Nichole A. Tower

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

High-throughput screening identifies a bisphenol inhibitor of SV40 large T antigen ATPase activity.

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose–response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.

Development of (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide, ML336: Novel 2-amidinophenylbenzamides as potent inhibitors of venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 = 0.8 μM), limited cytotoxic liability (CC50 > 50 μM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 μM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC50 = 0.02–0.04 μM, CC50 > 50 μM) while limiting in vitro viral replication (EC90 = 0.17 μM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg–1 day–1 and viral replication appeared to be inhibited through interference of viral nonstructural proteins.

(S)-N-(2,5-Dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2-carboxamide as a Small Molecule Inhibitor Probe for the Study of Respiratory Syncytial Virus Infection

A high-throughput, cell-based screen was used to identify chemotypes as inhibitors for human respiratory syncytial virus (hRSV). Optimization of a sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC₅₀ = 2.3 ± 0.8 μM) with marginal cytotoxicity (CC₅₀ = 30.9 ± 1.1 μM) and reduced viral titer by 100-fold. Compared to ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity index.

A BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus

Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

Lifting the Mask: Identification of New Small Molecule Inhibitors of Uropathogenic Escherichia coli Group 2 Capsule Biogenesis

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

Adapting High-Throughput Screening Methods and Assays for Biocontainment Laboratories

High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories.

Mycobacterium tuberculosis High-Throughput Screening

High-throughput screening is a valuable way to identify hit compounds that combined with a robust medicinal chemistry program could lead to the identification of new antibiotics. Here, we discuss our method for screening large compound libraries with virulent Mycobacterium tuberculosis, possibly one of the more difficult bacteria to use because of its slow growth and assignment to Biosafety Level-3 by the CDC and NIH. The principles illuminated here, however, are relevant to the execution of most bacteria high-throughput screens.

Abstract LB-055: High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers

By means of a phenomenon termed “the Warburg effect,” tumor cells shift their energy production by mitochondrial oxidative phosphorylation to aerobic glycolysis, resulting in the upregulation of glucose consumption and increased cellular oxidative and nitrosative stress. To compensate for such toxic levels of ROS/RNS, cancer cells rely heavily on their antioxidant defense mechanisms, which are largely controlled by the NAD(P)/NAD(P)H redox partners. We found that the modulation of NAD metabolism in vivo, specifically via the deletion of the NAD glycohydrolase CD38, resulted in increased production of intrinsic ROS and increased DNA damage following exposure to chemotherapeutics. Furthermore, in vitro experiments showed that CD38 knockdown in CD38-expressing tumor cells prevented the generation of stable transfectants, highlighting a role for CD38 in tumor cell survival. In light of these findings, we hypothesized that pharmacological inhibition of CD38 may be an effective therapy for the treatment of hematological cancers, in particular those which uniformly overexpress CD38, such as MM and chronic lymphocytic leukemia. Indeed, treatment of human MM cell lines LP-1 and KMS-12-PE with CD38 antagonists sensitized the cells to standard ROS-inducing chemotherapeutics. We conducted a high-throughput screening (HTS) campaign of over two hundred thousand unique and non-proprietary lead-like compounds using an optimized and miniaturized HTS based on a luminescent NAD quantitation platform. Five hundred active hits were analyzed for toxicity using a cell-based HTS assay purposely designed with CD38-negative HEK293 cells to avoid elimination of desirable compounds toxic to CD38-positive cells. Hits with non-specific properties, such as PAINS (Pan Assay Interference Compounds), were removed by computational filtering, and the remaining compounds were tested for inhibition of human CD38 activity in cells. The last phase of the compound progression pathway involved testing for non-selective inhibition of other NAD-consuming enzymes, namely Poly(ADP-ribose) polymerase-1, and Sirtuin-1, which led to the identification of two distinct chemical series that exhibit >10-fold selectivity for human CD38. Hit-to-lead chemistry is currently underway to synthesize key analogs by rational drug design. In summary, our data suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing hematological cancers. Our HTS campaign efforts are paving the way for the discovery and development of potent and selective small-molecule inhibitors of CD38. Citation Format: Davide Botta, Tulin Dadali, Betty J. Mousseau, Fen Zhou, Michael D. Schultz, Esther Zumaquero, Anna Manouvakhova, Melinda I. Sosa, Sara N. McKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E. Lucille White, Indira Padmalayam, Wei Zhang, Maaike Everts, Corinne E. Augelli-Szafran, James R. Bostwick, Mark J. Suto, Frances E. Lund. High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-055.

Abstract 1242: Modulating the cellular redox state by targeting the NAD glycohydrolase CD38: A novel therapeutic approach for chemosensitizing B-cell malignancies

The cellular redox state, which is controlled by the NAD/NADH and NADP/NADPH redox partners, is critical for numerous cellular activities including energy metabolism, signaling and transcription. Hematopoietic tumors have higher levels of intrinsic reactive oxygen species (ROS) due to increased NAD biosynthesis and metabolism required to further stimulate tumorigenesis. To compensate for such toxic levels of ROS, cancer cells rely heavily on their antioxidant defense mechanisms to prevent ROS-induced death/senescence and to promote neoplastic cell behavior. It has been postulated that pairing drugs that block the tumor antioxidant pathways with ROS-inducing chemotherapeutics may kill tumors more effectively. We hypothesized that the modulation of NAD metabolism dramatically alters the cellular redox state, and that drugs affecting NAD metabolism may be effective anti-cancer therapeutics. Using an in vivo model of Streptozotocin (STZ)-induced oxidative stress, we show that the NAD glycohydrolase CD38, which is expressed by many B-cell malignancies, acts as an antioxidant by lowering intrinsic ROS levels and by protecting cells from extrinsic ROS, DNA alkylating chemotherapeutics and ROS-induced senescence. In addition, we show that the overexpression of human CD38 increases the viability of murine Ba/F3 pro-B cells cultured in the absence of the survival factor IL-3. To examine the role of CD38 in tumor cell growth and survival, we reduced CD38 expression in human multiple myeloma KMS-12-PE cells by RNA interference (RNAi) targeting three separate regions of the CD38 coding sequence. The knockdown of CD38 expression resulted in a significant decrease in the number of stable transformants generated, suggesting that CD38 plays a key role in tumor cell survival. Lastly, we show that CD38 antagonists synergize with the chemotherapeutic agent Melphalan to block tumor cell survival in vitro. In summary, this work suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing B-cell malignancies. We are currently conducting a high-throughput screening (HTS) campaign of over 200 thousand small-molecule compounds to identify inhibitors of CD38 that can be used in combination with standard ROS-inducing chemotherapies to treat CD38+ B-cell malignancies, such as multiple myeloma and chronic lymphocytic leukemia. Citation Format: Davide Botta, Tulin Dadali, Betty Mousseau, Anna Manouvakhova, Melinda I. Sosa, Sara N. MKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E Lucile White, James R. Bostwick, Frances E. Lund. Modulating the cellular redox state by targeting the NAD glycohydrolase CD38: A novel therapeutic approach for chemosensitizing B-cell malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1242. doi:10.1158/1538-7445.AM2015-1242