Benzimidazole resistance-associated mutations improve the in silico dimerization of hookworm tubulin: an additional resistance mechanism?

doi:10.21203/rs.3.rs-4923236/v1

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Benzimidazole resistance-associated mutations improve the in silico dimerization of hookworm tubulin: an additional resistance mechanism?

https://doi.org/10.21203/rs.3.rs-4923236/v1

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Background

Benzimidazole resistance is conferred by mutations in the β-tubulin genes of helminths which result in diminished drug binding with the expressed protein. The impact of these resistance-associated mutations on tubulin dimerization has not been fully explored in soil-transmitted helminths. Hence, this research aims to determine the consequences of these mutations on the in silico dimerization of hookworm α- and β-tubulins using open-source bioinformatics tools.

Methods

The α- and β-tubulin amino acid sequences of Ancylostoma ceylanicum were used to predict the hookworm tubulin heterodimer fold via AlphaFold 3. Modeled complexes underwent several protein structure quality assurance checks. The complex’s binding free energies, overall binding affinity, dissociation constant, and interacting amino acids were determined. The dimer’s structural flexibility and motion were simulated through molecular dynamics.

Results

Benzimidazole resistance-associated amino acid substitutions in the β-tubulin isotype 1 protein of hookworms conferred alterations in tubulin dimerization. The E198K, E198V, and F200Y mutations provided the strongest and most stable binding between the α and β subunits, surpassing that of the wild-type. The opposite was observed in complexes with the Q134H and F200L mutations. The molecular dynamics simulations showed that both wild-type and mutated tubulin dimers shared similar dynamic behavior, except for slight alterations in those that have the F200L and E198K mutations.

Conclusion

Beyond hindering benzimidazole binding to the β-tubulin subunit, resistance-associated mutations enhance the hookworm's capacity to resist treatment through strengthening and stabilizing tubulin dimer interactions. Meanwhile, other mutations diminish the dimer’s interaction which could be to the detriment of the hookworm. Our results provide novel insights into helminth tubulin dimerization that are useful for research on new anthelmintics targeting tubulin dimerization.

Parasitology

Ancylostoma

Antimicrobial Resistance

Microtubules

Soil-transmitted helminths

Soi-transmitted helminths continue to be the most prevalent Neglected Tropical Disease globally [1, 2]. One of these helminths of public health concern, hookworms stand out due to the severity of their pathological consequences of infections [3]. Recent reports indicate that hookworm prevalence in endemic areas is around 32–79% and these infections cause 800,000 to 4 million disability-adjusted life years lost [4, 5]. These blood-sucking pathogens also cause significant infection burden and illness in poorly kept companion animals [6, 7]. To address this public and veterinary health concern, therapeutic interventions that eliminate and prevent infections are the best option currently being implemented [8, 9]. Among humans, the World Health Organization recommends the prolonged administration of benzimidazole drugs to susceptible populations to reduce prevalence and infection intensity and prevent reinfection [10]. In animals, routine veterinary deworming is done to achieve objectives similar to those in human interventions [11–13]. However, the repeated and protracted implementation of these pharmacological interventions has raised concerns about the emergence of resistance against the drugs used in these programs [14, 15].

Benzimidazoles are anthelmintics that cause microtubule disassembly through inhibition of tubulin dimerization by binding with the β-subunit [16]. Resistance against benzimidazoles is conferred by mutations that occur in the β-tubulin gene of helminths [17]. Single nucleotide mutations that occur in these genes result in amino acid substitutions that potentially alter the structure of the expressed β-tubulin which reduces the binding affinity of the benzimidazole drug ligands [18, 19]. Canonical amino acid substitutions associated with benzimidazole resistance include changes in positions 167 (TTC, TTT/ phenylalanine → TAC, TAT/tyrosine), 198 (GAG, GAA/ glutamic acid → GCG, GCA/alanine) and 200 (TTC/ phenylalanine → TAC/tyrosine) [20, 21]. Among hookworms, several variations of these mutations have also been reported: Q134H, E198K, E198V, and F200L [14, 22–25]. These mutations are currently widespread in Ancylostoma caninum in the United States and Canada and have also been reported in several hookworm species in Africa, South America, the Caribbean, New Zealand, and Australia [24, 26, 27]. The occurrence of these mutations in hookworm isolates from endemic countries presents a threat as they may undermine current control and elimination measures implemented to abate hookworm infections in humans and animals.

