Unraveling the Mechanisms of Benzimidazole Resistance in Hookworms: A Molecular Docking and Dynamics Study

Overview

Journal J Genet Eng Biotechnol

Specialty Biotechnology

Date 2025 Mar 12

PMID 40074446

Authors

Jan Clyden B Tenorio

Muhammad Fikri Heikal

Alok Kafle

Mark Andrian B Macalalad

Fredmoore L Orosco

Prasert Saichua

Sutas Suttiprapa

Affiliations

Soon will be listed here.

Abstract

Background: Benzimidazole resistance is an emerging challenge among parasitic helminths. It is caused by single nucleotide polymorphisms (SNPs) in specific loci in helminths' β-tubulin genes. Field studies and laboratory investigations reported resistance-associated SNPs in 4 codon locations with 7 allelic variations among hookworms. This study aimed to determine the effects of these mutations on the binding efficiency and behavior of the β-tubulin protein in four hookworm species against four benzimidazole drugs.

Methods: β-tubulin gene coding sequences of Ancylostoma caninum, A. duodenale, A. ceylanicum, and Necator americanus were retrieved, assessed phylogenetically, and used to construct the 3D structure models of the proteins. The modeled protein structures were verified and edited to contain the reported SNPs: Q134H, F167Y, E198A, E198K, E198V, F200L, and F200Y. Benzimidazole drugs such as albendazole (ABZ), fenbendazole (FBZ), mebendazole (MBZ) and oxfendazole (OBZ) were used as ligands. Molecular docking experiments were performed with the wild-type and mutated proteins. Molecular dynamics simulation assessed the dynamic behavior of the β-tubulin-benzimidazole complex.

Results: In silico docking assessments showed that various amino acid substitutions due to resistance-associated SNPs cause alterations in binding affinities and positions. E198K and Q134H in hookworm β-tubulins substantially weakened the binding affinities and altered the binding positions of benzimidazole drugs. Molecular dynamics analysis revealed that these mutations also caused marked reductions in the binding free energies owing to diminished hydrogen bond contacts with the benzimidazole ligands.

Conclusion: The evidence shown herein indicates that mutations at positions 198 and 134 are detrimental to conferring benzimidazole resistance among hookworms. The presence of these mutations may alter the efficacy of pharmacological interventions. Hence, further studies should be conducted to assess their emergence among hookworms in endemic areas with histories of chemotherapy.

References

Kim S, Thiessen P, Bolton E, Chen J, Fu G, Gindulyte A . PubChem Substance and Compound databases. Nucleic Acids Res. 2015; 44(D1):D1202-13. PMC: 4702940. DOI: 10.1093/nar/gkv951. View

Furtado L, de Paiva Bello A, Rabelo E . Benzimidazole resistance in helminths: From problem to diagnosis. Acta Trop. 2016; 162:95-102. DOI: 10.1016/j.actatropica.2016.06.021. View

Kalule F, Vudriko P, Nanteza A, Ekiri A, Alafiatayo R, Betts J . Prevalence of gastrointestinal parasites and molecular identification of beta-tubulin mutations associated with benzimidazole resistance in Haemonchus contortus in goats from selected districts of Uganda. Vet Parasitol Reg Stud Reports. 2023; 42:100889. DOI: 10.1016/j.vprsr.2023.100889. View

Ngetich A, Amoah I, Bux F, Kumari S . Anthelmintic resistance in soil-transmitted helminths: One-Health considerations. Parasitol Res. 2023; 123(1):62. PMC: 10730643. DOI: 10.1007/s00436-023-08088-8. View

. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2018; 47(D1):D506-D515. PMC: 6323992. DOI: 10.1093/nar/gky1049. View

Trott O, Olson A . AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2009; 31(2):455-61. PMC: 3041641. DOI: 10.1002/jcc.21334. View

