Novel, druglike 1,7-disubstituted 2,3,4,5-tetrahydro-1H-benzo[b]azepine-based selective inhibitors of human neuronal nitric oxide synthase (nNOS).

A novel class of 1,7-disubstituted 2,3,4,5-tetrahydro-1H-benzo[b]azepine derivatives was designed, synthesized and evaluated as human nitric oxide synthase (NOS) inhibitors. Structure-activity relationship studies based on various basic amine side chains attached at the 1-position of the 2,3,4,5-tetrahydro-1H-benzo[b]azepine ring led to the identification of several potent and highly selective inhibitors (17, 18, 25, (±)-39, and (±)-40) of human neuronal NOS. The potential therapeutic application of one of these new selective nNOS inhibitors (17) was demonstrated in an in vivo spinal nerve ligation model of neuropathic pain, and various in vitro safety pharmacology studies such as the hERG K(+) channel inhibition assay and high throughput broad screen (minimal activity at 79 receptors/transporters/ion channels).

Development of Antibiotics That Dysregulate the Neisserial ClpP Protease.

Evolving antimicrobial resistance has motivated the search for novel targets and alternative therapies. Caseinolytic protease (ClpP) has emerged as an enticing new target since its function is conserved and essential for bacterial fitness, and because its inhibition or dysregulation leads to bacterial cell death. ClpP protease function controls global protein homeostasis and is, therefore, crucial for the maintenance of the bacterial proteome during growth and infection. Previously, acyldepsipeptides (ADEPs) were discovered to dysregulate ClpP, leading to bactericidal activity against both actively growing and dormant Gram-positive pathogens. Unfortunately, these compounds had very low efficacy against Gram-negative bacteria. Hence, we sought to develop non-ADEP ClpP-targeting compounds with activity against Gram-negative species and called these activators of self-compartmentalizing proteases (ACPs). These ACPs bind and dysregulate ClpP in a manner similar to ADEPs, effectively digesting bacteria from the inside out. Here, we performed further ACP derivatization and testing to improve the efficacy and breadth of coverage of selected ACPs against Gram-negative bacteria. We observed that a diverse collection of Neisseria meningitidis and Neisseria gonorrhoeae clinical isolates were exquisitely sensitive to these ACP analogues. Furthermore, based on the ACP-ClpP cocrystal structure solved here, we demonstrate that ACPs could be designed to be species specific. This validates the feasibility of drug-based targeting of ClpP in Gram-negative bacteria.

Activators of cylindrical proteases as antimicrobials: identification and development of small molecule activators of ClpP protease.

ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.