The role of benzimidazole resistance-associated mutations in conferring resistance has been well-studied and well-established in vivo, in vitro, and in silico in helminths like Caenorhabditis elegans [28, 29], Haemonchus conturtos [30], Trichenella spiralis [31], among others. However, very little is known about the effect of carrying these resistance-associated mutations on the performance of normal tubulin function in soil-transmitted helminths. Experimental data shows that hookworms that carry some of these mutations and manifest benzimidazole resistance phenotypes pay the price of reduced survivability [23, 32]. In contrast, there are several evidence from field investigations showing that hookworms carrying resistance-associated mutations were able to resist benzimidazole treatment and continue to have such mutations post-treatment [22, 25, 33]. Varied survivability due to having benzimidazole-resistance-associated mutations could be due to unknown effects on tubulin function. To delve deeper into this issue, we conducted an in silico investigation to determine the consequences of these mutations on the dimerization of hookworm α- and β-tubulins using A. ceylanicum as our model hookworm. Our results indicate that several benzimidazole resistance-associated mutations enhance the hookworm's capacity to resist treatment through strengthening and stabilizing tubulin dimer interactions. Conversely, other mutations diminish inter-subunit interactions which could be to the detriment of the hookworm.

Prediction of hookworm tubulin dimer folding via AlphaFold3

Ancylostoma ceylanicum was used as the model hookworm for the current in silico investigation. The UniProt server was used to retrieve the amino acid sequences of α tubulin (UniProt ID: A0A016VPU0) and β tubulin (UniProt ID: A0A0D6MC88) proteins from A. ceylanicum (https://www.uniprot.org/) [34]. Benzimidazole (BZ) resistance-associated mutations occur in the β tubulin isotype 1 gene of several hookworm species [6]. Hence, the isotype 1 β tubulin of A. ceylanicum was used in this research. Known resistance-associated non-synonymous mutations were introduced to the β tubulin protein sequences via BioEdit Sequence Alignment Editor version 7.2.5 [35]. These amino acid substitutions include Q134H, F167Y, E198A, E198K, E198V, F200Y, and F200L [14, 22–25].

AlphaFold 3 was used to predict the tubulin heterodimer folding of the hookworm tubulin complex using the α and β tubulin amino acid sequences, both wild-type and mutated. AlphaFold 3 leverages artificial intelligence and extensive training on biomolecular data to predict 3D structures and interactions of biological molecules, including protein complexes [36] (https://golgi.sandbox.google.com). The amino acid sequences were uploaded to the server. One copy of each of α and β tubulin subunits was modeled as two separate proteins with the addition of two molecules of GTP (Guanosine-5'-triphosphate) as ligands. The addition of two GTP molecules was to simulate the molecule’s role in the dynamic instability of microtubules, which is reliant on GTP binding and hydrolysis within each tubulin subunit [37]. No post-translational modifications were added to either tubulin subunit. The 3D structure, the plDDT, ipTM, and pTM scores, and the predicted aligned error plots were documented and scrutinized. All models with ipTM and pTM scores of 0.8 and above were considered high-quality predictions and thus were downloaded. The .cis files from the server were further assessed using BIOVIA Discovery Studio Visualizer version 21.1.0.20298 (https://discover.3ds.com/discovery-studio-visualizer-download). These files were then saved in the .pdb format for downstream applications. The folded tubulin dimer was further refined using several open-source servers. GalaxyWEB’s GalaxyRefineComplex server was used to refine the protein-protein complex’s interface contact and inter-protein orientation through side-chain repacking and molecular dynamics relaxation (https://galaxy.seoklab.org/cgi-bin/submit.cgi?type=COMPLEX) [38] [Accessed: 17 May 2024].

Quality Assurance Checks of the Modeled Dimers

The quality of modeled and refined tubulin heterodimers was assessed using several assessment programs within the UCLA-DOE LAB — SAVES v6.0 platform (https://saves.mbi.ucla.edu/). VERIFY3D was used to assess the compatibility of the 3D protein structure with its amino acid sequence [39]. Models with scores higher than 80% were considered good-quality predictions of the 3D structure. PROCHECK’s Ramachandran Plot shows the allowed regions for the phi and psi backbones dihedral angles of the tubulin complex’s amino acid residues [40]. If more than 90% of the amino acid residues were in their most favored region, the modeled dimer was considered of good quality. Also, the ProSA (Protein Structure Analysis) server was used to identify errors in the 3D structure of proteins through statistical analysis of models against data of known proteins from databases (https://prosa.services.came.sbg.ac.at/prosa.php) [41]. The tubulin dimer models were uploaded to the server and each subunit was individually analyzed [Accessed: 18 May 2024]. Models with less than − 5.0 z-scores were considered of good quality. Additionally, the predicted wild-type tubulin dimer was subjected to pairwise structural alignment using the PDB Pairwise Structure Alignment Tool (https://www.rcsb.org/alignment) [Accessed: 18 May 2024]. The tubulin dimer of Bos taurus that was reported by Löwe et al. [42]was used as the template (PDB ID No.: 1JFF). The RMSD, TM-score, Identity, Aligned Residues, Sequence Length, and Modeled Residues were documented and evaluated.

