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Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration–approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections.

Programmed cell death (PCD) during neuronal development and disease has been shown to require de novo RNA synthesis. However, the time course and regulation of target genes is poorly understood. By using a brain-biased array of over 7,500 cDNAs, we profiled this gene expression component of PCD in cerebellar granule neurons challenged separately by potassium withdrawal, combined potassium and serum withdrawal, and kainic acid administration. We found that hundreds of genes were significantly regulated in discreet waves including known genes whose protein products are involved in PCD. A restricted set of genes was regulated by all models, providing evidence that signals inducing PCD can regulate large assemblages of genes (of which a restricted subset may be shared in multiple pathways).

SREBP-2, oxidosqualene cyclase (OSC) or lanosterol demethylase were identified as novel sterol pathway-associated targets that, when probed with chemical agents, can inhibit hepatitis C virus (HCV) replication. Using a combination chemical genetics approach, combinations of chemicals targeting sterol pathway enzymes downstream of and including OSC or protein geranylgeranyl transferase I (PGGT) produce robust and selective synergistic inhibition of HCV replication. Inhibition of enzymes upstream of OSC elicit proviral responses that are dominant to the effects of inhibiting all downstream targets. Inhibition of the sterol pathway without inhibition of regulatory feedback mechanisms ultimately results in an increase in HCV replication because of a compensatory upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression. Increases in HMGCR expression without inhibition of HMGCR enzymatic activity ultimately stimulate HCV replication through increasing the cellular pool of geranylgeranyl pyrophosphate (GGPP). Chemical inhibitors that ultimately prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced., Chemical inhibition of enzymes in either the cholesterol or the fatty acid biosynthetic pathways has been shown to impact viral replication, both positively and negatively (Su et al, 2002; Ye et al, 2003; Kapadia and Chisari, 2005; Sagan et al, 2006; Amemiya et al, 2008). FBL2 has been identified as a 50 kDa geranylgeranylated host protein that is necessary for localization of the hepatitis C virus (HCV) replication complex to the membranous web through its close association with the HCV protein NS5A and is critical for HCV replication (Wang et al, 2005). Inhibition of the protein geranylgeranyl transferase I (PGGT), an enzyme that transfers geranylgeranyl pyrophosphate (GGPP) to cellular proteins such as FBL2 for the purpose of membrane anchoring, negatively impacts HCV replication (Ye et al, 2003). Conversely, chemical agents that increase intracellular GGPP concentrations promote viral replication (Kapadia and Chisari, 2005). Statin compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the sterol pathway (Goldstein and Brown, 1990), have been suggested to inhibit HCV replication through ultimately reducing the cellular pool of GGPP (Ye et al, 2003; Kapadia and Chisari, 2005; Ikeda et al, 2006). However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. The use of statins for the treatment of HCV is likely to be complicated by the reported compensatory increase in HMGCR expression in vitro and in vivo (Stone et al, 1989; Cohen et al, 1993) in response to treatment. Enzymes in the sterol pathway are regulated on a transcriptional level by sterol regulatory element-binding proteins (SREBPs), specifically SREBP-2 (Hua et al, 1993; Brown and Goldstein, 1997). When cholesterol stores in cells are depleted, SREBP-2 activates transcription of genes in the sterol pathway such as HMGCR, HMG-CoA synthase, farnesyl pyrophosphate (FPP) synthase, squalene synthase (SQLS) and the LDL receptor (Smith et al, 1988, 1990; Sakai et al, 1996; Brown and Goldstein, 1999; Horton et al, 2002). The requirement of additional downstream sterol pathway metabolites for HCV replication has not been completely elucidated. To further understand the impact of the sterol pathway and its regulation on HCV replication, we conducted a high-throughput combination chemical genetic screen using 16 chemical probes that are known to modulate the activity of target enzymes relating to the sterol biosynthesis pathway (Figure 1). Using this approach, we identified several novel antiviral targets including SREBP-2 as well as targets downstream of HMGCR in the sterol pathway such as oxidosqualene cyclase (OSC) and lanosterol demethylase. Many of our chemical probes, specifically SR-12813, farnesol and squalestatin, strongly promoted replicon replication. The actions of both farnesol and squalestatin ultimately result in an increase in the cellular pool of GGPP, which is known to increase HCV replication (Ye et al, 2003; Kapadia and Chisari, 2005; Wang et al, 2005). Chemical combinations targeting enzymes upstream of squalene epoxidase (SQLE) at the top of the sterol pathway (Figure 4A) elicited Bateson-type epistatic responses (Boone et al, 2007), where the upstream agent's response predominates over the effects of inhibiting all downstream targets. This was especially notable for combinations including simvastatin and either U18666A or squalestatin, and for squalestatin in combination with Ro48-8071. Treatment with squalestatin prevents the SQLS substrate, farnesyl pyrophosphate (FPP) from being further metabolized by the sterol pathway. As FPP concentrations increase, the metabolite can be shunted away from the sterol pathway toward farnesylation and GGPP synthetic pathways, resulting in an increase in host protein geranylgeranylation, including FBL2, and consequently replicon replication. This increase in replicon replication explains the source of the observed epistasis over Ro48-8071 treatment. Combinations between probes targeting enzymes downstream of and including OSC produced robust synergies with each other or with a PGGT inhibitor. Figure 4B highlights examples of antiviral synergy resulting from treatment of cells with an OSC inhibitor in combination with an inhibitor of either an enzyme upstream or downstream of OSC. A combination of terconazole and U18666A is synergistic without similar combination effects in the host proliferation screen. Likewise, clomiphene was also synergistic when added to replicon cells in combination with U18666A. One of the greatest synergies observed downstream in the sterol pathway is a combination of amorolfine and AY 9944, suggesting that there is value in developing combinations of drugs that target enzymes in the sterol pathway, which are downstream of HMGCR. Interactions with the protein prenylation pathway also showed strong mechanistic patterns (Figure 4C). GGTI-286 is a peptidomimetic compound resembling the CAAX domain of a protein to be geranylgeranylated and is a competitive inhibitor of protein geranylgeranylation. Simvastatin impedes the antiviral effect of GGTI-286 at low concentrations but that antagonism is balanced by comparable synergy at higher concentrations. At the low simvastatin concentrations, a compensatory increase in HMGCR expression leads to increased cellular levels of GGPP, which are likely to result in an increase in PGGT enzymatic turnover and decreased GGTI-286 efficacy. The antiviral synergy observed at the higher inhibitor concentrations is likely nonspecific as synergy was also observed in a host viability assay. Further downstream, however, a competitive interaction was observed between GGTI-286 and squalestatin, where the opposing effect of one compound obscures the other compound's effect. This competitive relationship between GGTI and SQLE explains the epistatic response observed between those two agents. For inhibitors of targets downstream of OSC, such as amorolfine, there are strong antiviral synergies with GGTI-286. Notably, combinations with OSC inhibitors and GGTI-286 were selective, in that comparable synergy was not found in a host viability assay. This selectivity suggests that jointly targeting OSC and PGGT is a promising avenue for future HCV therapy development. This study provides a comprehensive and unique perspective into the impact of sterol pathway regulation on HCV replication and provides compelling insight into the use of chemical combinations to maximize antiviral effects while minimizing proviral consequences. Our results suggest that HCV therapeutics developed against sterol pathway targets must consider the impact on underlying sterol pathway regulation. We found combinations of inhibitors of the lower part of the sterol pathway that are effective and synergistic with each other when tested in combination. Furthermore, the combination effects observed with simvastatin suggest that, though statins inhibit HMGCR activity, the resulting regulatory consequences of such inhibition ultimately lead to undesirable epistatic effects. Inhibitors that prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced., The search for effective Hepatitis C antiviral therapies has recently focused on host sterol metabolism and protein prenylation pathways that indirectly affect viral replication. However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. Here, we present a combination chemical genetic study to explore how the sterol and protein prenylation pathways work together to affect hepatitis C viral replication in a replicon assay. In addition to finding novel targets affecting viral replication, our data suggest that the viral replication is strongly affected by sterol pathway regulation. There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway. We also show how pathway regulation frustrates potential hepatitis C therapies based on the sterol pathway, and reveal novel synergies that selectively inhibit hepatitis C replication over host toxicity. In particular, combinations targeting the downstream sterol pathway enzymes produced robust and selective synergistic inhibition of hepatitis C replication. Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.

