Louise Westbrook

Thiazolobenzimidazole: Biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

Antimycobacterial Activities of 2,4-Diamino-5-Deazapteridine Derivatives and Effects on Mycobacterial Dihydrofolate Reductase

ABSTRACT Development of new antimycobacterial agents for Mycobacterium avium complex (MAC) infections is important particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were to evaluate the in vitro activity of 2,4-diamino-5-methyl-5-deazapteridines (DMDPs) against MAC and to assess their activities against MAC dihydrofolate reductase recombinant enzyme (rDHFR). Seventy-seven DMDP derivatives were evaluated initially for in vitro activity against one to three strains of MAC (NJ168, NJ211, and/or NJ3404). MICs were determined with 10-fold dilutions of drug and a colorimetric (Alamar Blue) microdilution broth assay. MAC rDHFR 50% inhibitory concentrations versus those of human rDHFR were also determined. Substitutions at position 5 of the pteridine moiety included -CH3, -CH2CH3, and -CH2OCH3 groups. Additionally, different substituted and unsubstituted aryl groups were linked at position 6 through a two-atom bridge of either -CH2NH, -CH2N(CH3), -CH2CH2, or -CH2S. All but 4 of the 77 derivatives were active against MAC NJ168 at concentrations of ≤13 μg/ml. Depending on the MAC strain used, 81 to 87% had MICs of ≤1.3 μg/ml. Twenty-one derivatives were >100-fold more active against MAC rDHFR than against human rDHFR. In general, selectivity was dependent on the composition of the two-atom bridge at position 6 and the attached aryl group with substitutions at the 2′ and 5′ positions on the phenyl ring. Using this assessment, a rational synthetic approach was implemented that resulted in a DMDP derivative that had significant intracellular activity against a MAC-infected Mono Mac 6 monocytic cell line. These results demonstrate that it is possible to synthesize pteridine derivatives that have selective activity against MAC.

Vascular endothelial growth factor reduces tamoxifen efficacy and promotes metastatic colonization and desmoplasia in breast tumors.

Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A(165) in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors.

Prolonged Extracellular Signal-Regulated Kinase 1/2 Activation during Fibroblast Growth Factor 1- or Heregulin β1-Induced Antiestrogen-Resistant Growth of Breast Cancer Cells Is Resistant to Mitogen-Activated Protein/Extracellular Regulated Kinase Kinase Inhibitors

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin β1 (HRGβ1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGβ1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGβ1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGβ1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGβ1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGβ1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.

c-Abl-independent p73 stabilization during gemcitabine- or 4′-thio-β-d-arabinofuranosylcytosine–induced apoptosis in wild-type and p53-null colorectal cancer cells

Nucleoside anticancer drugs like gemcitabine (2′-deoxy-2′,2′-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4′-thio-β-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53−/− HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53−/− and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis. [Mol Cancer Ther 2006;5(2):400–10]

Antiviral activity of 9-beta-D-arabinofuranosyladenine (ara-A) against Gross murine leukemia virus in vitro.

Summary Ara-A, an antiviral agent with broad-spectrum activity against DNA viruses, was observed to inhibit Gross murine leukemia virus (an RNA tumor virus) in cell culture. Its activity was selective and was observed at concentrations well below cytotoxic levels.

Plant-derived and semi-synthetic calanolide compounds with in vitro activity against both human immunodeficiency virus type 1 and human cytomegalovirus.

Plant-derived and semi-synthetic calanolide compounds with anti-human immunodeficiency virus type 1 (HIV-1) activity were tested for anti-human cytomegalovirus (HCMV) activity in both cytopathic effect inhibition and plaque reduction assays. The results indicated that the anti-HCMV activity of calanolide compounds does not correlate with their activity against HIV-1. The semi-synthetic 12-keto derivatives tended to be more active against HCMV than the corresponding 12-OH congeners, which were more active against HIV-1. It appeared that the 7,8-unsaturated double bond in the chromene ring played a certain role in maintaining activities against both HCMV and HIV-1. Saturation of the double bond increased the EC50 values against both viruses, with concomitant increase in toxicity. The calanolide compounds reported here are the first non-nucleoside analogues capable of inhibiting both HIV-1 and HCMV and, therefore, may be useful chemoprophylactic agents for HCMV in HIV-infected people or vice versa.

An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus

Abstract A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Biological activities and modes of action of 9-α-d-arabinofuranosyladenine and 9-α-d-arabinofuranosyl-8-azaadenine

Abstract From earlier studies it is known that 9-α- d -arabinofuranosyladenine (α-araA) and 9-α- d -arabinofuranosyl-8-azaadenine (α-ara-8-azaA) bave in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in vivo antiviral activities of these compounds and their loci of action with those of 9-β- d -arabinofura-nosyladenine (β-araA). the latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas β-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, α-araA and α-ara-8-azaA were therapeutically ineffective. α-AraATP at a concentration of ~1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-α from mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerase-β from mouse embryo cells was inhibited to a small extent by 10−4 M α-araATP. In contrast, all of these enzymes were inhibited by β-araATP at a concentration of 10−5M (as shown in these or in earlier studies). the reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by αaraATP (~10−3M), whereas β-araATP produced 70–80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, α-ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. α-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of [14C] formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and [14C] formate. These results indicate that the α-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of β-araA.

Inhibition of Gross Murine Leukemia Virus Replication by Rifamycin SV and Certain of Its Derivatives in vitro

Rifamycin SV and several of its derivatives were examined for their ability to inhibit the replication of Gross murine leukemia virus in intact Swiss mouse embryo cells growing in culture. Antiviral activity was observed with rifamycin SV, 3-formyl rifamycin SV: dipropylhydrazone and benzyl dimethyl rifampicin, when the compounds were tested against this RNA tumor virus by means of the UV-XC plaque-reduction procedure. However, several of the rifamycin derivatives which strongly inhibited the viral RNA-directed DNA polymerase reaction in cell-free systems failed to inhibit virus replication in intact cells at noncytotoxic concentrations.