This was due to a slower dissociation as compared with the natural ligand. These data further underline the potential of monoclonal antibodies as targeting agents, and offer new insights into the possibility of F105 as a targeting moiety for the delivery of antiretroviral drugs to HIV-1 infected cells.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells.

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Stimulated trans-acting factor of 50 kDa (Staf50) inhibits HIV-1 replication in human monocyte-derived macrophages.

In order to identify cellular genes which interfere with HIV-1 replication in monocyte-derived macrophages (MAC), cells were stimulated with interferon (IFN) or lipopolysaccharide (LPS) leading to a pronounced inhibition of HIV-1 infection in these cells, and the resulting gene expression was analyzed. Using the microarray technology we identified a gene named Stimulated Trans-Acting Factor of 50 kDa (Staf50), which is known to repress the activity of the HIV-1 LTR. Analysis of the Staf50 expression by real-time PCR showed an overexpression in IFNalpha (up to 20-fold) and LPS (up to 10-fold)-stimulated MAC as well as in infected cells (up to 3-fold). For stable overexpression, 293 T cells and primary macrophages were transduced with Staf50-IRES-GFP bicistronic pseudotype viruses. After transduction, 293 T CD4/CCR5 and MAC were infected with HIV-1, and virus replication was monitored by p24 ELISA. Overexpression of Staf50 inhibited the HIV-1 infection between 50% and 90% in 293 T CD4/CCR5 as well as in MAC. Our findings suggest that host genetic effects in combination with viral properties determine the susceptibility of an appropriate target cell for HIV-1 infection as well as the replication potential of the virus in the cell resulting in an overall productive infection.

The non-nucleoside antiviral, BAY 38-4766, protects against cytomegalovirus (CMV) disease and mortality in immunocompromised guinea pigs.

New antiviral drugs are needed for the treatment of cytomegalovirus (CMV) infections, particularly in immunocompromised patients. These studies evaluated the in vitro and in vivo activity of the non-nucleosidic CMV inhibitor, BAY 38-4766, against guinea pig cytomegalovirus (GPCMV). Plaque reduction assays indicated that BAY 38-4766 was active against GPCMV, with an IC(50) of 0.5muM. Yield reduction assays demonstrated an ED(90) and ED(99) of 0.4 and 0.6muM, respectively, of BAY 38-4766 against GPCMV. Guinea pigs tolerated oral administration of 50mg/kg/day of BAY 38-4766 without evidence of biochemical or hematologic toxicity. Plasma concentrations of BAY 38-4766 were high following oral dosing, with a mean peak level at 1-h post-dose of 26.7mg/ml (n=6; range, 17.8-35.4). Treatment with BAY 38-4766 reduced both viremia and DNAemia, as determined by a real-time PCR assay, following GPCMV infection of cyclophosphamide-immunosuppressed strain 2 guinea pigs (p<0.05, Mann-Whitney test). BAY 38-4766 also reduced mortality following lethal GPCMV challenge in immunosuppressed Hartley guinea pigs, from 83% (20/24) in placebo-treated guinea pigs, to 17% (4/24) in BAY 38-4766-treated animals (p<0.0001, Fisher's exact test). Mortality differences were accompanied by reduction in DNAemia in Hartley guinea pigs. Based upon its favorable safety, pharmacokinetic, and therapeutic profiles, BAY 38-4766 warrants further investigation in the GPCMV model.

Antivirals against DNA viruses (hepatitis B and the herpes viruses).

Antiviral drugs against DNA viruses are widely used for the management of diseases caused by infections with the Herpes viruses and have recently been introduced for Hepatitis B. There are also several emerging treatments (i.e. those that are in clinical development) and novel treatments that are still in the preclinical phase. Although the majority of emerging drugs are nucleoside analogues, there is a trend towards the development of non-nucleosidic drugs with unique mechanisms of action, in the hope that efficacy will be maximised and drug resistance and viral rebound minimised.