Tubulin Heterodimer Interactions

The folded hookworm tubulin dimer’s protein-protein interactions were initially investigated using the HawkDock server (http://cadd.zju.edu.cn/hawkdock/). Specifically, HawkDock’s MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) analysis platform was utilized to estimate the dimer’s binding free energy (ΔG_bind), which reflects the thermodynamic favorability of the protein-protein interaction [43]. The previously energy minimized .pdb files were uploaded to the server noting that chain A (i.e., α tubulin) is the receptor and chain B (i.e., β tubulin) is the ligand. The protein complex’s binding free energy (in kcal/mol) and the top 10 interacting amino acid residues with their corresponding binding free energies (also in kcal/mol) were determined, documented, and assessed.

The tubulin dimer’s overall binding affinity (ΔG) and dissociation constant (K_d) were estimated using the PRODIGY (PROtein binDIng enerGY prediction) server (https://bianca.science.uu.nl/prodigy/). Utilizing a protein complex's 3D structure, this server utilizes intermolecular contacts and properties derived from the non-interface surface to predict its binding affinity [44]. The energy minimized .pdb files were uploaded to the server noting that chain A (i.e., α tubulin) is Interactor 1 and chain B (i.e., β tubulin) is Interactor 2. Analyses were set at 25 ℃. The ΔG (in kcal/mol) and K_d (in M) of the tubulin heterodimers were documented and analyzed. The volume of the benzimidazole binding site of the wild-type and mutated β-tubulin subunits using CAVER Web v.1.2 server (https://loschmidt.chemi.muni.cz/caverweb/) [45]. The β-tubulin subunit was extracted from the tubulin dimer file. It was uploaded to the server. The predicted binding site that included the amino acid positions that contain the resistance-associated mutations (i.e., 134, 167, 198, and 200) were selected.

Molecular Dynamics

Molecular dynamics simulation to assess the behavior of the protein complex was done using the WebGro server (https://simlab.uams.edu/index.php). GROMOS96 43a1 forcefield was applied in a Triclinic system with SPC water and 0.15 M neutralized NaCl salt model. Energy minimization at 5000 steps was done using steep descent algorithm. NVT/NPT equilibration at 300 K and 1.0 bar pressure, using the Leap-frog integrator, was applied to 50 ns simulations at 1000 frames per simulation. RMSD (Root Mean Square Deviation), RMSF (Root Mean Square Fluctuation), radius of gyration, and SASA plots as used to assess the dynamics simulation.

AlphaFold 3 satisfactorily predicted the folding of hookworm tubulin dimer

AlphaFold 3, through its deep learning algorithms, predicted the protein-protein interactions of the hookworm tubulin heterodimers based solely on the provision of the subunits’ amino acid sequences. Moreover, the post hoc complex refinement and energy minimization improved the quality of the prediction (Fig. 1). All folded tubulin dimers, both wild-type and those with BZ resistance-associated mutations, had pTM and ipTM scores above 0.8. All folded dimers passed the VERIFY 3D assessment; all scores were more than 80%. Further, the Ramachandran plots showed that more than 90% of the residues in all models were on their most favorable conformation. The dimer models were judged to have stable and correct folding based on having z-scores of more than − 8.0. Lastly, the pairwise structural alignment showed that the subunits of the predicted hookworm tubulin have a high degree of structural similarity to the crystal structure of its mammalian homolog (PDB ID No.: 1JFF) (Fig. 2). When taken together, the quality assurance checks both from AlphaFold 3 and the external verification servers indicate that the protein folding prediction of hookworm tubulin heterodimers by the aforementioned artificial intelligence server is accurate and reliable.

BZ resistance-associated mutations affect hookworm tubulin dimerization

Benzimidazole resistance-associated mutations in the β tubulin isotype 1 amino acid sequences of hookworms alter the binding properties of the two tubulin subunits. Among these alterations are changes in the ΔG_bind between the subunits (Fig. 3). Mutations in amino acid position 198 substantially increased the ΔG_bind. These findings are affirmed by the marked increase in ΔG and reduction in K_d in dimers with E198K, E198V, and F200Y (Table 1). Conversely, the Q134H and F200L mutations resulted in slightly diminished ΔG but a marked increase in the K_d indicating weaker and unstable interactions. BZ resistance-associated mutations also cause alterations in the interacting amino acids and their corresponding ΔG_bind between the α-and β-tubulin subunits (Table 2). These results mean that mutations that confer amino acid substitution in position 198 may result in more stable microtubule structures which can make it less susceptible to benzimidazole-induced disruption and hence aid resistance. Diminished binding values (i.e., ΔG_bind, ΔG, and K_d) caused by other mutations may lead to weaker interactions between the tubulin subunits and, thus unstable microtubules that could be detrimental to the hookworm. The binding site pocket volume in the β-tubulin subunit was determined using the CAVER Web v.1.2. Most of the mutations reduced the binding site’s volume (Fig. 4). Only F167Y increased the volume. A marked reduction was observed when E198A and F200Y were introduced to the β-tubulin subunits.

