Karl J. Okolotowicz

Chemical synthesis of two series of nerve agent model compounds and their stereoselective interaction with human acetylcholinesterase and human butyrylcholinesterase

Both G and V type nerve agents possess a center of chirality about phosphorus. The S(p) enantiomers are generally more potent inhibitors than their R(p) counterparts toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To develop model compounds with defined centers of chirality that mimic the target nerve agent structures, we synthesized both the S(p) and the R(p) stereoisomers of two series of G type nerve agent model compounds in enantiomerically enriched form. The two series of model compounds contained identical substituents on the phosphorus as the G type agents, except that thiomethyl (CH(3)-S-) and thiocholine [(CH(3))(3)NCH(2)CH(2)-S-] groups were used to replace the traditional nerve agent leaving groups (i.e., fluoro for GB, GF, and GD and cyano for GA). Inhibition kinetic studies of the thiomethyl- and thiocholine-substituted series of nerve agent model compounds revealed that the S(p) enantiomers of both series of compounds showed greater inhibition potency toward AChE and BChE. The level of stereoselectivity, as indicated by the ratio of the bimolecular inhibition rate constants between S(p) and R(p) enantiomers, was greatest for the GF model compounds in both series. The thiocholine analogues were much more potent than the corresponding thiomethyl analogues. With the exception of the GA model compounds, both series showed greater potency against AChE than BChE. The stereoselectivity (i.e., S(p) > R(p)), enzyme selectivity, and dynamic range of inhibition potency contributed from these two series of compounds suggest that the combined application of these model compounds will provide useful research tools for understanding interactions of nerve agents with cholinesterase and other enzymes involved in nerve agent and organophosphate pharmacology. The potential of and limitations for using these model compounds in the development of biological therapeutics against nerve agent toxicity are also discussed.

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-κB 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-κB activation.

Direct detection of the hydrolysis of nerve agent model compounds using a fluorescent probe.

Nerve agents are highly toxic organophosphorus compounds (OPs) that are used as chemical warfare agents. Developing a catalytic bioscavenger to efficiently detoxify nerve agents in the bloodstream of affected individuals has been recognized as an attractive approach to prevent nerve agent toxicity. However, the search for nerve agent catalysts has been hindered by the lack of efficient direct assays for nerve agent hydrolysis. In addition, authentic nerve agents are restricted and access to use for experiments by the general research community is prohibited. Herein we report development of a method that combines use of novel nerve agent model compounds possessing a thiocholine leaving group that reacts with the fluorescent thio-detection probe, BES-Thio, to afford detection of sub-micromolar amounts of nerve agent model compounds hydrolysis products. The detection sensitivity of BES-Thio assay was approximately 10 times better than the Ellman assay. This developed method is useful as a direct, sensitive screening method for evaluating OP hydrolysis efficiency from catalytic cholinesterases. When the assay was assembled in the presence of oxime, OP-inhibited cholinesterases that were able to be reactivated by specific oxime showed oxime-assisted enzyme-mediated OP hydrolysis. Therefore, this method is also useful to screen oxime analogs to identify novel agents that can reactivate OP-inhibited cholinesterases or to screen various enzymes to identify pseudo-catalytic bioscavengers that can be readily reactivated by clinically approved oximes.

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.

Chemical Biology Strategy Reveals Pathway-Selective Inhibitor of NF-κB 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.

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.

Selective benzimidazole inhibitors of the antigen receptor-mediated NF-κB 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.

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/β-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/β-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072-83. ©2018 AACR.