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

Abstract

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

Keywords: FRET; c-di-GMP; diguanylate cyclase inhibitors; high-throughput screening; structure-activity relationships.

Figure 1:

FRET assay for c-di-GMP. A) Kinetics of fluorescence emission ration change (527/480nm) measured in a 384 well plate format. Affinity purified YcgR FRET biosensor was added in presence of 20 nM DgcA enzyme and 20μM GTP substrate (closed circles) or in absence of GTP (open circles). B) Corresponding increase in c-di-GMP concentration derived from the change in fluorescence emission ration (535/470nm). Above 800 nM c-di-GMP, allosteric product inhibition decreases DGC activity of DgcA. Each graph shows the average of three independent measurements. The reaction volume per well was 20 μl in a 384 well Corning low volume flat bottom plate. C) Histogram of the fluorescence emission ratio after 3 hours incubation of 20 nM DgcA with 20 μM GTP in presence of 50μg/ml compounds and 1% DMSO. Wells without GTP substrate added were used as positive controls (red), wells with exogenous c-di-GMP (5 μM) were used as negative controls (blue). D) Plate uniformity of the HT screening. For every well, background fluorescence signal of the compound has been measured prior addition of the FRET-biosensor. Compounds with auto fluorescence exceeding 7% of the FRET biosensor signal (535/470nm) were discarded.

Figure 2:

Inhibitors of the DGC enzyme DgcA. A) Structure of the small molecular inhibitors 1,2, 3, 4, 5, 6 and 7 identified in the FRET-screening with IC₅₀ values below 50 μM. Out of them, 4 compounds 1, 2, 3 and 4 exhibit IC₅₀ values <10 μM. Scaffolds 1 and 2 have been selected for subsequent SAR studies. B) The concentration response curve of compound 1 (IC₅₀ 4 μM) reveals complete inhibition against DgcA. C) Compound 2 (IC₅₀ μM) acts as partial inhibitor for DgcA. The corresponding concentration response curve of 1 is shown as dashed line.

Figure 3:

Examples of silent (SAM), negative (NAM) and positive (PAM) allosteric modulators of the DGC enzyme DgcA. A) Structure and concentration response curve of the 1,3-benzoxazole-6-carboxylate derivative 1g, a silent allosteric regulator of DgcA. B) Representative example of an allosteric activator of the scaffold 1. The 4-nitrobenzoic acid derivative of 1h acts as a positive allosteric regulator with AC₅₀ of 55.5 μM. The concentration response curves of the parental 1,3-benzothiazole-6-carboxylate 1 are shown as dashed lines in panels A and B. C) Structure and concentration response curve of 2a. Alteration of the methyl substituents from 2,5 to 2,4 in the 2 scaffold increases residual activity of the enzyme-substrate-inhibitor complex from 31.4% in 2 (dashed line) to 69.8% in 2a (closed circles). D) The 2-methoxy, 5-sulfonylmorpholine derivative 2b is a positive allosteric regulator with AC₅₀ of 12.5 μM. Concentration response curves of the parental 2 are shown as dashed lines in panels C and D.

Figure 4:

Effect of 1 and 2 on substrate binding and maximal reaction rate of DgcA. Corresponding plot of the initial velocity versus GTP concentration. A) Presence of 1 decreases affinity for substrate binding and maximal rate of DGC reaction (Table S3). B) In contrast to 1, binding of 2 affects only the Km for GTP but does not affect maximal velocity (Table S3).

Scheme 1.. Synthesis of 1v.

a). benzyloxyacetyl chloride, ether 0°C - r.t. 1.5h. b). Pd-C, EtOH, DMF, H₂

Scheme 2.. General method A.

a). 5-benzothiazole-carboxylic acid, EDCI, DMAP, CH₂Cl₂, DMF r.t. overnight

Scheme 3.. Synthesis of 1x.

a).CBr4, triphenylphospine, CH₂Cl₂, r.t. overnight.

Scheme 4.. Synthesis of 1s.

a). PCl₅, ether, −15C - 0C, 3h. b). TMS-2-pyrrolidinone, ether, 0°C-r.t. 1.25h. c). TFA, thioanisole, TMSBr, r.t. 1.5h. d). benzohiazole-6-carboxylic acid, HBTU, HOAt, DIEA, DMF, CH₂Cl₂, r.t. overnight.

Scheme 5.. Synthesis of 1t.

a). 2-bromopropionyl bromide, ether 0°C - r.t. 1.25h. b). benzothiazole-6-carboxylic acid, Cs₂CO₃, DMF r.t. overnight.

Scheme 6.. Synthesis of 1u.

a). t-butyl 3-hydroxypropionate, EDCI, DMAP, CH₂Cl₂, DMF, r.t. overnight. b). Pd-C, EtOH, DMF, H₂ c). biphenyl-4-carboxylic acid, EDCI, DMAP, CH₂Cl₂, DMF, r.t. overnight.