Reengineering an Antiarrhythmic Drug Using Patient hiPSC Cardiomyocytes to Improve Therapeutic Potential and Reduce Toxicity.

Modeling cardiac disorders with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes is a new paradigm for preclinical testing of candidate therapeutics. However, disease-relevant physiological assays can be complex, and the use of hiPSC-cardiomyocyte models of congenital disease phenotypes for guiding large-scale screening and medicinal chemistry have not been shown. We report chemical refinement of the antiarrhythmic drug mexiletine via high-throughput screening of hiPSC-CMs derived from patients with the cardiac rhythm disorder long QT syndrome 3 (LQT3) carrying SCN5A sodium channel variants. Using iterative cycles of medicinal chemistry synthesis and testing, we identified drug analogs with increased potency and selectivity for inhibiting late sodium current across a panel of 7 LQT3 sodium channel variants and suppressing arrhythmic activity across multiple genetic and pharmacological hiPSC-CM models of LQT3 with diverse backgrounds. These mexiletine analogs can be exploited as mechanistic probes and for clinical development.

A Novel Small Molecule Inhibits Tumor Growth and Synergizes Effects of Enzalutamide on Prostate Cancer.

Prostate cancer (PCa) is the second leading cause of cancer-related death for men in the United States. Approximately 35% of PCa recurs and is often transformed to castration-resistant prostate cancer (CRPCa), the most deadly and aggressive form of PCa. However, the CRPCa standard-of-care treatment (enzalutamide with abiraterone) usually has limited efficacy. Herein, we report a novel molecule (PAWI-2) that inhibits cellular proliferation of androgen-sensitive and androgen-insensitive cells (LNCaP and PC-3, respectively). In vivo studies in a PC-3 xenograft model showed that PAWI-2 (20 mg/kg per day i.p., 21 days) inhibited tumor growth by 49% compared with vehicle-treated mice. PAWI-2 synergized currently clinically used enzalutamide in in vitro inhibition of PCa cell viability and resensitized inhibition of in vivo PC-3 tumor growth. Compared with vehicle-treated mice, PC-3 xenograft studies also showed that PAWI-2 (20 mg/kg per day i.p., 21 days) and enzalutamide (5 mg/kg per day i.p., 21 days) inhibited tumor growth by 63%. Synergism was mainly controlled by the imbalance of prosurvival factors (e.g., Bcl-2, Bcl-xL, Mcl-1) and antisurvival factors (e.g., Bax, Bak) induced by affecting mitochondrial membrane potential/mitochondria dynamics. Thus, PAWI-2 utilizes a distinct mechanism of action to inhibit PCa growth independently of androgen receptor signaling and overcomes enzalutamide-resistant CRPCa. SIGNIFICANCE STATEMENT: Castration-resistant prostate cancer (CRPCa) is the most aggressive human prostate cancer (PCa) but standard chemotherapies for CRPCa are largely ineffective. PAWI-2 potently inhibits PCa proliferation in vitro and in vivo regardless of androgen receptor status and uses a distinct mechanism of action. PAWI-2 has greater utility in treating CRPCa than standard-of-care therapy. PAWI-2 possesses promising therapeutic potency in low-dose combination therapy with a clinically used drug (e.g., enzalutamide). This study describes a new approach to address the overarching challenge in clinical treatment of CRPCa.

Inhibition of invasive pancreatic cancer: restoring cell apoptosis by activating mitochondrial p53.

Pancreatic ductal adenocarcinoma (PDAC), constitutes >90% of pancreatic cancers (PC) and is one of the most aggressive human tumors. Standard chemotherapies for PDAC (e.g., gemcitabine, FOLFIRINOX, etc.) has proven to be largely ineffective. Herein, we report a novel molecule (i.e., compound 1) that potently inhibits proliferation and induces apoptosis of PDAC cells. As we observed in other cancer types (i.e., colorectal, breast cancer), the effect of 1 against PDAC cells is also related to microtubule destabilization and DNA damage checkpoint activation. However, in PDAC cells, the inhibitory effect of 1 was mainly controlled by mitochondrial p53-dependent apoptosis. Compound 1 worked with cells of different p53 mutant status and affected p53 activation/phosphorylation not simply by stabilizing p53 protein but through antagonizing anti-apoptotic effects of Bcl-xL and restoring p53 to activate mitochondrial-apoptotic pathways (i.e., cytochrome c release, caspase activation and PARP cleavage). Compound 1 was more efficient than a typical PDAC combination therapy (i.e., gemcitabine with paclitaxel) and showed synergism in inhibiting PDAC cell proliferation with gemcitabine (or gemcitabine with paclitaxel). This synergism varied between different types of PDAC cells and was partially controlled by the phosphorylation of p53 on Serine15 (phospho-Ser15-p53). In vivo studies in an orthotopic syngeneic murine model showed that 1 (20 mg/kg/day, 28 days, i.p.) inhibited tumor growth by 65% compared to vehicle-treated mice. No apparent acute or chronic toxicity was observed. Thus, compound 1 utilizes a distinct mechanism of action to inhibit PC growth in vitro and in vivo and is a novel anti-PDAC compound.