Table 1

The ΔG (overall binding affinity) and K_d (dissociation constant) of the predicted hookworm tubulin dimers that were estimated through the PRODIGY server (PRODIGY (PROtein binDIng enerGY prediction) (https://bianca.science.uu.nl/prodigy/).
α-β Tubulin Dimer	ΔG (kcal/mol)	K_d (M)
Wild type α-β Tubulin	-16.1	1.4e-12
α-β Q134H	-14.7	1.5e-11
α-β F167Y	-16.1	1.4e-12
α-β E198A	-14.0	5.6e-11
α-β E198K	-17.1	2.9e-13
α-β E198V	-17.0	3.3e-13
α-β F200L	-14.9	1.2e-11
α-β F200Y	-16.4	9.9e-13

Table 2

The top five interacting amino acids of the α and β tubulin subunits in the folded dimers and their corresponding ΔG_bind (binding free energies) that were determined via HawkDock’s MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) platform (http://cadd.zju.edu.cn/hawkdock/).
α-β Tubulin Dimer	α Tubulin Subunit		β Tubulin Subunit
α-β Tubulin Dimer	Interacting Amino Acid	ΔG_bind (in kcal/mol)	Interacting Amino Acid	ΔG_bind (in kcal/mol)
Wild type α-β Tubulin	LYS 397	-8.96	ARG 2	-7.1
	ARG 217	-8.13	LEU 436	-618
	ARG 210	-7.73	LYS 252	-6.04
	TRP 403	-7.54	ARG 251	-4.27
	ARG 398	-7.42	TRP 344	-4.09
α-β Q134H	ARG 398	-7.65	LYS 252	-7.49
	TRP 403	-7.61	ARG 2	-5.66
	ARG 210	-7.32	ASN 347	-5.06
	PHE 400	-6.78	ARG 324	-4.6
	VAL 177	-6.33	LEU 436	-4.59
α-β F167Y	ARG 398	-9.97	ARG 251	-9.31
	TRP 403	-8.22	ARG 324	-8.11
	PHE 400	-7.53	ASN 347	-6.64
	VAL 177	-7.25	ARG 2	-5.2
	ARG 210	-6.96	TRP 344	-4.76
α-β E198A	LYS 397	-8.43	ARG 251	-8.81
	TRP 403	-7.69	ARG 324	-8.75
	VAL 177	-7.44	ARG 2	-8.37
	PHE 400	-6.98	ASN 347	-5.14
	ARG 217	-6.68	TRP 311	-4.3
α-β E198K	ARG 398	-13.74	ARG 251	-9.53
	LYS 387	-9.22	ARG 324	-8.04
	ARG 217	-8.17	ARG 2	-5.62
	PHE 400	-7.8	TRP 344	-4.28
	ARG 210	-7.44	TYR 445	-4.26
α-β E198V	ARG 398	-10.02	ARG 251	-6.87
	ARG 210	-7.83	ARG 162	-5.84
	TRP 403	-7.76	ARG 2	-5.32
	PHE 400	-7.27	LEU 436	-4.25
	VAL 177	-6.62	PHE 260	-3.99
α-β F200L	ARG 217	-8.43	ARG 251	-8.64
	TRO 403	-8.14	ARG 324	-7.71
	VAL 177	-7.51	ARG 2	-4.43
	ARG 398	-7.14	TRP 344	-4.15
	PHE 400	-6.64	VAL 255	-3.64
α-β F200Y	ARG 217	-8.03	ARG 2	-10.9
	VAL 177	-7.22	ARG 324	-8.19
	PHE 400	-6.92	LYS 252	-5.09
	ARG 398	-6.81	TYR 445	-4.85
	TRP 403	-6.79	MET 433	-4.49

Molecular Dynamics Simulations

Molecular dynamics simulation showed comparable behavior between folded tubulin dimers despite containing resistance-associated mutations. The RMSD plots show the dimers containing F200Y, Q134H, and E198A in the β subunit were relatively more stable than others including the wildtype (Fig. 5A). The dimers that had F167Y, E198K, and E198V showed similar dynamic behavior as the wildtype. Meanwhile, the dimer that had the F200L mutation displayed increased RMSD deviations at 25 ns onwards. This deviation may indicate that dimer binding may not be as stable tending towards dissociation. This is supported by the lowered ΔG_bind and k_d for this mutation. The RMSF plots showed similar trends with regard to the flexibility of the dimer backbone relative to its constituting residue (Fig. 5B). This indicates that the presence of resistance-associated mutations does not significantly alter the flexibility of the dimer backbone. The increased RMSF value at residues ~ 440 to ~ 450 indicates the hinge between the dimers. A similar tendency is shown by the solvent-accessible surface area (SASA) plot where values of the tubulin complexes do not significantly change despite the presence of resistance-associated mutations (Fig. 5C). Lastly, the radius of the gyration plot shows that all mutated and wildtype complexes remain relatively compact during the simulation (Fig. 5D). The dimer with the E19K mutation showed increased but insignificant gyration at 10 to 30 ns simulation time indicating slight conformational changes during this period of simulation. Overall, the molecular dynamics simulation results show that tubulin dimers behave in a similar manner regardless of the presence of benzimidazole resistance-associated mutations in the tubulin dimer.

