Unveiling the ATPase activity of Abelson kinase and its implications for the development and validation of assays for antileukemic drug screening

Abstract

The relevance of protein kinases as therapeutic targets is widely recognized, owing to their central role in regulating cellular processes and in the pathogenesis of various diseases. Among them, Abelson kinase (Abl) is a widely studied target since its dysregulation leads to the development of chronic myeloid leukemia (CML). Despite the success of ATP-competitive inhibitors, drug resistance and side effects necessitate the discovery of new inhibitors. However, the assessment of Abl activity still relies on expensive, complex methodologies or those that generate radioactive waste, highlighting the need for more accessible and economical methods. In this context, this thesis aims to bridge this gap through the development of colorimetric assays for monitoring the catalytic activity of Abl. During the initial stage of this work, we made a significant discovery: Abl exhibits an intrinsic ATPase activity even in the absence of a peptide substrate, with this work being the first to report such activity. We confirmed this activity and its enzymatic origin using ³¹P Nuclear Magnetic Resonance spectroscopy (³¹P NMR) and demonstrated that this ATPase activity is inhibited by classical Abl kinase inhibitors, confirming its direct correlation with the canonical phosphotransferase activity. Based on this discovery, we developed, optimized, and validated a malachite green-based colorimetric assay to monitor phosphate formation. The method was validated for its figures of merit: linearity, accuracy, precision, and limits of detection and quantification. Its performance in determining kinetic parameters showed excellent agreement with the reference ³¹P NMR method. To improve the proposed colorimetric method, we investigated the role of surfactants in stabilizing the chromogenic complex formed by malachite green and 12-molybdophosphoric acid, using Dynamic Light Scattering (DLS) and Zeta Potential techniques. We identified that the stabilization of the chromogenic complex by surfactants occurs via steric mechanisms, with Kolliphor P 188 preventing nanoparticle aggregation and precipitation. Further expanding the limits of accessibility, we implemented a digital image colorimetry strategy using different cameras and applied it to monitor Abl inhibition as a proof of concept. In conclusion, this work makes a dual contribution: it reveals a new fundamental catalytic activity of a widely studied kinase and translates this discovery into a suite of accessible technologies. The developed spectrophotometric and digital methods offer low-cost alternatives for inhibitor screening, holding significant potential to democratize research and accelerate drug discovery efforts targeting Abl and potentially other kinases.