Thioredoxin Reductase as a Target for Antibacterial Gold Compounds in Burkholderia cenocepacia: Disclosing the Molecular Basis of Enzyme Inhibition

Molecular insights into inhibitor action on the catalytic activity of Mycobacterium tuberculosis cystathionine β-synthase enzyme.

Tuberculosis (TB) remains a major global health threat, with Mycobacterium tuberculosis (Mtb) infecting nearly a quarter of the global population. Drug-resistant TB and HIV-TB co-infections emphasize the need for novel therapeutic approaches targeting essential metabolic pathways. Here, we investigated Mtb cystathionine β-synthase (MtbCBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme critical for sulfur metabolism and redox regulation, owing to its potential as a therapeutic target. Despite growing efforts to develop novel therapeutics, the widely used inhibitor aminooxy acetic acid (AOAA) is a non-specific inhibitor of all PLP-dependent enzymes, and the precise structural and mechanistic basis for its activity and specificity remains poorly understood. We present the high-resolution cryo-EM structure of full-length tetrameric MtbCBS in complex with AOAA, revealing a stable PLP-inhibitor adduct stabilized by two highly conserved active-site residues, T75 and Q147. This integrated approach employs cryo-EM, molecular dynamics (MD) simulations, Density Functional Theory (DFT) calculations, and comparative inhibition studies to reveal the molecular basis and determinants governing PLP-enzyme MtbCBS inhibition by AOAA. Through molecular mimic studies, we identified precise structural and electronic features of the inhibitor candidate that are critical for inhibition efficiency. These findings provide a mechanistic rationale for MtbCBS inhibition, and the unexplored roles of these key residues can be considered in the design of next-generation inhibitors targeting CBS enzymes implicated in infectious diseases, cancer, and neurological disorders.

Apo and substrate-bound dihydropteroate synthase crystal structures from Thermus thermophilus HB8.

A cryptic aromatic binding pocket in the highly thermostable GpgS from Mycobacterium hassiacum hints at MGLP pathway crosstalk

The findings establish M. hassiacum as a valuable model for structural enzymology in mycobacteria and highlight GpgS as a potential drug target, opening new avenues for the development of inhibitors targeting this key, potentially pleiotropic enzyme.

Structural and mechanistic basis for the heterotetrameric benzaldehyde synthase from petunia

Benzaldehyde is a widespread volatile compound produced by plants. Its final biosynthetic step is catalyzed by benzaldehyde synthase (BS), a peroxisomal enzyme composed of α and β subunits, both belonging to the short-chain dehydrogenase/reductase (SDR) family. Here, we report the crystal structure of Petunia hybrida BS, which reveals an α2β2 heterotetrameric arrangement. Structural and biochemical analyses show that the α subunits contain the canonical catalytic site, whereas the β subunits have lost catalytic activity but are essential for heterotetramer assembly. Notably, the C terminus of the β subunit extends into the diagonally positioned α subunit, contributing to the formation of the composite benzoyl-CoA substrate-binding pocket. Site-directed mutagenesis and subunit-mixing experiments support noncooperative, additive contributions of protomers within the heterotetramer. This work establishes BS as a rare heterotetrameric plant SDR and demonstrates how subunit specialization and intersubunit arrangement enable function, providing principles for understanding and engineering multimeric enzyme complexes.

Antibacterial Activity of α‐Sulfamidophosphonate Derivatives and Their In Silico Assessment as Dihydropteroate Synthase Binders

In this study, we report the first antibacterial evaluation of a focused library of 12 previously synthesized α‐sulfamidophosphonate derivatives (4a–4l) against a panel of nine Gram‐positive and Gram‐negative strains, including ESBL‐ and carbapenemase‐producing Enterobacterales. The overall hit rate was modest, as most compounds were inactive at the highest tested concentration (MIC ≥ 256 µg/mL), indicating a narrow and strain‐selective profile. Nevertheless, selected derivatives showed noteworthy activity against individual isolates: 4 g inhibited Staphylococcus aureus ATCC29213 at 4 µg/mL, 4c inhibited KPC‐3‐producing Klebsiella pneumoniae at 2 µg/mL, and 4e and 4l inhibited VIM‐producing Escherichia coli at 4 µg/mL. Density functional theory (DFT) calculations were used to examine substituent‐dependent electronic features, whereas docking and 100 ns molecular dynamics (MD) simulations were employed to test whether the scaffold can adopt plausible binding modes in a dihydropteroate synthase (DHPS) model. Because no biochemical DHPS assay was performed, the computational results should be regarded as hypothesis‐generating rather than mechanistic proof. In silico ADMET predictions were used as an exploratory triage step and similarly await experimental validation. Overall, the present work identifies α‐sulfamidophosphonates as preliminary antibacterial hits for further optimization, while emphasizing the need for broader microbiology, cytotoxicity, bactericidal, and target‐validation studies.

Discovery of Novel LRPPRC Inhibitors Featuring a Biphenyl-Acylhydrazone Scaffold with Potent Broad-Spectrum Antitumor Activity.

Leucine-rich pentatricopeptide repeat containing (LRPPRC), a critical regulator of mitochondrial gene expression, is overexpressed in various malignancies and sustains oxidative phosphorylation (OXPHOS)-dependent adenosine triphosphate (ATP) production essential for tumor growth, chemoresistance, and stem cell survival, rendering it a promising therapeutic target. Herein, using an aptamer-assisted fluorescence polarization platform, we identified acylhydrazone-skeleton inhibitors targeting LRPPRC's RNA-binding domain, leading to the design and synthesis of over 60 derivatives. Lead compound 3o exhibited excellent LRPPRC inhibitory activity (92% at 6.25 μM vs 38% for gossypol acetate (GAA)) and induced robust LRPPRC degradation. Notably, 3o downregulated downstream OXPHOS subunits and ATP synthase, eliciting broad antiproliferative effects, particularly in refractory and drug-resistant A549, BXPC-3, and NCI-H1975 cells (IC50 = 0.54, 0.27, and 1.39 μM, respectively). In PC9 and HCT116 xenografts, 3o achieved tumor growth inhibition (TGI) rates of 73 and 49% with favorable safety profiles. Overall, we developed novel biphenyl-acylhydrazone LRPPRC inhibitors as potent antitumor agents acting via OXPHOS modulation, providing a valuable lead compound for cancer therapy.