Computational structure modeling, functional characterization, and identification of potential inhibitors for the cyclic-di-GMP mediated biofilm forming membrane protein in Vibrio cholerae.

Abstract

Cholera, caused by Vibrio cholerae, continues to pose a serious global public health challenge, with its impact worsened by rising antibiotic resistance associated with bacterial biofilm formation. This study explicitly describes the role of the uncharacterised protein (UP) TYC33605.1 in cyclic-di-GMP (c-di-GMP)-mediated biofilm regulation and identifies natural computationally predicted inhibitors to disrupt this mechanism. Functional annotation revealed TYC33605.1 as a membrane-associated diguanylate cyclase (DGC) with GGDEF and sensory domains, a potential driver for c-di-GMP synthesis and biofilm persistence. Homology modelling and molecular dynamics (MD) simulations supported a plausible, stable predicted 3D structure (C-score: -1.22, Ramachandran favoured regions: 91.1%) and dynamic behaviour (average RMSD: 8.55 Å). Moreover, virtual molecular docking screening of 1,092 natural compounds identified Luteolin (CID 5280445) and Sativanone (CID 13886678) as top candidates, exhibiting strong binding affinities (- 9.1 and - 9.0 kcal/mol, respectively) and forming stable hydrogen bonds, π-cation, and hydrophobic interactions with key residues (Glu293, Arg364, Ala176). MD simulations (100 ns) were consistent with complex stability, with Luteolin and Sativanone showing lower RMSD fluctuations (7.78 and 8.13 Å) compared to the control and apoprotein. The ADME/Tox predictions indicated favourable pharmacodynamics (PD), pharmacokinetics (PK), high gastrointestinal absorption, no hepatotoxicity, and drug-likeness (Lipinski compliance). Besides, principal component, probability density function, and free energy analyses were consistent with ligand-induced conformational stability. This study proposes the molecular characterisation of the protein and the bioactive compounds Luteolin and Sativanone as promising inhibitors targeting TYC33605.1, offering a novel strategy to combat biofilm-mediated antibiotic resistance and a framework for analogous antimicrobial discovery in Vibrio cholerae.

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Supplementary information: The online version contains supplementary material available at https://doi.org/10.1007/s40203-026-00719-z.