Novel tertiary sulfonamides as potent anti-cancer agents.

For adult women in the United States, breast cancer is the most prevalent form of cancer. Compounds that target dysregulated signal transduction can be efficacious anti-cancer therapies. A prominent signaling pathway frequently dysregulated in breast cancer cells is the Wingless-related integration site (Wnt) pathway. The purpose of the work was to optimize a "hit" from a screening campaign. 76,000 compounds were tested in a Wnt transcription assay and revealed potent and reproducible "hit," compound 1. Medicinal chemistry optimization of 1 led to more potent and drug-like molecules, 19, 24 and 25 (i.e., Wnt pathway IC50 values = 11, 18 and 7 nM, respectively). The principal results showed compounds 19, 24 and 25 were potent anti-proliferative agents in breast cancer cell lines, MCF-7 (i.e., IC50 values = 10, 7 and 4 nM, respectively) and MDA-MB 231 (i.e., IC50 values = 13, 13 and 16 nM, respectively). Compound 19 synergized anti-proliferation with chemotherapeutic Doxorubicin in vitro. A major conclusion was that compound 19 enhanced anti-proliferation of Doxorubicin in vitro and in a xenograft animal model of breast cancer.

A Novel Inhibitor Targets Both Wnt Signaling and ATM/p53 in Colorectal Cancer.

For 2017, the estimated lifetime risk of developing colorectal cancer was 1 in 22. Even though preventative colonoscopy screening and standard-of-care surgery, radiation, and chemotherapy have decreased the death rate from colorectal cancer, new therapies are needed for metastatic colorectal cancer. Here, we developed a novel small molecule, compound 2, that inhibited proliferation and viability of human colorectal cancer cells (HCT-116, DLD-1, SW480, and 10.1). Compound 2 inhibited cell migration, invasion, and epithelial-mesenchymal transition processes and potently increased cell apoptosis in human colorectal cancer cells. Compound 2 also modulated mitotic stress signaling, leading to both inhibition of Wnt responsiveness and stabilization and activation of p53 to cause cell-cycle arrest. In mouse xenografts, treatment with compound 2 (20 mg/kg/day, i.p.) induced cell death and inhibited tumor growth more than four-fold compared with vehicle at day 34. Neither acute cytotoxicity nor toxicity in animals (up to 1,000 mg/kg, i.p.) were observed for compound 2 To our knowledge, compound 2 is the first reported potent small molecule that inhibits Wnt/ß-catenin signaling, activates p53 signaling regardless of p53 mutation status, and binds microtubules without detectable toxicity. Thus, compound 2 offers a novel mechanism of action and a new strategy to treat colorectal cancer.Significance: These findings identify a potent small molecule that may be therapeutically useful for colon cancer that works by inhibiting Wnt/ß-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072-83. ©2018 AACR.

1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells.

Cardiomyopathy is the leading cause of death worldwide. Despite progress in medical treatments, heart transplantation is one of the only current options for those with infarcted heart muscle. Stem cell differentiation technology may afford cell-based therapeutics that may lead to the generation of new, healthy heart muscle cells from undifferentiated stem cells. Our approach is to use small molecules to stimulate stem cell differentiation. Herein, we describe a novel class of 1,5-disubstituted benzimidazoles that induce differentiation of stem cells into cardiac cells. We report on the evaluation in vitro for cardiomyocyte differentiation and describe structure-activity relationship results that led to molecules with drug-like properties. The results of this study show the promise of small molecules to direct stem cell lineage commitment, to probe signaling pathways and to develop compounds for the stimulation of stem cells to repair damaged heart tissue.

