Chemical library screen for novel inhibitors of Kaposi's sarcoma-associated herpesvirus processive DNA synthesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and certain lymphoproliferative disorders. The role of KSHV lytic replication has been implicated in the tumor pathogenesis. A highly specific molecular complex formed by the KSHV DNA polymerase (POL8) and processivity factor (PF8) is indispensable for lytic viral DNA synthesis and may serve as an excellent molecular anti-KSHV target. The majority of conventional nucleoside-based anti-herpetic DNA synthesis inhibitors require intracellular phosphorylation/activation before they can exert inhibitory activity as competitive substrates for viral DNA polymerases. Novel and more potent inhibitors of KSHV DNA synthesis may be discovered through POL8/PF8-targeted high throughput screening (HTS) of small molecule chemical libraries. We developed a microplate-based KSHV POL8/PF8-mediated DNA synthesis inhibition assay suitable for HTS and screened the NCI Diversity Set that comprised 1992 synthetic compounds. Twenty-eight compounds exhibited greater than 50% inhibition. The inhibitory activity was confirmed for 25 of the 26 hit compounds available for further testing, with the 50% inhibitory concentrations ranging from 0.12+/-0.07 microM (mean+/-S.D.) to 10.83+/-4.19 microM. Eighteen of the confirmed active compounds efficiently blocked KSHV processive DNA synthesis in vitro. One of the hit compounds, NSC 373989, a pyrimidoquinoline analog, was shown to dose-dependently reduce the levels of KSHV virion production and KSHV DNA in lytically induced KSHV-infected BCBL-1 cells, suggesting that the compound blocked lytic KSHV DNA synthesis. HTS for KSHV POL8/PF8 inhibitors is feasible and may lead to discovery of novel non-nucleoside KSHV DNA synthesis inhibitors.

Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2'-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean +/- standard deviation: 0.08 +/- 0.03, 0.42 +/- 0.07, and 0.96 +/- 0.49 microM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5'-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 +/- 0.08 microM (mean +/- standard deviation) compared to CDV-diphosphate (14.70 +/-2.47 microM) or GCV-TP (24.59 +/- 5.60 microM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4.

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.

Synthesis and biological properties of amino acid amide ligand-based pyridinioalkanoyl thioesters as anti-HIV agents.

Hyper-mutable retroviruses such as HIV can become rapidly resistant to drugs used to treat infection. Strategies for coping with drug-resistant strains of virus include combination therapies, using viral protease and reverse transcriptase inhibitors. Another approach is the development of antiviral agents that attack mutationally nonpermissive targets that have functions essential for viral replication. Thus, the highly conserved nucleocapsid protein, NCp7, was chosen as a prime target in our search for novel anti-HIV agents that can overcome the problem of viral drug resistance. Recently, we reported (J. Med. Chem. 1999, 42, 67) a novel chemotype, the pyridinioalkanoyl thioesters (PATEs), based on 2-mercaptobenzamides as the thiol component and having its amide nitrogen substituted with various phenylsulfonyl moieties. These compounds were identified as relatively nontoxic anti-HIV agents in the XTT cytoprotection assay. In this study, we wish to report a separate genre of active PATEs wherein the thiol component consists of an N-2-mercaptobenzoyl-amino acid derivative. Active derivatives (EC(50) < 10 microM) reported herein were confined to amino acid primary amides or methyl amides having side chains no larger than isobutyl. Amino acids terminating in free carboxyl or carboxylic acid ester groups were mostly inactive. Selected compounds were shown to be active on chronically infected CEM/SK-1, TNFalpha-induced U1, ACH-2 cells and virucidal on cell-free virus, latently infected U1 cells and acutely infected primary peripheral blood mononuclear cells (PBMCs).