Design of novel anti-quorum sensing peptides targeting LuxO to combat Vibrio cholerae pathogenesis.

Vibrio cholerae, the Gram-negative bacterium abruptly colonizes the human intestine causing diarrhea, employing quorum sensing (QS) system as the major survival technique for mediating biofilm formation, virulence, competence, etc. Two distinct QS systems coordinated by the auto-inducer molecules, cholera-specific CqsA/S system and universal LuxS/PQ system, operate in parallel converging into a common phosphorelay pathway involving LuxU and LuxO. The master regulatory proteins of the QS system, AphA and HapR regulate the biofilm formation based on cell density, whose expression is controlled by the global response regulator LuxO. At low cell density, activated LuxO indirectly represses HapR, favoring the virulence cascade expression. Rather at high cell densities, LuxO represses AphA expression subsequently blocking the expression of virulence factors. Hence, targeting LuxO would be a promising strategy to downregulate the virulence pathway and modulate the QS system. With this insight, the present study has been designed to intrude the interaction between LuxU and LuxO through in silico design of novel peptides and validation of these peptides through molecular simulations. QS peptides were designed using QSPred server by altering the template sequence representing the LuxU-LuxO interaction, among which PEP8 exhibited better interaction and stability with the target protein LuxO. These in silico designed novel peptides would serve as potential target-specific molecules that would inhibit the LuxU-LuxO interaction and modulate the QS system to restrict Vibrio cholerae pathogenesis. However, further in vitro validations would substantiate the efficacy of these novel QS peptides. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00172-2.

Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 µm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly folded secretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.