ABSTRACT

Background and Aim: Benzimidazoles (BZ) remain the cornerstone of soil-transmitted helminth control programs; however, the emergence of resistance-associated mutations in the β-tubulin gene threatens their long-term effectiveness. Information on BZ resistance markers in hookworms from Southeast Asia remains limited. This study investigated the occurrence of β-tubulin isotype 1 mutations in hookworms from the Philippines and Indonesia and evaluated the potential effects of detected variants on BZ binding and tubulin dimerization using in silico analyses. 

Materials and Methods: Eighteen archived hookworm-positive fecal samples collected from the Philippines (n = 13) and Indonesia (n = 5) were analyzed. Molecular speciation was performed using species-specific genetic markers, followed by amplification and sequencing of the β-tubulin isotype 1 gene regions that encompass known BZ resistance codons. Identified variants were subjected to in silico molecular docking, molecular dynamics simulations, binding free-energy calculations, and protein–protein interaction analyses to assess their effects on drug binding and tubulin dimer formation. 

Results: Fifteen samples were identified as Necator americanus and three as Ancylostoma ceylanicum. No canonical BZ resistance-associated mutations were detected at codons 134, 167, 198, or 200. However, two A. ceylanicum isolates from the Philippines carried a novel V236I substitution resulting from a GTC→ATC nucleotide change. Molecular docking demonstrated minimal alterations in docking poses, whereas molecular dynamics simulations revealed a marked reduction in BZ-binding favorability in the mutant protein. The V236I variant exhibited substantially lower MM-GBSA binding free-energy with albendazole than the wild-type protein (−13.34 vs. −33.42 kcal/mol), accompanied by reduced hydrogen-bond formation. Structural analyses suggested that the bulkier isoleucine side chain may introduce steric hindrance within the binding region, thereby weakening ligand–protein interactions. In contrast, the mutation produced negligible effects on tubulin dimer stability, binding affinity, and conformational dynamics. 

Conclusion: A novel V236I substitution was identified in A. ceylanicum from Southeast Asia and was predicted to reduce BZ-binding efficiency through steric and energetic effects while preserving tubulin dimer integrity. These findings highlight V236I as a potential BZ resistance-associated variant and emphasize the need for continued molecular surveillance and experimental validation of emerging resistance markers in hookworm populations. 

Keywords: albendazole, Ancylostoma ceylanicum, benzimidazole resistance, β-tubulin, hookworm, molecular dynamics, soil-transmitted helminths, Southeast Asia.