Our study aimed to assess the consequences of BZ resistance-associated mutations on the in silico dimerization of hookworm tubulin α and β subunits. Using A. ceylanicum as the model organism, the folding and interaction of the tubulin heterodimers were predicted using AlphaFold 3. Folding predictions were considered accurate and reliable based on several quality assurance checks. Assessment of the dimer’s interactions showed that the introduction of resistance-associated mutations into the β tubulin altered the binding properties of the dimer. Likewise, the mutations also modified the amino acids interacting in the α and β subunits and their corresponding binding free energies. However, molecular dynamics simulations of atomic flexibility and motions revealed that both wildtype and mutated heterodimers had comparable structural flexibility and mobility. To our knowledge, this is the first study on in silico dimerization of hookworm tubulins concerning benzimidazole resistance. These results provide several implications for the study of benzimidazole resistance in hookworms.

Our in silico dimerization of wild-type and mutated hookworm tubulins revealed several changes in dimer interactions due to the addition of the benzimidazole resistance-associated amino acid substitutions in the β subunit. E198K, E198V, and F200Y mutations increased the dimer binding energies. A higher binding energy (i.e., higher ΔG_bind and ΔG, lower K_d) between the mutated α- and β-tubulin suggests a stronger interaction between these subunits. This finding is corroborated by previous reports indicating that certain mutations in the α- and β-tubulin improve the stiffness, rupture force, and interface interaction energy of the formed dimer [46]. If the mutated β-tubulin binds more tightly to its α counterpart, the benzimidazole molecule might have difficulty binding to its target site on the β subunit [47]. This is perhaps exemplified by our results showing reduced binding pocket volume in β tubulins with resistance-associated mutations. Moreover, stronger interactions between the subunits can prevent the curving of tubulin conformation observed in crystal structures bound to colchicine [48]. Therefore, the heightening of tubulin subunit binding could assist in conferring benzimidazole resistance. This notion is supported by laboratory studies indicating that E198V, E198K, F167Y, and F200Y conferred marked resistance in C. elegans against Fenbendazole and Albendazole [28]. Furthermore, in silico docking studies have shown that mutated tubulins having amino acid shifts at position 198 had reduced binding affinities with benzimidazoles in Trichuris trichuria, Ancylostoma duodenale, Ascaris lumbricoides, and Haemonchus contortus [18, 30, 49]. Therefore, benzimidazole resistance-associated mutations enhance the hookworm's capacity to resist treatment through strengthening and stabilizing tubulin dimer interactions, beyond hindering benzimidazole binding to the β-tubulin subunit.

On the other hand, Q134H and F200L lowered the binding energies of the predicted tubulin dimer. Lowered binding energies indicate a weaker interaction between the mutated β- and wild-type α-tubulin. Therefore, other mutations could result in a less favorable microtubule structure prone to disassembly and thus detrimental to the hookworm [50, 51]. This notion is perhaps shown by laboratory studies showing that the acquisition of resistance phenotype due to these mutations in helminths is at the cost of survivability [23, 29]. Potential alterations in tubulin heterodimer binding dynamics due to resistance-associated mutations have also been reported by other studies concerned with anticancer drugs. Natarajan and Senapati [52] reported that T237I, R282Q, and Q292E mutations in mammalian β-tubulin can modulate tubulin dimerization through allosteric changes in the N-domain, which is involved in microtubule assembly. Moreover, mutations in the α-tubulin (e.g., W407X, G43V, T145P, and A383T) resulted in reduced hydrogen bonding with its β counterpart and significant departures of RMSD and RMSF values thereby causing tubulin heterocomplex instability [53]. These results, together with ours, indicate that mutations in either β- or α-tubulin subunits confer binding alterations that have consequences on tubulin dimer formation and the subsequent microtubule assembly, which either can assist in drug resistance or be detrimental to the parasite.

