Aminodeoxychorismate Synthase Inhibitors from One-Bead One-Compound Combinatorial Libraries:  “Staged” Inhibitor Design

Abstract

4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

Comparative studies of the catalytic mechanisms of two chorismatases: CH‐fkbo and CH‐Hyg5

“…Due to the lack of chorismate pathway in mammals, these enzymes have been exploited as attractive targets for many potential herbicides and antibiotic agents . In recent years, a new class of bacterial chorismate‐converting enzymes, named as chorismatases (CH), have been recognized, which catalyze the hydrolysis of chorismate into pyruvate and (dihydro)‐benzoic acid derivatives.…”

Spectral measurements of large particles by flow cytometry

“…The analysis of whole organisms(1,2), assemblies of cells(3,4), and solid phase chemistry resins(5,6) represent emerging applications for flow cytometry and are driving instrument developments. Spectral measurements of fluorescence and Raman labels are also providing new possibilities for multiplexed and multiparameter flow cytometry.…”

“…One aspect of this evolution is the adaptation of flow cytometry to measure large particles approaching a millimeter in size. These include whole organisms such as C. elegans (1,2), multicellular structures such as pancreatic islets(3,4), and polymer resins bearing combinatorial libraries(5,6). Custom and commercial instruments(7,8,9,10) have been developed to meet these challenges, and have opened up flow cytometry to new communities of biological researchers.…”

Inhibition of p-Aminobenzoate and Folate Syntheses in Plants and Apicomplexan Parasites by Natural Product Rubreserine

“…Until now, searches of inhibitors for this class of enzymes were only achieved using prokaryotic systems. They involved docking studies and design of chorismate analogous compounds (22)(23)(24)(25), combinatorial chemistry approaches (26,27), and a specific screening of a microorganism extract collection using growth inhibition of test bacteria as a marker of activity (28,29). Several compounds were identified by these different methods but appeared to be relatively weak inhibitors of ADCS, although some of them could be quite potent against other chorismate-utilizing enzymes (22)(23)(24)26).…”