Axltide

The constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20-30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro, a mutant Abl kinase harboring the "gatekeeper" mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant.

A method to study Abl1 tyrosine kinase inhibitors (TKIs) by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was developed and validated. Chromatographic separation was achieved on a Symmetry(®) C-18 column using a gradient. The detection was performed by selected ion monitoring (SIM) mode via positive ESI interface. The limit of quantification (LOQ) was 40.8 nM for p-Abltide [product, KKGEAIpYAAPFA-NH2] and 26.7 nM for Abltide (substrate, KKGEAIYAAPFA-NH2). The residual plot of linearity calibration curve indicated a good fit with a linear model. Intra- and inter-day precision was less than 10% and accuracy was from -6.93% to +0.15%. Matrix effect was not significant in this method. The validated method was applied to an Abl1 TKIs study. Imatinib mesylate (IM) and dasatinib were used to evaluate this method and the IC50 values were 202.1 nM and 925.1 pM, respectively. Two natural products (-)- epigallocatechingallate (EGCG) and caffeic acid were tested with this model. The IC50 value of EGCG was found at 64.03 nM and caffeic acid showed fluctuant inhibitory activity from 26% to 55% in the concentration range from 1 nM to 1mM. The IC50 value of a dimethylpyrrole hydroxylbenzoic acid derivative (MPB) was 1.915 μM.

Abelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide. The phosphorylation reactions were carried out by incubating ABL1, ATP and the substrate abltide. Adsorption at the glassy carbon electrode surface in either reaction mixtures or control solutions, followed by differential pulse voltammetry in buffer allowed detection of the variation of abltide tyrosine residue oxidation peak reflecting the occurrence of the phosphorylation reaction. The effect of abltide, ATP and ABL1 concentrations as well as the time course of the phosphorylation reaction were studied. The influence of co-adsorption of ABL1, ATP and phosphorylated abltide was evaluated and the conditions for the electrochemical detection of ABL1-catalysed phosphorylation optimised. The Michaelis-Menten constant for abltide binding KM~4.5 μM, turnover number kcat~11 s(-1) and enzyme efficiency kcat/KM~2.3 s(-1) μM(-1) were calculated. The inhibition of ABL1 by imatinib mesylate and danusertib was also electrochemically investigated and IC50 values of 0.53 and 0.08 μM determined.