Computer-aided design of a cyclic di-AMP synthesizing enzyme CdaA inhibitor

Abstract

Cyclic di-AMP (c-di-AMP) is an essential secondary messenger regulating cell wall homeostasis and myriads of physiological processes in several Gram-positive and mycobacteria, including human pathogens. Hence c-di-AMP synthesizing enzymes (DACs) have become a promising antibacterial drug target. To overcome a scarcity of small molecule inhibitors of c-di-AMP synthesizing enzyme CdaA, a computer-aided design of a new compound that should block the enzyme has been performed. This has led to the identification of a molecule comprising two thiazole rings and showing inhibitory potential based on ITC measurements. Thiazole scaffold is a good pharmacophore nucleus known due to its various pharmaceutical applications. It is contained in more than 18 FDA-approved drugs as well as in dozens of experimental drugs. Hence, the designed inhibitor can serve as a potent lead compound for further development of inhibitor against CdaA.

A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA

“…These two conserved hydrogen bonds have been observed in several CdaA complex structures (ADP, ATP and c-di-AMP), regardless of the postcatalytic or precatalytic state, the well described Tyr187 backrub motion and its �-� stacking interactions with aromatic rings of bound ligands (Fig. 6; Neumann et al, 2023). Superposition of ATP-bound CdaA (PDB entry 8c4o) with the structure containing fragment D07 (Figs.…”

“…Remarkably, the aforementioned structural pattern was also used as a strong donor and acceptor constraint for the development of the bi-thiazole inhibitor (compound 7; Fig. 6), which binds to CdaA in the micromolar range and with a K d �8-fold lower than that of the natural substrate ATP (Neumann et al, 2023). Superposition of the crystal structure of the CdaA-compound 7 complex (PDB entry 8c4p) with those of the B04 and D07 fragment complexes reported here (Fig.…”

“…The previously reported crystallization condition for apo CdaA (Heidemann et al, 2019) was optimized in order to yield crystals that were more suited for fragment screening, i.e. had better diffraction properties (Neumann et al, 2023). Crystallization trials were performed at 20 � C using a concentration of 6 mg ml À 1 �100CdaA in 2 ml droplets and a 1:1 protein:reservoir ratio.…”

“…Protein expression and purification were performed as described previously (Heidemann et al, 2019;Neumann et al, 2023). Briefly, the plasmid pGEXpBP33, originating from the pGEX-6P-1 (Cytiva) vector and encoding a truncated Lm CdaA protein (�100CdaA) with an N-terminal GST tag, was transformed into E. coli BL21 (DE3) cells.…”

“…The two active sites (one per monomer) are exposed to solvent, which makes them accessible to small-molecule ligands and favors soaking experiments. Two of the identified fragments that have been localized in the ATP-binding site of CdaA could serve as potential starting points for antibiotic drug discovery and for 'growing' the computationally designed lead candidate comprising two substituted thiazole rings (compound 7;Neumann et al, 2023). This in silico-designed compound showed inhibitory potential based on ITC measurements and is the first structurally characterized CdaA inhibitor to date.…”