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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PCAGED-KI (Photocaged Kinase Inhibitors as Molecular Tools for Investigating Neurodegenerative Diseases)

Teaser

Summary

When a drug is administered to a biological system, we have no control over when or where the drug will exhibit its therapeutic activity. This intrinsic lack of control combined with poor knowledge of how the drug works at a cellular level, often results in toxic side effects, significantly impeding current drug development and validation programs. As such, the utilisation of light as an external stimulus to control the pharmacological activity of drugs in cells and tissue, with high spatiotemporal resolution, has the potential to provide valuable insight into the fundamental molecular events of complex cellular processes.
Protein kinase enzymes play a critical role in a number of cellular processes. More recently, the aberrant regulation of lymphocyte-specific protein tyrosine kinases (LCK) has been associate with the over activation of microglia cells (important immune effector cells that reside in the central nervous system, CNS) and in turn, the development of Alzheimerâ€™s disease (AD). Unfortunately, the detail of LCK\'s dynamic function and the importance of quantitative, spatial and time-dependent parameters regarding microglia activation are poorly understood. As such, the ability to manipulate LCK activity using light would result in temporal control of enzymatic activity, thus serving as a valuable approach to probe the function of LCK in microglia cells and in turn, further our understanding of AD and related neurodegenerative disorders.
While such studies cannot be performed using conventional LCK inhibitors, this project aims to develop a â€˜light-responsiveâ€™ kinase inhibitor, through the introduction of a photolabile caging moiety onto a fluorescent LCK inhibitor, which will function to both mask its therapeutic activity while rendering the inhibitor non-fluorescent. In doing so, we can (i) choose when to activate the inhibitorâ€™s therapeutic properties by simply exposing it to light; and (ii) employ â€˜OFF-ONâ€™ fluorescent changes as a means to report both the release of the active kinase inhibitor and its binding to the target of interest. Hence, the time and location of the photoactivation of the LCK inhibitor can be controlled by the researcher.
A potent LCK inhibitor that exhibits favourable fluorescent properties for cellular imaging has been developed. The inclusion of appropriate photolabile caging groups and their effects on kinase activity and fluorescence properties has been thoroughly explored. To date, three photocaged substrates that function as â€˜light-responsiveâ€™ LCK inhibitors have been identified, and in turn deemed suitable candidates for advanced biological studies in whole cells.
In contrast to classical inhibitors, the development of such â€˜light-responsiveâ€™ kinase inhibitors will serve as a valuable means to study in real time the intracellular events of LCK activity in healthy and diseased cells and tissue. Such studies will help us understand the events that lead to the decline of brain function that are observed in many CNS-based disorders, which today are poorly understood.

Work performed

Potential small molecule kinase inhibitors with innate fluorescent properties were developed via the integration of a highly fluorescent fluorophore (Prodan) into the pharmacophore of a kinase inhibitor. Following a small structure-activity-relationship (SAR) study and detailed photophysical evaluation, the most promising candidates were further evaluated in a kinome screen of 65 enzymes. Based on the selectivity profile and their ability to bind LCK, a LCK inhibitor that exhibits inherent fluorescent properties was identified.
The fluorescent properties of the LCK inhibitor are highly environment-sensitive with regards to changes in polarity. Fluorescence titration experiments of the inhibitor with the LCK enzyme demonstrate that more than a 100-fold increase in emission intensity and blue-shift in emission maxima is clearly observed upon binding LCK â€“ suggesting that one should be able to distinguish between bound and unbound molecules in a cellular setting. Furthermore, microscopy experiments in combination with flow cytometry demonstrate both rapid cellular uptake of the inhibitor and that fluorescence intensity can be correlated with LCK concentration.
Following the development of the fluorescent LCK inhibitor, the inclusion of appropriate photolabile caging groups to render the inhibitor both inactive and non-fluorescent was pursued. A series of photocaged LCK inhibitors were developed and their ability to bind LCK evaluated. For those compounds which exhibited poor LCK activity, their corresponding photo-induced decaging reactions to trigger the release of the active LCK inhibitor were thoroughly evaluated. During the course of these experiments, three photocaged substrates that can function as â€˜light-responsiveâ€™ LCK inhibitors have been identified, and in turn deemed suitable candidates for advanced biological studies in whole cells.
To date, results acquired through the duration of this project have been presented at five international conferences, and has led to two publications in peer-reviewed journals (J. Am. Chem. Soc., 2018 and Angew. Chem. Int. Ed, 2019), as well as an invited mini review on light-controlled kinase inhibition (ChemPhotoChem, 2019).

Final results

Despite the advantages obtained from the use of â€˜light-responsiveâ€™ bioactives in biological settings, to date only a handful of photocaged kinase inhibitors have been reported, and to the best of our knowledge, the use of fluorescence as a â€˜reporting mechanismâ€™ for kinase activation and binding has yet to be fully explored. Conventional methods of fluorescent tagging of bioactives (typically achieved by conjugating a bulky fluorescent probe to the drug of interest), invariably leads to dramatic increases in molecular weight and changes in pharmacokinetic properties. Changes in these properties can have a deleterious effect on therapeutic action. As such, this project focused on the development of a novel system in which fluorescence is utilised as a means to report both the occurrence of the decaging process as well as the binding of the active kinase inhibitor. The fluorescent properties were achieved by the integration of a fluorophore into the pharmacophore of the kinase inhibitor, giving rise to a small molecule LCK inhibitor with innate fluorescent properties.
The fluorescent environment-sensitive LCK inhibitor will serve as a valuable molecular tool to study in real-time the intracellular mechanisms of LCK signalling and will be of significant interest to those studying signal transduction pathways in healthy and diseased cells and tissue. Furthermore, our approach of merging the fluorophore with the pharmacophore of type I kinase inhibitors can be applied to the development of other fluorescent and solvatochromic small molecule kinase inhibitors, in which kinase selectivity could be tailored through subtle structural modification. Furthermore, in contrast to conventional kinase inhibitors, the development of â€˜light responsiveâ€™ kinase inhibitors will allow us to gain spatiotemporal control over the activity of kinase enzymes, visualise the decaging process and subcellular localisation of the active kinase inhibitor and in turn, increase our understanding of LCK function in complex cellular pathways. Such studies will help us further understand the underlying events of neurodegenerative diseases, which today are poorly understood.