Recent advancements in protein folding predictions and protein-protein interaction modeling are a welcome development for anthelmintic resistance studies, particularly those involving benzimidazoles. Through deep learning algorithms trained on data from protein databases, AlphaFold 3 was able to reliably and accurately predict the dimerization of hookworm α- and β-tubulins. It is important to note that the crystal structure of hookworm α-and β-tubulin subunits and their dimerized conformation has yet to be elucidated by crystallography or spectroscopy. However, our structural alignment comparison with the ascertained mammalian tubulin crystal structure showed that our fold predictions from hookworm sequences bore a very close structural resemblance. This finding is also backed by the results of our external quality assurance checks. The lack of available ascertained tubulin crystal structures among soil-transmitted helminths has hampered the study of benzimidazole resistance, which should be addressed urgently. Also, the capacity to conduct in silico dimerization of hookworm tubulins with relative ease provides additional insights into previously reported β tubulin-benzimidazole docking studies [18, 30, 49]. However, our dimerization model does not include the docked benzimidazole ligand as the AlphaFold 3 server cannot incorporate such molecules in its predictions as of writing [54]. Hence, future in silico studies like ours should consider the dimerization of hookworm tubulins with the bound benzimidazole ligand to gain more insights regarding the tubulin disruption action of the drug. However, our research does highlight the use of open-source platforms in the in silico study of drug resistance. Our research presents a free pipeline that applies to those who do not have access to high-end computers and costly software.

Benzimidazole resistance-associated amino acid substitutions in the β-tubulin isotype 1 protein of hookworms confer in silico alterations in the tubulin heterodimer interaction. The protein folding prediction of hookworm tubulin heterodimers by AlphaFold 3 was accurate and reliable. Mutations that occur in positions 198 and 200 (i.e., E198K, E198V, and F200Y) caused better interactions between the α and β subunits resulting in more stable tubulin dimers and assisting in benzimidazole resistance. The opposite was observed in mutated β-tubulins with the Q134H and F200L mutations. The structural flexibility and motion dynamics of the wild-type and mutated tubulin heterodimers were not markedly deviated—mutated tubulin complexes behave similarly to their wild-type counterparts. Beyond hindering benzimidazole binding to the β-tubulin subunit, resistance-associated mutations enhance the hookworm's capacity to resist treatment through strengthening and stabilizing tubulin dimer interactions. Meanwhile, other mutations diminish the dimer’s interaction which could be to the detriment of the hookworm. Our results provide novel insights into helminth tubulin dimerization that may be useful for drug design and the discovery of new anthelmintics. These results should be affirmed by in vitro and/or in vivo investigations.

Data availability

Data generated in this study can be made available upon request from the corresponding author.

Acknowledgments

Not applicable.

Authorship Statement

Competing interest

The authors have no conflict of interest to declare.

Ethical standards

This research does not require ethical approval for it did not use animal or human subjects.

