Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs.

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.

Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations.

Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy.

Immune escape is a crucial feature of cancer progression about which little is known. Elevation of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) in tumor cells can facilitate immune escape. Not known is how IDO becomes elevated or whether IDO inhibitors will be useful for cancer treatment. Here we show that IDO is under genetic control of Bin1, which is attenuated in many human malignancies. Mouse knockout studies indicate that Bin1 loss elevates the STAT1- and NF-kappaB-dependent expression of IDO, driving escape of oncogenically transformed cells from T cell-dependent antitumor immunity. In MMTV-Neu mice, an established breast cancer model, we show that small-molecule inhibitors of IDO cooperate with cytotoxic agents to elicit regression of established tumors refractory to single-agent therapy. Our findings suggest that Bin1 loss promotes immune escape in cancer by deregulating IDO and that IDO inhibitors may improve responses to cancer chemotherapy.

Targeted deletion of the suppressor gene bin1/amphiphysin2 accentuates the neoplastic character of transformed mouse fibroblasts.

The Bin1/Amphiphysin2 gene encodes several alternately spliced BAR adapter proteins that have been implicated in membrane-associated and nuclear processes. Bin1 expression is often attenuated during tumor progression and Bin1 splice isoforms that localize to the nucleus display tumor suppressor properties. While these properties may reflect the ability of these isoforms to interact with and suppress the cell transforming activity of c-Myc, the effects of Bin1 deletion on the oncogenicity of c-myc or other transforming genes has not been gauged directly. Here we report that targeted deletion of Bin1 enhances the neoplastic character of primary murine embryo fibroblasts (MEFs) cotransformed by c-myc and mutant grasg. Specifically, Bin1 loss accentuated the spindle morphology of transformed cells, increased anchorage-independent proliferation, and promoted tumor formation in syngeneic hosts. These effects were specific as they were not recapitulated in cells transformed by viral oncoproteins and mutant ras. Although some Bin1 splice isoforms associate with endocytotic complexes the effects of Bin1 loss were not correlated with a generalized defect in receptor-mediated endocytosis. However, Bin1 loss increased sensitivity to paclitaxel, a drug that can affect endocytotic trafficking by disrupting microtubule dynamics. In E1A?transformed MEFs, Bin1 loss reduced the susceptibility to apoptosis triggered by tumor necrosis factor-alpha, an effect that was associated with precocious nuclear trafficking of NF-kappaB. These findings offer a novel line of support for the hypothesized role of Bin1 in limiting malignant growth, possibly as a negative modifier or anti-progression gene.

Cyclin B1 is a critical target of RhoB in the cell suicide program triggered by farnesyl transferase inhibition.

Farnesyl transferase inhibitors (FTIs) have displayed limited efficacy in clinical trials, possibly because of their relatively limited cytotoxic effects against most human cancer cells. Therefore, efforts to leverage the clinical utility of FTIs may benefit from learning how these agents elicit p53-independent apoptosis in mouse models of cancer. Knockout mouse studies have established that gain of the geranylgeranylated isoform of the small GTPase RhoB is essential for FTI to trigger apoptosis. Here we demonstrate that Cyclin B1 is a crucial target for suppression by RhoB in this death program. Steady-state levels of Cyclin B1 and its associated kinase Cdk1 were suppressed in a RhoB-dependent manner in cells fated to undergo FTI-induced apoptosis. These events were not derivative of cell cycle arrest, because they did not occur in cells fated to undergo FTI-induced growth inhibition. Mechanistic investigations indicated that RhoB mediated transcriptional suppression but also accumulation of Cyclin B1 in the cytosol at early times after FTI treatment, at a time before the subsequent reduction in steady-state protein levels. Enforcing Cyclin B1 expression attenuated apoptosis but not growth inhibition triggered by FTI. Moreover, enforcing Cyclin B1 abolished FTI antitumor activity in graft assays. These findings suggest that Cyclin B1 suppression is a critical step in the mechanism by which FTI triggers apoptosis and robust antitumor efficacy. Our findings suggest that Cyclin B1 suppression may predict favorable clinical responses to FTI, based on cytotoxic susceptibility, and they suggest a rational strategy to address FTI nonresponders by coinhibition of Cdk1 activity.

Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation.

The mammalian Bin1/Amphiphysin II gene encodes an assortment of alternatively spliced adapter proteins that exhibit markedly divergent expression and subcellular localization profiles. Bin1 proteins have been implicated in a variety of different cellular processes, including endocytosis, actin cytoskeletal organization, transcription, and stress responses. To gain insight into the physiological functions of the Bin1 gene, we have disrupted it by homologous recombination in the mouse. Bin1 loss had no discernible impact on either endocytosis or phagocytosis in mouse embryo-derived fibroblasts and macrophages, respectively. Similarly, actin cytoskeletal organization, proliferation, and apoptosis in embryo fibroblasts were all unaffected by Bin1 loss. In vivo, however, Bin1 loss resulted in perinatal lethality. Bin1 has been reported to affect muscle cell differentiation and T-tubule formation. No striking histological abnormalities were evident in skeletal muscle of Bin1 null embryos, but severe ventricular cardiomyopathy was observed in these embryos. Ultrastructurally, myofibrils in ventricular cardiomyocytes of Bin1 null embryos were severely disorganized. These results define a developmentally critical role for the Bin1 gene in cardiac muscle development.

hob1+, the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress.

BAR (Bin/Amphiphysin/Rvs) adapter proteins have been suggested to regulate endocytosis, actin organization, apoptosis, and transcription, but their precise roles are obscure. There are at least five mammalian genes that encode BAR adapter proteins, including the evolutionarily conserved and ubiquitously expressed Bin1/Amphiphysin-II and Bin3 genes. Bin1 holds special interest as certain splice isoforms localize to the nucleus, interact with the c-Abl and c-Myc oncoproteins, and display tumor suppressor properties. To obtain functional insights, we embarked upon a genetic analysis of the two BAR adapter proteins expressed in the fission yeast Schizosaccharomyces pombe. In a previous work, a role in actin organization and cytokinesis was identified for the Bin3 homolog hob3+. In this study, a role in stress signaling was defined for the Bin1 homolog, hob1+. Notably, hob1+ was dispensable for endocytosis, actin organization, or osmotic sensitivity. Instead, mutation of hob1+ led to slight cell elongation and faulty cell cycle arrest upon nutrient starvation. These defects were complemented by Bin1, but not by Amphiphysin-I, arguing that these genes have distinct functions despite their structural similarity. hob1 delta mutant cells were also hypersensitive to genotoxic stress. This was not related to a faulty checkpoint response, but mutation in the checkpoint gene rad3(+) further exacerbated the sensitivity of hob1 delta mutant cells. Interestingly, mutation of the cell cycle regulator wee1+ partially relieved the sensitivity defect, suggesting that hob1+ may influence the efficiency of DNA repair or checkpoint release after DNA damage. Genetic and biochemical evidence indicated that hob3+ is epistatic to hob1+ in the response to genotoxic stress. Our findings indicate that the Bin1 homolog hob1+ participates in DNA damage signaling and they suggest a novel role for BAR adapter proteins in stress response processes.