Biophysica

Hexokinase-I anchors to voltage-dependent anion channel 1 (VDAC1) at a membrane-buried glutamate, E73, and disrupting this contact is an established anticancer objective. The site-directed docking of natural product libraries into such hotspots routinely identifies disruptors, yet these candidates are rarely tested for either site specificity or ligand specificity. We screened a verified 43-compound library from Camptotheca acuminata against the VDAC1 E73 groove and then applied five orthogonal controls, calibrated against an empirical uncertainty budget. The conventional workflow was persuasive; ursolic acid ranked first at −7.25 kcal/mol, redocked reproducibly, remained associated with the groove through 100 ns of POPC-embedded dynamics, and returned −42.7 kcal/mol by Molecular Mechanics/Generalized Born Surface Area (MM-GBSA). Replicate calculations then showed that score uncertainty was dominated not by stochastic search, which contributes a standard deviation of 0.007 kcal/mol, but by the arbitrary definition of the site, 0.379 kcal/mol, and by receptor conformation, 0.550 kcal/mol. Against this scale, the controls rule out the candidates. Blind docking identified alternative sites with scores equal to or better than E73. A scan of twenty-four surface sites placed E73 within one box placement standard deviation of several unrelated positions. Neutralizing the E73 carboxylate changed affinity by −0.30 to +0.04 kcal/mol across fifteen compounds. Cholesterol, a membrane lipid, scored within the same uncertainty, and outscored the lead ligand under a second scoring function and in four of six relaxed receptor conformations. Contact occupancies reached 29% for the lead ligand compared to 100% for the reference ligand, NADH. We propose using the controls as a checklist, as illustrated by this case study.

Accurate background subtraction is critical in the analysis of X-ray fiber diffraction patterns from striated muscle. Diffuse scattering and low signal-to-noise ratio complicate the separation of signal from background. Existing methods are applied heuristically, without guidance on parameter selection, leading to variability in following analyses. We propose a framework for background removal in muscle fiber diffraction images that evaluates methods and selects optimal parameters. It combines quantitative metrics and validation using synthetic features to guide the optimization. Minimizing the aggregate loss produced visually plausible results in the representative cases examined by the authors; independent blinded validation was not performed. The loss metric and its components allow currently used approaches to be compared within a single reproducible scheme. Individual metrics show trade-offs among approaches, such as preservation of detail or oversubtraction. Additionally, we evaluate parametric fitting of the equatorial streak and isotropic general background, which may be combined with the optimization framework. The framework improves the analysis of fiber diffraction patterns by providing a reproducible quantitative basis for the choice of background subtraction method and its parameters.

Bromelain is a widely used as model for papain-like cysteine proteases and shares significant sequence homology with bacterial and viral counterparts, making it a suitable target for computational screening of potential modulators. In this study, we performed molecular docking and protein–ligand interaction profiling for eight synthetic derivatives containing 6-chloroquinolone (compounds 1–4), 1,3,5 triazine (compounds 5–7) or furopyrrole (8) scaffolds. In the quinoline series, the position of the methyl group on the piperidine ring appeared to influence efficacy (4 methyl > unsubstituted > 2 methyl). Molecular docking and PLIP interaction profiling revealed that the highest computed binding affinities (up to –12.4 kcal/mol for 5) did not directly correlate with experimental inhibition, but identified the residues predicted by docking and PLIP to contribute to ligand interactions: Lys18, Phe29, Ile163, Trp180, Tyr185, and Ile186. Ligands 6 and 7 formed H-bonds with catalytic residues His158 and Cys26, respectively. Sequence alignment revealed 42–49% identity between bromelain and bacterial cysteine proteases from Klebsiella pneumoniae and Acinetobacter baumannii, suggesting that bromelain may serve as a tentative model system for the preliminary study of pathogen cysteine proteases, although direct extrapolation would require experimental validation. To experimentally assess these predictions, we conducted a preliminary activity screen using an azocasein proteolytic assay. Compound 1 exhibited a moderate inhibitory effect, whereas compounds 2–8 produced only marginal changes, which lacked statistical significance after correction for multiple comparisons. These findings provide a structural basis for the rational design of new protease inhibitors and identify compound 1 as a preliminary screening hit and a possible starting point for further optimization.