Agrawal R, Pattnaik S, Kshatri JS, Kanungo S, Mandal N, Palo SK, Pati S (2024) Prevalence and correlates of soil-transmitted helminths in schoolchildren aged 5 to 18 years in low- and middle-income countries: a systematic review and meta-analysis. Front Public Health 12:1283054
World Health Organization (2023) Soil-transmitted helminth infections. https://www.who.int/news-room/fact-sheets/detail/soil-transmitted-helminth-infections. Accessed 16 Sep 2023
Jourdan PM, Lamberton PHL, Fenwick A, Addiss DG (2018) Soil-transmitted helminth infections. Lancet 391(10117):252–265
Clements ACA, Alene KA (2022) Global distribution of human hookworm species and differences in their morbidity effects: a systematic review. Lancet Microbe 3:e72–e79
GBD 2017 Disease and Injury Incidence and Prevalence Collaborators (2018) 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 393(10190):e44
Tenorio JCB, Tabios IKB, Inpankaew T, Ybañez AP, Tiwananthagorn S, Tangkawattana S, Suttiprapa S (2024) Ancylostoma ceylanicum and other zoonotic canine hookworms: neglected public and animal health risks in the Asia–Pacific region. Anim Dis 4:11
Zibaei M, Nosrati MRC, Shadnoosh F, Houshmand E, Karami MF, Rafsanjani MK, Majidiani H, Ghaffarifar F, Cortes HCE, Dalvand S, Badri M (2020) Insights into hookworm prevalence in Asia: a systematic review and meta-analysis. Trans R Soc Trop Med Hyg 114(3):141–154
Brooker SJ (2018) Soil-transmitted helminth treatment: multiple-drug regimens. Lancet Infect Dis 18(7):698–699
Mationg MLS, Tallo VL, Williams GM, Gordon CA, Clements ACA, McManus DP, Gray DJ (2021) The control of soil-transmitted helminthiases in the Philippines: the story continues. Infect Dis Poverty 10(1):85
World Health Organization (2011) Helminth control in school-age children : a guide for managers of control programmes. World Health Organization
Traversa D (2012) Pet roundworms and hookworms: A continuing need for global worming. Parasit Vectors 5:91
TroCCAP (2019) Guidelines for the diagnosis, treatment and control of canine endoparasites in the tropics, 2nd ed
TroCCAP (2019) Guidelines for the diagnosis, treatment and control of feline endoparasites in the tropics, 2nd ed
Ng’etich AI, Amoah ID, Bux F, Kumari S (2023) Anthelmintic resistance in soil-transmitted helminths: One-Health considerations. Parasitol Res 123(1):62
Tinkler SH (2020) Preventive chemotherapy and anthelmintic resistance of soil-transmitted helminths – Can we learn nothing from veterinary medicine? One Health 9:100106
Monga J, Ghosh NS, Rani I, Singh R, Deswal G, Dhingra AK, Grewal AS (2024) Unlocking the Pharmacological Potential of Benzimidazole Derivatives: A Pathway to Drug Development. Curr Top Med Chem 24(5):437–485
Furtado LFV, De Paiva Bello ACP, Rabelo ÉML (2016) Benzimidazole resistance in helminths: From problem to diagnosis. Acta Trop 162:95–102
Jones BP, van Vliet AHM, LaCourse EJ, Betson M (2022) In Silico Docking of Nematode β-Tubulins With Benzimidazoles Points to Gene Expression and Orthologue Variation as Factors in Anthelmintic Resistance. Front Trop Dis 3:898814
Lacey E, Gill JH (1994) Biochemistry of benzimidazole resistance. Acta Trop 56:245–262
Diawara A, Halpenny CM, Churcher TS, Mwandawiro C, Kihara J, Kaplan RM, Streit TG, Idaghdour Y, Scott ME, Basáñez MG, Prichard RK (2013) Association between Response to Albendazole Treatment and β-Tubulin Genotype Frequencies in Soil-transmitted Helminths. PLoS Negl Trop Dis 7:e2247
Jones BP, Kozel K, Alonte AJI, Llanes KKR, Juhász A, Chaudhry U, Roose S, Geldhof P, Belizario VY, Nejsum P, Stothard JR, LaCourse EJ, van Vliet AHM, Paller VGV, Betson M (2024) Worldwide absence of canonical benzimidazole resistance-associated mutations within β-tubulin genes from Ascaris. Parasit Vectors 17(1):225
Evason MD, Weese JS, Polansky B, Leutenegger CM (2023) Emergence of canine hookworm treatment resistance: Novel detection of Ancylostoma caninum anthelmintic resistance markers by fecal PCR in 11 dogs from Canada. Am. J. Vet. Res. 84(9):ajvr.23.05.0116
Medeiros CDS, Furtado LFV, Miranda GS, Da Silva VJ, Santos D, Rabelo TR, É.M.L (2022) Moving beyond the state of the art of understanding resistance mechanisms in hookworms: confirming old and suggesting new associated SNPs. Acta Trop 233:106533
Stocker T, Scott I, Šlapeta J (2023) Unambiguous identification of Ancylostoma caninum and Uncinaria stenocephala in Australian and New Zealand dogs from faecal samples. Aust Veterinary J 101(10):373–376
Venkatesan A, Castro PDJ, Morosetti A, Horvath H, Chen R, Redman E, Dunn K, Collins JB, Fraser JS, Andersen EC, Kaplan RM, Gilleard JS (2023) 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 19(3):e1011146
Castro PDJ, Durrence K, Durrence S, Gianechini LS, Collins J, Dunn K, Kaplan RM (2023) Multiple anthelmintic drug resistance in hookworms (Ancylostoma caninum) in a Labrador breeding and training kennel in Georgia, USA. J Am Vet Med Assoc 261(3):342–347
Furtado LFV, Dos Santos TR, de Oliveira VNGM, Rabelo ÉML (2020) Genotypic profile of benzimidazole resistance associated with SNP F167Y in the beta-tubulin gene of Necator americanus helminths obtained from Brazilian populations. Infect Genet Evol 86:104594
Dilks CM, Hahnel SR, Sheng Q, Long L, McGrath PT, Andersen EC (2020) Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles. Int J Parasitol Drugs Drug Resist 14:28–36
