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The preclinical antitumor agent RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan, NSC 652287), an analog of the natural product α-terthiophene, failed during the development phase due to acute pulmonary toxicity in animal models. A series of synthetic modifications to RITA's heterocyclic scaffold resulted in activity ranging from broadly cytotoxic to highly selective. In the NCI 60-cell line screen, these “hyperselective” agents (e.g., imatinib) are rare. A selectivity index (SI) was developed to quantify this desirable feature, which is 20 for imatinib, whereas RITA's SI is only 0.10. One of the described hyperselective RITA analogs (SI = 7.9) completely lost activity in the presence of a known SULT1A1 inhibitor. These results, coupled with previous evidence that RITA is a SULT1A1 substrate, suggest that carbinol modification by a sulfate leaving group and subsequent formation of a reactive carbocation may explain RITA's broad cytotoxicity. Although SULT1A1 expression is required for susceptibility, hyperselective analogs exhibited reduced association of activity with SULT1A1 mRNA expression compared with RITA, apparently requiring some additional target(s). In support of this hypothesis, there is a strong correlation (P < 0.01, r = 0.95) between quantum mechanically calculated energy barriers for carbocation formation from sulfonated analogs and SI, indicating that hyperselective RITA analogs generate reactive carbocations less readily after sulfate activation. Importantly, narrowing the cytotoxicity profile of RITA did not eliminate its analogs' in vivo antitumor activity, as several new hyperselective agents, NSC 773097 (1), 773392 (2), and 782846 (6), displayed impressive activity against A498 xenografts in mice.

Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins’ proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin’s enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.

An assessment of the total protein composition of filovirus (ebolavirus and marburgvirus) virions is currently lacking. In this study, liquid chromatography-linked tandem mass spectrometry of purified ebola and marburg virions was performed to identify associated cellular proteins. Host proteins involved in cell adhesion, cytoskeleton, cell signaling, intracellular trafficking, membrane organization, and chaperones were identified. Significant overlap exists between this data set and proteomic studies of disparate viruses, including HIV-1 and influenza A, generated in multiple cell types. However, the great majority of proteins identified here have not been previously described to be incorporated within filovirus particles. Host proteins identified by liquid chromatography-linked tandem mass spectrometry could lack biological relevance because they represent protein contaminants in the virus preparation, or because they are incorporated within virions by chance. These issues were addressed using siRNA library-mediated gene knockdown (targeting each identified virion-associated host protein), followed by filovirus infection. Knockdown of several host proteins (e.g. HSPA5 and RPL18) significantly interfered with ebolavirus and marburgvirus infection, suggesting specific and relevant virion incorporation. Notably, select siRNAs inhibited ebolavirus, but enhanced marburgvirus infection, suggesting important differences between the two viruses. The proteomic analysis presented here contributes to a greater understanding of filovirus biology and potentially identifies host factors that can be targeted for antiviral drug development.

Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

The vast majority of cancer treatments currently administered to patients consist of combinations of more than one drug via routine infusions that adhere to specific dosing schedules. It is thought that this multi-arm and time dependent approach will kill not only the tumor cells within the primary site, but also any metastatic lesions, and importantly, any circulating tumor cells (CTCs) which may still exist in the blood. Combination therapies have also been developed as a means to reduce general cytotoxic side effects and prevent resistance and recurrence. Our labs have recently developed a high throughput screening platform to test compounds in pair-wise combinations to rapidly and systematically identify additive, synergistic and antagonistic drug combinations. This HTS capability can easily generate hundreds of dose response matrices in a single study and can increase significantly when applied to multiple cell lines. We are using this combination screening platform with in vitro models from both established cell lines and primary patient material, and we expect it will serve as a very valuable tool and a starting point when designing clinical trials after these combinations show promise within in vivo models. In a proof of concept study, we tested combinations of compounds that effectively kill 2 established lines of the ABC sub-type of diffuse large B-cell lymphoma (DLBCL); TMD8 and HBL1. We will present the infrastructure and methods that we have developed to implement the combination screens, visualize data from the combination dose response comparisons and numerically compare combinations in terms of their response matrices. We will also describe how this approach allows us to investigate putative polypharmacological effects that play a role in compound combination responses. Finally, we will show the results of a combination screen with TMD8 and HBL1 cells, including the identification of a novel drug-drug combination for the BTK inhibitor ibrutinib (PCI-32765) which is of both basic and translational interest for the treatment of DLBCL. Citation Format: Lesley A. Mathews, Rajarshi Guha, Paul Shinn, Ryan M. Young, Kian-Huat Lim, Jonathan Keller, Dongbo Liu, Adam Yasgar, Crystal McKnight, Matthew B. Boxer, Damien Y. Duveau, Jian-kang Jiang, Sam Michael, Bryan T. Mott, Paresma R. Patel, William Leister, David J. Maloney, Christopher A. LeClair, Ganesha Rai, Ajit Jadhav, Brian D. Peyser, Christopher P. Austin, Scott Martin, Anton Simeonov, Marc Ferrer, Louis Staudt, Craig J. Thomas. High-throughput combination screening identifies novel drug-drug pairings for a Bruton's tyrosine kinase inhibitor against the ABC subtype of diffuse large B-cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4543. doi:10.1158/1538-7445.AM2013-4543

SummaryEbola virus (EBOV) infection of humans is a lethal but accidental dead-end event. Understanding resistance to EBOV in other species may help establish the basis of susceptibility differences among its hosts. Although rodents are resistant to EBOV, a murine-adapted variant is lethal when injected intraperitoneally into mice. We find that mice expressing reduced levels of the tyrosine phosphatase CD45 are protected against EBOV, whereas wild-type, CD45-deficient, or enzymatically inactive CD45-expressing mice succumbed to infection. Protection was dependent on CD8+ T cells and interferon γ. Reduced CD45-expressing mice retained greater control of gene expression and immune cell proliferation following EBOV infection, which contributed to reduced apoptosis, enhanced viral clearance, and increased protection against the virus. Together, these findings suggest that host susceptibility to EBOV is dependent on the delicate balance of immune homeostasis, which, as demonstrated here, can be determined by the levels of a single regulator.