Preclinical studies of noncharged oxime reactivators for organophosphate exposure.

A countermeasure that protects the brain from organophosphate toxicity is an unmet need. Few small molecule reactivators that can cross the blood brain barrier and reactivate brain acetyl cholinesterases have been reported. Herein, we describe preclinical investigations of a new class of amidine-oxime reactivator of cholinesterases with improved potency and blood brain barrier permeability. (Z)-N-((E)-1-(Dimethylamino)-2-(hydroxyimino)ethylidene)butan-1-aminium chloride, 1, is zwitterionic at physiological pH but possesses increased oxime nucleophilicity because of the adjacent amidine functionality. The amidine-oximes reported herein were observed to be nontoxic (up to 200 mg/kg in vivo) and are chemically and metabolically stable. The results presented herein show that uncharged amidine-oxime reactivators such as 1 can penetrate the blood brain barrier in animals and protect from the toxicity of nerve agent model compounds.

Inhibition of Bfl-1 with N-aryl maleimides.

High-throughput screening of 66,000 compounds using competitive binding of peptides comprising the BH3 domain to anti-apoptotic Bfl-1 led to the identification of 14 validated 'hits' as inhibitors of Bfl-1. N-Aryl maleimide 1 was among the validated 'hits'. A chemical library encompassing over 280 analogs of 1 was prepared following a two-step synthesis. Structure-activity studies for inhibition of Bfl-1 by analogs of N-aryl maleimide 1 revealed a preference for electron-withdrawing substituents in the N-aryl ring and hydrophilic amines appended to the maleimide core. Inhibitors of Bfl-1 are potential development candidates for anti-cancer therapeutics.

Inhibition of protein kinase C-driven nuclear factor-kappaB activation: synthesis, structure-activity relationship, and pharmacological profiling of pathway specific benzimidazole probe molecules.

A unique series of biologically active chemical probes that selectively inhibit NF-kappaB activation induced by protein kinase C (PKC) pathway activators have been identified through a cell-based phenotypic reporter gene assay. These 2-aminobenzimidazoles represent initial chemical tools to be used in gaining further understanding on the cellular mechanisms driven by B and T cell antigen receptors. Starting from the founding member of this chemical series 1a (notated in PubChem as CID-2858522), we report the chemical synthesis, SAR studies, and pharmacological profiling of this pathway-selective inhibitor of NF-kappaB activation.

Chemical biology strategy reveals pathway-selective inhibitor of NF-kappaB activation induced by protein kinase C.

Dysregulation of NF-kappaB activity contributes to many autoimmune and inflammatory diseases. At least nine pathways for NF-kappaB activation have been identified, most of which converge on the IkappaB kinases (IKKs). Although IKKs represent logical targets for potential drug discovery, chemical inhibitors of IKKs suppress all known NF-kappaB activation pathways and thus lack the selectivity required for safe use. A unique NF-kappaB activation pathway is initiated by protein kinase C (PKC) that is stimulated by antigen receptors and many growth factor receptors. Using a cell-based high-throughput screening (HTS) assay and chemical biology strategy, we identified a 2-aminobenzimidazole compound, CID-2858522, which selectively inhibits the NF-kappaB pathway induced by PKC, operating downstream of PKC but upstream of IKKbeta, without inhibiting other NF-kappaB activation pathways. In human B cells stimulated through surface immunoglobulin, CID-2858522 inhibited NF-kappaB DNA-binding activity and expression of endogenous NF-kappaB-dependent target gene, TRAF1. Altogether, as a selective chemical inhibitor of the NF-kappaB pathway induced by PKC, CID-2858522 serves as a powerful research tool and may reveal new paths toward therapeutically useful NF-kappaB inhibitors.

Selective benzimidazole inhibitors of the antigen receptor-mediated NF-kappaB activation pathway.

Dysregulated antigen receptor-mediated NF-kappaB activation can contribute to development of autoimmunity, chronic inflammation, and malignancy. A chemical biology screening strategy has identified a substituted benzimidazole that selectively inhibits antigen receptor-mediated NF-kappaB activation without blocking other NF-kappaB activation pathways. A library of analogs was synthesized and the structure-activity relationship and metabolic stability for the series is presented.