Unmasking the Black Box: Computational Evidence Identifying Mitochondrial ATP Synthase Subunit β as a High-Confidence Target of Glycyrrhetinic Acid Analogs Against Brugia malayi

<title>Abstract</title> <p> Lymphatic filariasis (LF), a devastating tropical disease caused by filarial nematodes such as Brugia malayi, remains an urgent public health concern due to the microfilaricidal limitations of frontline treatments. While Kalani et al. (2013) identified compound 6a, a semi-synthetic benzyl amide analog of glycyrrhetinic acid (GA), as a highly potent in vitro macrofilaricide (IC ₅₀  = 8.8 <italic>µ</italic> <bold>M), its molecular mechanism of action has remained an unmapped black box. In this study, we bridged functional genomics and structural biology by conducting a proteome-wide reverse molecular docking screen against 395 high-confidence AlphaFold2 models (average pLDDT</bold>   <italic>≥</italic>   <bold>70) selected from the</bold> 589 essential filarial protein targets pre-filtered by Kumar et al. (2007). Mitochondrial ATP synthase subunit β (UniProt: A0A4E9FU14) was selected for detailed investigation using a multi-criteria consensus approach integrating reproducible high-affinity binding, biological essentiality, and the availability of a structurally comparable human ortholog (UniProt: Q08ET0) for selectivity assessment. Independent docking performed using twenty replicates to ensure stochastic search convergence demonstrated consistent preferential binding of compound 6a ( ∆ <italic>G</italic> _{<bold>best</bold>}  =−9.011 <bold>kcal/mol</bold> , ∆ <italic>G</italic> _{<bold>mean</bold>}  =−8.818  <italic>±</italic>  0.122 <bold>kcal/mol) over parent GA (</bold> ∆ <italic>G</italic> _{<bold>best</bold>}  =−7.125 <bold>kcal/mol) and Diethylcarbamazine (</bold> ∆ <italic>G</italic> _{<bold>best</bold>}  =−4.365 <bold>kcal/mol). The Protein-Ligand Interaction Profiler (PLIP v2.2.2) and independent pocket predictions (PrankWeb and fpocket) supported the structural plausibility of the proposed binding pocket on the solvent-accessible catalytic</bold> <italic>F</italic> ₁ <bold>domain. Although the predicted affinity difference between the parasite and human ortholog (</bold> ∆∆ <italic>G ≈</italic>  0.89 <bold>kcal/mol) is modest and falls within the known systematic uncertainty of empirical docking scoring functions (</bold> <italic>±</italic>  2.0 <bold>kcal/mol), the directional trend remained highly consistent across independent docking runs. Because no experimentally determined ligand-bound structure of</bold> <bold>B. malayi</bold> <bold>ATP synthase</bold> <italic>β</italic> <bold>is currently available, conventional re-docking validation was not feasible; therefore, reproducibility analyses, ortholog comparisons, independent pocket predictions, and protein-ligand interaction profiling were employed as complementary computational validation strategies. All receptor and ligand preparations were performed using standard physiological protonation states, and the results should be regarded as computational evidence supporting ATP synthase subunit</bold> <italic>β</italic> <bold>as a high-confidence candidate molecular target of compound 6a, pending further validation through molecular dynamics simulations and experimental studies.</bold> </p>