Comparative Analyses of theβ-Tubulin Gene and Molecular Modeling Reveal Molecular Insight into the Colchicine Resistance in Kinetoplastids Organisms

Abstract

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the β-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania β-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the β-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α-helix structure and displacement to the inside of the pocket of one β-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.

Colchicine treatment reversibly blocks cytokinesis but not mitosis in Trypanosoma cruzi epimastigotes

“…By in silico analysis of β-tubulin sequences from Trypanosoma brucei and Leishmania guyanensis, some researchers have hypothesized that differences in amino acid composition between trypanosomes and bovine tubulin proteins at key positions are responsible for conformational changes that prevent colchicine interaction with its putative binding pocket (Lama et al 2012;Luis et al 2013). However, some reports with experiments incubating trypanosomatid parasite cultures in the presence of colchicine have shown an inhibitory growth effect (Ochola et al 2002).…”

“…This reversibility in the effect observed with colchicine treatment on different cell types may be the direct result of the intrinsic dynamic nature of microtubule assembly-disassembly (Desai and Mitchison 1997). However, the different colchicine concentrations needed to maintain this reversibility could be in relation to the level of drug affinity for tubulin isotypes from different organisms (Luis et al 2013). In addition, it is important to note that the piperine and vinka alkaloid treatments referred above also exhibited a reversible effect (Freire-de-Lima et al 2008;Grellier et al 1999), in which blockage of cytokinesis was reversed upon drug removal.…”

Emergence of Tubulin as a Vaccine against Parasitic Infections

“…Inherent factors of parasitic tubulin, such as changes in structure, conformation, existence of multigene families, amino-acid substitutions, etc. are considered to block drug access [60,61]. Differential expression of microtubule-associated proteins (MAPs) has also been speculated as one of the causes that obstructs drug binding and therefore leads to drug resistance [62].…”

“…Differential expression of microtubule-associated proteins (MAPs) has also been speculated as one of the causes that obstructs drug binding and therefore leads to drug resistance [62]. Emergence of multi-drug resistant parasites has led to therapeutic failure for many parasitic ailments [63]; where majority of the structural alterations leading to resistance have been found to be localised in beta tubulin [61].…”

In SilicoStudy ofLeishmania donovaniα-βTubulin and Inhibitors

“…Currently, an orally administered drug, miltefosine, is known; however, it has teratogenic effects and high cost [4]. Microtubules are crucial to cell division and intracellular organelle transport, hence, recognized as an important target for therapy for several diseases [6]. They are made of repeated -tubulin heterodimers, which are associated in parallel forming a wall of microtubules, giving rise to the polymer [7].…”