Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• Cyclic diguanosine monophosphate (c-di-GMP) is a crucial bacterial second messenger regulating vital cellular processes.
• Dysregulation of c-di-GMP signaling contributes to chronic infections due to increased antibiotic tolerance associated with biofilm formation.
• Diguanylate cyclases (DGCs), enzymes producing c-di-GMP, are potential drug targets for controlling biofilm-related infections.

Purpose of the Study:

• To identify and characterize small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus.
• To explore the structure-activity relationships of identified modulators.
• To develop chemical genetic tools for dissecting c-di-GMP signaling pathways.

Main Methods:

• A FRET-based biochemical high-throughput screening (HTS) assay was employed to identify modulators.
• Structure-activity relationship (SAR) studies were conducted on identified small-molecule scaffolds.
• Enzymatic activity assays were used to quantify modulation effects.

Main Results:

• Seven small molecules regulating DgcA activity were identified in the low-micromolar range.
• SAR studies revealed diverse modulatory behaviors, with some compounds showing reversed effects (inhibition to activation) upon minor chemical modification.
• The identified compounds represent novel chemotypes.

Conclusions:

• The identified small molecules are potential chemical genetic tools for studying c-di-GMP networks and phenotypes.
• Detailed mechanism-of-action studies are essential for understanding c-di-GMP signaling inhibitors.
• Synthetic small molecules exhibit complex interactions with DGC regulatory mechanisms.