Novel phorbol esters exert dichotomous effects on inhibition of HIV-1 infection and activation of latent HIV-1 expression

Two new phorbol esters, NPB-11 (12-O-methoxymethylphorbol-13-decanoate) and NPB-15 (12-O-benzyloxymethylphorbol-13-decanoate) were synthesized. The compounds exhibited potent anti-HIV-1 activity and low cytotoxicity in MT-4 cells by MTT assay even at a high concentration [50% cytotoxic concentrations (CC50) were 8.32 and 4.39 microg/ml, respectively]. Two inhibitors strongly suppressed HIV-1 (IIIB strain) replication in MT-4 cells with a 50% effective concentration (EC50) of 1.3 and 0.27 ng/ml, respectively. NPB-11 efficiently blocked replication of both X4 and R5 HIV-1 in PHA-activated peripheral blood mononuclear cells and MT-4 cells as revealed by p24 assay. The antiviral activity appeared to be mediated, at least partially, by the down-regulation of the expression of CD4 and the HIV-1 co-receptors, CXCR4 and CCR5. The compounds were also capable of selectively up-regulating HIV-1 expression in a variety of latently infected cell lines and inducing cell death in HIV-1 infected cells. The effect of NPBs on the induction of HIV-1 was specifically blocked by nontoxic doses of a protein kinase C blocker, staurosporine. NPB-11 blocked the spread of HIV-1 released from latently infected ACH-2 cells to MT-4 cells in a co-culture system. When combined with AZT, NPB-11 synergistically inhibited HIV-1 replication in MTT assay using MT-4 cells. These data suggest that these agents might be useful in reducing persistent viral reservoirs in patients and as adjuvant therapy in patients treated with HAART.     

Drug efflux by breast cancer resistance protein is a mechanism of resistance to the benzimidazole insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924

Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism.     

In vitro inhibition of human immunodeficiency virus (HIV) type 1 replication by C2 symmetry-based HIV protease inhibitors as single agents or in combinations.

C2 symmetry-based human immunodeficiency virus (HIV) protease inhibitors were examined in vitro as single agents or in combination with 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine for activity against HIV type 1 (HIV-1). Ten C2 symmetry-based or pseudo-C2 symmetry-based HIV protease inhibitors were active against a laboratory strain (HIV-1IIIB) in the HIV-1 cytopathic effect inhibition assay. Three inhibitors, A75925, A76928, and A77003, selected to represent a range of aqueous solubility and antiviral activity, were active against four different HIV-1 strains tested. These three inhibitors exhibited a significant inhibition of the cytopathic effect of HIV-1 against the CD4+ ATH8 cell line, with 90% inhibitory concentrations ranging from 0.1 to 4 microM. Cellular toxicity was negligible at up to 20 microM. Furthermore, they completely inhibited the replication of monocytotropic strain HIV-1Ba-L in purified monocytes and macrophages at 0.75 to 2 microM. Potent inhibitory activity against a primary HIV-1 isolate and an AZT-resistant HIV-1 variant was also observed for all three inhibitors in phytohemagglutinin-activated peripheral blood mononuclear cells. When these three HIV protease inhibitors and AZT or 2',3'-dideoxyinosine were used in combinations against a primary HIV isolate in phytohemagglutinin-activated peripheral blood mononuclear cells and the results were analyzed with the COMBO program package, their antiviral activities were identified to be synergistic in some cases and additive in others. The present data warrant further investigations of these compounds as potential antiviral agents for the therapy of HIV infections.