Heinrich J. Schostarez

Novel Riboswitch-Binding Flavin Analog That Protects Mice against Clostridium difficile Infection without Inhibiting Cecal Flora

ABSTRACT Novel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD)—an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis. In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that 5FDQD has potent and rapidly bactericidal activity against Clostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-induced C. difficile infection (CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little activity against representative strains from the Bacteroides, Lactobacillus, Bifidobacterium, Actinomyces, and Prevotella genera. Accordingly, a single oral dose of 5FDQD caused less alteration of culturable cecal flora in mice than the comparators. Collectively, these data suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI cure with a low likelihood of infection recurrence. Future studies will seek to assess the role of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our results indicate that riboswitch-binding antibacterial compounds can be discovered and optimized to exhibit activity profiles that merit preclinical and clinical development as potential antibacterial therapeutic agents.

Cyanoguanidine bioisosteres in potassium channel openers: Evaluation of 3,4-disubstituted-1,2,5-thiadiazole-1-oxides

Abstract Bioisosteric substitution of the cyanoguanidine group found in pinacidil ( 1 ) with a 3,4-diamino-1,2,5-thiadiazole-1-oxide moiety and replacement of the 4-aminopyridine group with a 3,5-dichlorophenyl group has resulted in a new structural class of potassium channel opener (PCO).

An experimental method for the determination of enzyme-competitive inhibitor dissociation constants from displacement curves: application to human renin using fluorescence energy transfer to a synthetic dansylated inhibitor peptide.

We developed a facile procedure for the determination of enzyme-competitive inhibitor dissociation constants over a wide range of potencies at any ratios of enzyme, labeled ligand, and inhibitor. The assay uses displacement curves and a fluorescent-labeled ligand to allow experimental determination of dissociation constants (Kds) of inhibitors of human renin, a highly specific enzyme, for which numerous high affinity (up to 100 pM) inhibitors have been synthesized. The procedure involves binding a dansylated competitive inhibitor, U80215, followed by its displacement by an unlabeled inhibitor of renin. Binding of U80215 is monitored by fluorescent energy transfer from the renin tryptophans to the dansyl moiety; displacement of U80215 by an unlabeled inhibitor is monitored by a reversal of this process. The procedures may be used to determine the potencies of unlabeled inhibitors up to 100 pM affinities and to determine kinetic binding constants. The concepts described should also be useful in other protein/ligand systems.

Structure-Activity Relationship of Flavin Analogues That Target the Flavin Mononucleotide Riboswitch.

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.

Exploiting the Molecular Template of Angiotensinogen in the Discovery and Design of Peptidyl, Pseudopeptidyl and Peptidemimetic Inhibitors of Human Renin: A Structure-Activity Perspective

The design of potent and pharmacologically effective, substrate-related inhibitors of renin has been the subject of intensive pharmaceutical discovery research for about one decade. Milestone achievements in synthetic tailoring of fragment analogs of angiotensinogen (ANG; Figure 1) have been documented in terms of identifying renin inhibitors of subnanomolar potency, sustained in vivo hypotensive activity, stability towards proteolytic degradation, and, more recently, oral bioavailability and decreased systemic clearance.1 By chemical modification of ANG-based derivatives, structure-activity analysis, and computer-assisted molecular modeling of peptidyl, pseudopeptidyl and peptidemimetic inhibitors using 3-D structural models of human renin, there currently exists a rather sophisticated wealth of information of relevance to the “rational” design of prototypic renin-targeted cardiovascular therapeutic agents. Such efforts have bridged biochemistry, medicinal chemistry, computational and biophysical chemistry, and in vivo pharmacology including, in a few cases, clinical evaluation in humans.