8559 Background: Signaling via PI3K-δ and PI3K-γ has distinct and complementary effects on the tumor cell/ tumor microenvironment in hematologic malignancies (HM). Duvelisib (IPI-145) is an orally active inhibitor of the PI3K-δ and PI3K-γ isoforms in clinical development in HM. To gain mechanistic insights into the cellular response to duvelisib and identify novel pairings for duvelisib in HM, a high-throughput in vitro combination screen was conducted. Methods: Duvelisib was evaluated alone and in combination with 35 compounds comprising a diverse panel of standard-of-care agents and emerging drugs in development for HM. These compounds were tested in 20 cell lines including diffuse large B-cell (DLBCL), follicular, T-cell, and mantle cell lymphomas, and multiple myeloma. Growth inhibition (GI) was measured by ATPLite (Perkin Elmer) in a 6x6 or 9x9 dose combination matrix. Results: Single agent activity was seen in 14 cell lines treated with duvelisib, with a median GI50 of 0.59 µM. A scalar measure of t...

Background NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. Methods CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. Results Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH2-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. Conclusion The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein. © 2006 International Society for Analytical Cytology

The NARC 1 gene encodes a novel proteinase K family proteinase. The domain structure of rat Narc 1 resembles that of the subtilisin-like proprotein convertases (SPCs), except that rNarc 1 lacks the canonical P-domain of SPCs, retaining only the RGD motif as part of what might be a cryptically functioning P-domain. Narc 1 undergoes autocatalytic intramolecular processing at the site LVFAQ/, resulting in the cleavage of its prosegment and the generation of an active proteinase with a broad alkaline pH optimum and no apparent calcium requirement for activity. Both primary and secondary structural determinants influence Narc 1 substrate recognition. Our functional characterization of Narc 1 reinforces the inference drawn from the analysis of its predicted structure that this enzyme is most closely related to representatives of the proteinase K family, but that it is also sufficiently different to warrant its possible classification in a separate sub-family.

PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-kappa B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1 beta. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-kappa B and cytokine processing.

Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a mechanism distinct from most other antitubulin therapeutics, is clinically used in the United States for treatment of certain patients with locally advanced or metastatic breast cancer. Eribulin has broad anticancer activity in a wide variety of preclinical cancer models; as a result, numerous clinical trials are ongoing to investigate efficacy in other non-breast tumor types. We have examined eribulin sensitivity in a panel of 25 human cancer cell lines representing a variety of tumor types albeit with a preponderance of breast and lung cancer cell lines. As expected, the cell lines vary in sensitivity to eribulin at clinically relevant concentrations. To identify combination drugs capable of increasing anticancer effects in patients already responsive to eribulin, as well as inducing de novo anticancer effects in non-responders, we performed a combinatorial high throughput screen to identify drugs that combine with eribulin to selectively kill tumor cells. Among other observations, we found that inhibitors of ErbB1/ErbB2 (lapatinib, BIBW-2992, erlotinib), MEK (E6201, trametinib), PI3K (BKM-120), mTOR (AZD 8055, everolimus), and PI3K/mTOR (BEZ 235), and a BCL2 family antagonist (ABT-263) show combinatorial activity with eribulin. In addition, antagonistic pairings with other agents, such as topoisomerase I inhibitors (topotecan hydrochloride), an HSP-90 inhibitor (17-DMAG), and gemcitabine and cytarabine, were also identified. In summary, the preclinical studies described here have identified several combination drugs that have the potential to either augment or antagonize eribulin's anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be important for identifying valuable therapeutic partners for eribulin. Citation Format: Toshimitsu Uenaka, Richard Rickles, Yasuhiro Funahashi, Ping Zhu, Jill M. Grenier, Janine Steiger, Nanding Zhao, Bruce A. Littlefield, Junji Matsui, Kenichi Nomoto. Identification of drugs with eribulin combinatorial activity that kill both eribulin-sensitive and eribulin-insensitive tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1698. doi:10.1158/1538-7445.AM2014-1698

SAR3419, a novel antibody-drug conjugate (ADC), is composed of a humanized monoclonal IgG1 anti-CD19 antibody (huB4) attached by a cleavable linker to DM4, a derivative of cytotoxic agent maytansine (an inhibitor of microtubule polymerization). CD19 expression is restricted to B-cells and treatment of CD19-positive human lymphoma cells with SAR3419 can result in cell cycle arrest followed by apoptosis. As traditional monotherapies can rarely treat cancer effectively, particularly in non-solid tumours, a combination high throughput screen (cHTS) was performed where SAR3419 was combined with two hundred potential enhancer compounds across a panel of twenty cell lines using a 6x6 combination dose response matrix. The enhancer compounds compose a library of approved, emerging oncology drugs, and well-defined molecular probes. Robust synergies were observed between SAR3419 and PI3K inhibitors and examined further. Target/pathway inhibitor redundancy for the class of compounds served to validate PI3K inhibition as important for combination synergy. Surprisingly, for several of the cell lines, the molecular mechanism important for SAR3419-dependent combination activity is distinct. For example, for the Daudi and REC-1 tumor cell lines, PI3K inhibitor synergy can be attributed to the toxin DM4. In contrast for SU-DHL4 and SU-DHL6 cell lines, little combination activity is observed with toxin alone while the conjugate is strongly synergistic. By examining three-way combination activities (PI3K inhibitor x DM4 x huB4) using high resolution matrices, we show that the anti-CD19 antibody huB4 contributes to the synergy in the SU-DHL6 cell line and linkage of DM4 to huB4 (SAR3419) shifts both the potency and maximum activity for PI3K inhibitor combinations. The results demonstrate that the antibody component of an ADC can contribute to combination activity for specific drug pairings in a drug and cell-type specific manner and has wide ranging implications with respect to combination drug design. Citation Format: Richard J. Rickles, Thomas P. Giordano, Shakira F. Cotard, Jill M. Grenier, Angela Romanelli, Ti Cai. Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2014-4765

PYRIN-containing Apaf-1-like proteins (PYPAFs) are a recently identified family of proteins thought to function in apoptotic and inflammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN-CARD protein ASC and coordinate the activation of NF-kappaB and pro-caspase-1. To determine if other PYPAF family members function in pro-inflammatory signaling pathways, we screened five other PYPAF proteins (PYPAF2, PYPAF3, PYPAF4, PYPAF5 and PYPAF6) for their ability to activate NF-kappaB and pro-caspase-1. Co-expression of PYPAF5 with ASC results in a synergistic activation of NF-kappaB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T-cells, indicating a role for this protein in inflammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF-kappaB. PYPAF5 also synergistically activated caspase-1-dependent cytokine processing when co-expressed with ASC. These findings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro-inflammatory signals to the activation of NF-kappaB and pro-caspase-1.