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One of the major challenges for
targeted cancer therapies like tyrosine kinase inhibitors (TKIs) is drug
resistance. It can bring about by different factors such as point mutations in
the kinase receptor 13, 34, 35. The
impact of point mutations can be evaluated in different ways, both
experimentally and theoretically. Computational techniques can be utilized to
both assessment of experimental data and predict probable results in clinical
practice 31, 36, 37. By
theoretical studies, instances like the dynamics of proteins 38-40, the
shape and location of binding pockets 41-43 and
the anticipated interactions between the protein and ligand 44-46 can
be inspected. In drug resistance studying, mutated structures would be compared
to native states to identify remarkable differences 47, 48 which could consist of loss of interactions or change of
conformation which could be as a result of ligand binding 31, 49. In the present study, computational methods such as molecular
dynamics simulation 50, 51 to study the impact of point mutations on the BCR-ABL kinase and
how these mutations confer resistance have been applied.

Based on previous studies, there are
possible mechanisms of mutational impacts on ligand resistance including
eliminating important hydrogen bonds or preventing BCR-ABL from achieving a
specific conformation required for high-affinity ligand binding or mutations in
regulatory motifs like activation loop which may stabilize an active
conformation that is inaccessible to ligand 52.

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In order to investigate structural
behavior and flexibility of the wild-type and BCR-ABL mutants, all lead
complexes were analyzed by Gromacs ….. and MD simulation was performed for 50 ns
for each complexes. Root mean square deviations (RMSD) of the mentioned
complexes were calculated against their initial structure and to compare the
flexibility of the backbone graphs were created. During the simulation period,
no significant fluctuations in the backbone of all lead compounds were observed
indicating not only binding of ligand to the active site is stable and strong
but also the protein backbone stability does not interrupted.

The RMSF data of the wild-type and
mutants reveals the mutational impact on the mobility of different regions
ranging from N- to C-terminal which is implying on global impact of mutations
on the structure of the protein. The more common mutations occur in hot spots
within the BCR-ABL kinase domain including the ATP binding P-loop (amino acids
248-256), the catalytic domain (amino acids 350-363) and the activation
(A)-loop (amino acids 381-402). From these regions, the A-loop is a critical
regulator of BCR-ABL kinase activity through adopting a closed (inactive) or
open (active) conformation and mutation in this region often destabilize the
inactive conformation, the situation which is necessary for ligand binding 53. In our study, L364I brings about an increasing flexibility in the
A-loop region including amino acids 394-398. The higher flexibility of this
region likely weakens the interaction between imatinib and BCR-ABL. Furthermore,
based on MMPBSA data, the more negative binding energy, the higher affinity of
ligand and protein and would result in more stability of the structure. As
mentioned in Table 1, all mutants unveil more positive value compared to
wild-type implying the binding of imatinib to the protein is weaker. Also,
analysis of hydrogen bonds interactions disclosing reduction of hydrogen bonds
between mutated states of the protein and imatinib that likely may bring about
disruption of the interactions between imatinib and BCR-ABL and as a consequence,
drug binding is impaired or totally abolished 54. In addition, SASA data shows in all mutations this parameter has
increased compared to native state indicating mutation may result in a
conformational change such as partial unfolding which increases the solvent
accessible surface area. Therefore, catalytic domain mutations studied in our
experiment can cause a global impact on the overall structure of the protein
and may bring about drug resistance.

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