Pallotto LM, Dilks CM, Park YJ, Smit RB, Lu BT, Gopalakrishnan C, Gilleard JS, Andersen EC, Mains PE (2022) Interactions of Caenorhabditis elegans β-tubulins with the microtubule inhibitor and anthelmintic drug albendazole. Genetics 221:iyac093
Aguayo-Ortiz R, Méndez-Lucio O, Romo-Mancillas A, Castillo R, Yépez-Mulia L, Medina-Franco JL, Hernández-Campos A (2013) Molecular basis for benzimidazole resistance from a novel β-tubulin binding site model. J Mol Graph Model 45:26–37
Aguayo-Ortiz R, Méndez-Lucio O, Medina-Franco JL, Castillo R, Yépez-Mulia L, Hernández-Luis F, Hernández-Campos A (2013) Towards the identification of the binding site of benzimidazoles to β-tubulin of Trichinella spiralis: Insights from computational and experimental data. J Mol Graph Model 41:12–19
Furtado LFV, de Aguiar PHN, Zuccherato LW, Teixeira TTG, Alves WP, da Silva VJ, Gasser RB, Rabelo ÉML (2019) Albendazole resistance induced in Ancylostoma ceylanicum is not due to single-nucleotide polymorphisms (SNPs) at codons 167, 198, or 200 of the beta-tubulin gene, indicating another resistance mechanism. Parasitol Res 118:837–849
George S, Suwondo P, Akorli J, Otchere J, Harrison LM, Bilguvar K, Knight JR, Humphries D, Wilson MD, Caccone A, Cappello M (2022) Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene. Sci Rep 12(1):11459
The UniProt Consortium (2019) UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res 47(D1):D506–D515
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series 41(11):95–98
Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, Bodenstein SW, Evans DA, Hung CC, O’Neill M, Reiman D, Tunyasuvunakool K, Wu Z, Žemgulytė A, Arvaniti E, Beattie C, Bertolli O, Bridgland A, Cherepanov A, Congreve M, Cowen-Rivers AI, Cowie A, Figurnov M, Fuchs FB, Gladman H, Jain R, Khan YA, Low CMR, Perlin K, Potapenko A, Savy P, Singh S, Stecula A, Thillaisundaram A, Tong C, Yakneen S, Zhong ED, Zielinski M, Žídek A, Bapst V, Kohli P, Jaderberg M, Hassabis D, Jumper JM (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630(8016):493–500
Nogales E (2000) Structural insights into microtubule function. Annu Rev Biochem 69:277–302
Heo L, Lee H, Seok C (2016) GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking. Sci Rep 6:32153
Eisenberg D, Lüthy R, Bowie JU (1997) VERIFY3D: Assessment of protein models with three-dimensional profiles. In: Methods in Enzymology. Academic Press, pp 396–404
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291
Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35:W407–W410
Löwe J, Li H, Downing KH, Nogales E (2001) Refined structure of αβ-tubulin at 3.5 Å resolution1. J Mol Biol 313(5):1045–1057
Weng G, Wang E, Wang Z, Liu H, Zhu F, Li D, Hou T (2019) HawkDock: a web server to predict and analyze the protein–protein complex based on computational docking and MM/GBSA. Nucleic Acids Res 47(W1):W322–W330
Xue LC, Rodrigues JP, Kastritis PL, Bonvin AM, Vangone A (2016) PRODIGY: a web server for predicting the binding affinity of protein–protein complexes. Bioinformatics 32:3676–3678
Stourac J, Vavra O, Kokkonen P, Filipovic J, Pinto G, Brezovsky J, Damborsky J, Bednar D (2019) Caver Web 1.0: identification of tunnels and channels in proteins and analysis of ligand transport. Nucleic Acids Res 47(W1):W414–W422
Liu N, Pidaparti R, Wang X (2017) Effect of amino acid mutations on intra-dimer tubulin–tubulin binding strength of microtubules. Integr Biol 9:925–933
Robinson MW, McFerran N, Trudgett A, Hoey L, Fairweather I (2004) A possible model of benzimidazole binding to beta-tubulin disclosed by invoking an inter-domain movement. J Mol Graph Model 23:275–284
Ravelli RBG, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, Knossow M (2004) Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 428:198–202
Jones BP, van Vliet AHM, LaCourse EJ, Betson M (2022) Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations. Sci Rep 12:13725
Bueno O, Estévez Gallego J, Martins S, Prota AE, Gago F, Gómez-SanJuan A, Camarasa MJ, Barasoain I, Steinmetz MO, Díaz JF, Pérez-Pérez MJ, Liekens S, Priego EM (2018) High-affinity ligands of the colchicine domain in tubulin based on a structure-guided design. Sci Rep 8:4242
Vega LR, Fleming J, Solomon F (1998) An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins. MBoC 9:2349–2360
Natarajan K, Senapati S (2012) Understanding the Basis of Drug Resistance of the Mutants of αβ-Tubulin Dimer via Molecular Dynamics Simulations. PLoS ONE 7:e42351
Ganne A, Balasubramaniam M, Ayyadevara H, Kiaei L, Reis S, Varughese RJ, Kiaei KI, M (2023) In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration. Sci Rep 13:2096
Thompson B, Petrić Howe N (2024) Alphafold 3.0: the AI protein predictor gets an upgrade. Nature. 10.1038/d41586-024-01385-x

The authors declare no competing interests.

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Benzimidazole resistance-associated mutations improve the in silico dimerization of hookworm tubulin: an additional resistance mechanism?

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Prediction of hookworm tubulin dimer folding via AlphaFold3

Quality Assurance Checks of the Modeled Dimers

Tubulin Heterodimer Interactions

Molecular Dynamics

Results

AlphaFold 3 satisfactorily predicted the folding of hookworm tubulin dimer

BZ resistance-associated mutations affect hookworm tubulin dimerization

Molecular Dynamics Simulations

Discussion

Conclusion

Declarations

References

Additional Declarations

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