Prichard R . Markers for benzimidazole resistance in human parasitic nematodes?. Parasitology. 2007; 134(Pt 8):1087-92. DOI: 10.1017/S003118200700008X. View

von Samson-Himmelstjerna G, Blackhall W, McCarthy J, Skuce P . Single nucleotide polymorphism (SNP) markers for benzimidazole resistance in veterinary nematodes. Parasitology. 2007; 134(Pt 8):1077-86. DOI: 10.1017/S0031182007000054. View

Jimenez Castro P, Durrence K, Durrence S, Sicalo Gianechini L, Collins J, Dunn K . Multiple anthelmintic drug resistance in hookworms (Ancylostoma caninum) in a Labrador breeding and training kennel in Georgia, USA. J Am Vet Med Assoc. 2022; 261(3):342-347. DOI: 10.2460/javma.22.08.0377. View

10.

Venkatesan A, Jimenez Castro P, Morosetti A, Horvath H, Chen R, Redman E . Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel β-tubulin benzimidazole resistance mutation. PLoS Pathog. 2023; 19(3):e1011146. PMC: 10013918. DOI: 10.1371/journal.ppat.1011146. View

11.

Dilks C, Koury E, Buchanan C, Andersen E . Newly identified parasitic nematode beta-tubulin alleles confer resistance to benzimidazoles. Int J Parasitol Drugs Drug Resist. 2021; 17:168-175. PMC: 8503852. DOI: 10.1016/j.ijpddr.2021.09.006. View

12.

Tenorio J, Heikal M, Kafle A, Saichua P, Suttiprapa S . Benzimidazole Resistance-Associated Mutations in the β-tubulin Gene of Hookworms: A Systematic Review. Parasitol Res. 2024; 123(12):405. DOI: 10.1007/s00436-024-08432-6. View

13.

Liu Y, Chen X, Jiang J, Hamada M, Yin Y, Ma Z . Detection and dynamics of different carbendazim-resistance conferring β-tubulin variants of Gibberella zeae collected from infected wheat heads and rice stubble in China. Pest Manag Sci. 2013; 70(8):1228-36. DOI: 10.1002/ps.3680. View

14.

Tenorio J, Heikal M, Kafle A, Saichua P, Suttiprapa S . Benzimidazole resistance-associated mutations improve the dimerization of hookworm tubulin: An additional resistance mechanism. Vet World. 2025; 17(12):2736-2746. PMC: 11784061. DOI: 10.14202/vetworld.2024.2736-2746. View

15.

Doyle S, Laing R, Bartley D, Morrison A, Holroyd N, Maitland K . Genomic landscape of drug response reveals mediators of anthelmintic resistance. Cell Rep. 2022; 41(3):111522. PMC: 9597552. DOI: 10.1016/j.celrep.2022.111522. View

16.

Robinson M, McFerran N, Trudgett A, Hoey L, Fairweather I . A possible model of benzimidazole binding to beta-tubulin disclosed by invoking an inter-domain movement. J Mol Graph Model. 2004; 23(3):275-84. DOI: 10.1016/j.jmgm.2004.08.001. View

17.

Aguayo-Ortiz R, Mendez-Lucio O, Romo-Mancillas A, Castillo R, Yepez-Mulia L, Medina-Franco J . Molecular basis for benzimidazole resistance from a novel β-tubulin binding site model. J Mol Graph Model. 2013; 45:26-37. DOI: 10.1016/j.jmgm.2013.07.008. View

18.

. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018; 392(10159):1789-1858. PMC: 6227754. DOI: 10.1016/S0140-6736(18)32279-7. View

19.

Jones B, Van Vliet A, LaCourse E, Betson M . Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations. Sci Rep. 2022; 12(1):13725. PMC: 9374697. DOI: 10.1038/s41598-022-16765-4. View

20.

Schwede T, Kopp J, Guex N, Peitsch M . SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 2003; 31(13):3381-5. PMC: 168927. DOI: 10.1093/nar/gkg520. View