New therapeutic approaches and novel alternatives for organophosphate toxicity.

Organophosphate compounds (OPCs) are commonly used as pesticides and were developed as nerve agents for chemical warfare. Exposure to OPCs results in toxicity due to their covalent binding and inhibition of acetylcholinesterase (AChE). Treatment for toxicity due to OPC exposure has been largely focused on the reactivation of AChE by oxime-based compounds via direct nucleophilic attack on the phosphorous center. However, due to the disadvantages to existing oxime-based reactivators for treatment of OPC poisoning, we considered non-oxime mechanisms of reactivation. A high throughput screen of compound libraries was performed to discover previously unidentified reactivation compounds, followed by studies on their analogs. In the process, we discovered multiple non-oxime classes of compounds, the most robust of which we have already reported [1]. Herein, we report other classes of compounds we identified in our screen that are efficient at reactivation. During biochemical characterization, we also found some compounds with other activities that may inspire novel therapeutic approaches to OPC toxicity. Specifically, we found compounds that [1] increase the rate of substrate hydrolysis by AChE and, [2] protect the enzyme from inhibition by OPC. Further, we discovered that a subset of reactivator compounds recover activity from both AChE and the related enzyme butyrylcholinesterase (BuChE). We now report these compounds, their activities and discuss how each relates to therapeutic approaches that would provide alternatives to traditional oxime-based reactivation.

Discovery of New Classes of Compounds that Reactivate Acetylcholinesterase Inhibited by Organophosphates.

Acetylcholinesterase (AChE) that has been covalently inhibited by organophosphate compounds (OPCs), such as nerve agents and pesticides, has traditionally been reactivated by using nucleophilic oximes. There is, however, a clearly recognized need for new classes of compounds with the ability to reactivate inhibited AChE with improved in vivo efficacy. Here we describe our discovery of new functional groups--Mannich phenols and general bases--that are capable of reactivating OPC--inhibited AChE more efficiently than standard oximes and we describe the cooperative mechanism by which these functionalities are delivered to the active site. These discoveries, supported by preliminary in vivo results and crystallographic data, significantly broaden the available approaches for reactivation of AChE.

Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors.

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure-activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.

Discovery of potent non-urea inhibitors of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC(50)s of the most potent compounds range from micromolar to low nanomolar.

Discovery of novel small molecule cell type-specific enhancers of NF-kappaB nuclear translocation.

An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.

Constrained NBMPR analogue synthesis, pharmacophore mapping and 3D-QSAR modeling of equilibrative nucleoside transporter 1 (ENT1) inhibitory activity.

Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors. In our previous study [J. Med. Chem. 2003, 46, 831-837], novel regioisomeric nitro-1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of NBMPR were synthesized and evaluated as ENT1 ligands. 7-NO(2)-1,2,3,4-Tetrahydroisoquino-2-yl purine riboside was identified as the analogue with the nitro group in the best orientation at the NBMPR binding site of ENT1. In the present study, further conformational constraining was introduced by synthesizing 5'-O,8-cyclo derivatives. The flow cytometrically determined binding affinities indicated that the additional 5'-O,8-cyclo constraining was unfavorable for binding to the ENT1 transporter. The structure-activity relationship (SAR) acquired was applied to pharmacophore mapping using the PHASE program. The best pharmacophore hypothesis obtained embodied an anti-conformation with three hydrogen-bond acceptors, one hydrophobic center, and two aromatic rings involving the 3'-OH, 4'-oxygen, the NO(2) group, the benzyl phenyl and the imidazole and pyrimidine portions of the purine ring, respectively. A PHASE 3D-QSAR model derived with this pharmacophore yielded an r(2) of 0.916 for four (4) PLS components, and an excellent external test set predictive r(2) of 0.78 for 39 compounds. This pharmacophore was used for molecular alignment in a comparative molecular field analysis (CoMFA) 3D-QSAR study that also afforded a predictive model with external test set validation predictive r(2) of 0.73. Thus, although limited, this study suggests that the bioactive conformation for NBMPR at the ENT1 transporter could be anti. The study has also suggested an ENT1 inhibitory pharmacophore, and established a predictive CoMFA 3D-QSAR model that might be useful for novel ENT1 inhibitor discovery and optimization.

Synthesis and flow cytometric evaluation of novel 1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of nitrobenzylmercaptopurine riboside (NBMPR) designed for probing its conformation when bound to the es nucleoside transporter.

Novel regioisomers of conformationally constrained analogues of the potent es nucleoside transporter ligand, nitrobenzylmercaptopurine riboside (NBMPR), designed for probing its bound (bioactive) conformation, were synthesized and evaluated as es transporter ligands by flow cytometry. Purine 6-position 5, 6, 7, or 8-nitro-1,2,3,4-tetrahydroisoquinolylpurine ribosides, in which the nitrobenzyl moiety in NBMPR has been locked into the nitro-1,2,3,4-tetrahydroisoquinoline system, were synthesized by reaction of the appropriate nitro-1,2,3,4-tetrahydroisoquinoline with 6-chloropurine riboside. Flow cytometry was performed using 5-(SAENTA)-X8-fluorescein as the competitive ligand. A high degree of variation in the es transporter binding capacity of the target compounds was observed, with the K(i) values ranging from 0.45 nM for the most tightly bound compound (4) to 300 nM for the least tightly bound compound (5). The K(i) of NBMPR was 0.70 nM, a little higher than that of compound 4. Compound 4 is the isomer that has the nitro group in the best orientation at the es transporter binding site compared to the other three compounds, 2, 3, and 5.