研究目的
To identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus using a FRET-based high-throughput screening approach and structure–activity studies, aiming to develop tools for controlling biofilm formation and understanding c-di-GMP signaling.
研究成果
The research successfully identified small-molecule modulators of DgcA with diverse effects, including inhibitors, silent modulators, and activators, through FRET-based HTS and SAR studies. These compounds represent new chemotypes for probing c-di-GMP signaling networks and have potential as leads for anti-biofilm agents. The findings highlight the complexity of allosteric regulation in DGCs and the importance of detailed mechanism-of-action studies.
研究不足
The study is limited to in vitro biochemical assays with purified DgcA enzyme; in vivo efficacy and specificity for other DGC enzymes were not fully explored. The compounds may have issues with stability (e.g., ester bonds susceptible to hydrolysis) and potential cytotoxicity. The HTS may miss compounds due to autofluorescence or other interference. Further optimization is needed for pharmaceutical development.
1：Experimental Design and Method Selection:
A FRET-based biochemical high-throughput screening (HTS) assay was developed to monitor c-di-GMP production. The assay uses a FRET biosensor consisting of the c-di-GMP binding domain of the PilZ protein YcgR tagged with mCYPet and mYPet fluorescent proteins. Binding of c-di-GMP alters FRET efficiency, allowing detection of c-di-GMP levels. The assay was performed in 384-well plates with a 20 μL reaction volume.
2：Sample Selection and Data Sources:
The DgcA enzyme from Caulobacter crescentus was used as the model enzyme. A chemical library of 27,502 commercially available small molecules was screened, sourced from companies like Asinex Corporation, Enamine Ltd., and Life Chemicals.
3：List of Experimental Equipment and Materials:
Equipment includes a PlateMate Plus liquid handler (Hamilton), Microflo plate dispenser (Biotek), Corning Low Volume 384 Well Black Flat Bottom Polystyrene Microplates (Product #3821BC), and an Envision 2104 fluorescence plate reader (PerkinElmer) with specific filters. Proteins were expressed in Escherichia coli BL21 (DE3) strains and purified using affinity chromatography. Reagents include GTP, c-di-GMP, DMSO, and various buffers.
4：Experimental Procedures and Operational Workflow:
For screening, DgcA enzyme was preincubated with compounds (200 nL, 5 mg/mL in DMSO) in 384-well plates. Fluorescence emission was measured before and after adding the FRET biosensor and GTP substrate. FRET was measured at 470 and 535 nm after 3 hours incubation. Hits were selected based on c-di-GMP levels below 300 nM, consistency in duplicates, and low autofluorescence. Secondary assays involved dose-response (IC50) measurements and structure–activity relationship (SAR) studies with synthesized derivatives.
5：Data Analysis Methods:
Data were analyzed in Excel. FRET ratio was calculated as Em527 nm / Em470 nm. Z' factor was used for assay quality control. IC50 and AC50 values were fitted using Hill equations. Initial velocities were determined from Michaelis-Menten kinetics.
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