Kinase inhibitors and degraders

: tools for discovery of novel antiplatelet therapies

Abstract

Platelets are a fundamental cellular component of blood, playing key roles in both primary and secondary haemostasis. They are essential for the formation of a wound healing clot and prevention of blood loss at the site of vascular damage. However, under pathological conditions, such as these seen in cardiovascular disease (CVD), they can become involved in untimely clot formation and its subsequent embolism, which can lead to catastrophic vaso-occlusive events and complications, including stroke and myocardial infarction. CVD is a leading cause of morbidity and mortality in the developed countries. It is predicted to remain the predominant cause of death in the coming years, due to the increasing prevalence of diabetes and obesity within the general population, which are major predisposing factors for the development of CVD. These are the reasons why CVD is one of the most researched diseases around the world, with a focus placed on finding new, advanced ways of its prevention and treatment. Antiplatelet therapies are at the moment an integral component of the mitigation and treatment strategy for CVD. Unfortunately, not all patients respond to the most commonly used ‘gold-standard’ antiplatelet drugs, which inhibit thromboxane A2 (TxA2)- and ADP- mediated activation pathways. These patients remain at risk of thrombotic events. The other drawbacks of current antiplatelet therapies include serious side effects, such as excessive bleeding. In consequence, there is a growing need for a better understanding of platelet physiology, and investigation into the effect of known and innovative compounds on their function, in order to support development of novel antiplatelet treatment.
The studies described in this thesis are therefore focused on testing the effect of several structurally and functionally different compounds on platelet function, and on dissecting the underlying mechanisms of their action. The main aim of this PhD project was to discover novel antiplatelet therapy targets and to investigate whether a group of drugs presently used in the clinic for treatment of other diseases, could be repurposed for platelet inhibition.
Firstly, as described in Chapter 3, the effect of a mammalian target of rapamycin complex 1 (mTORC1) inhibitor Sirolimus (also known as rapamycin) on platelet signalling and function was assessed. Interestingly, it was found that the compound diminishes platelet procoagulant responses, including phosphatidylserine exposure, membrane ballooning and concurrent thrombin generation. Furthermore, the study demonstrates that rapamycin suppresses platelet procoagulant responses via FK506-binding protein (FKBP)-mediated protection of mitochondrial integrity, in a manner that is independent of its inhibitory action on mTORC1.
Secondly, as outlined in Chapter 4, the effect of the apoptosis signal-regulating kinase (ASK1) inhibitor Selonsertib (GS-4997) on thromboxane A2 synthesis and amplification of platelet activity was investigated. The importance of these pathway was previously described in murine ASK1-/- models. However, the results presented in thesis show that the effect seen in mice does not translate to human platelets, indicating that ASK1 signalling is redundant for TxA2 formation and human platelet activity.
Finally, as described in Chapter 5, the use of innovative small-molecule proteolysis targeting chimeras (PROTACs) for selective degradation of kinases in human platelets was explored. Not only do the findings confirm that platelets can perform proteasome-mediated proteolysis, but they also demonstrate for the first time that a variety of tyrosine protein kinases expressed in platelets can be targeted for degradation using PROTAC-induced ubiquitination. Such a methodology can be used in the future not only to study kinase function in human platelets, by allowing to produce ‘knockdown’ human platelets in vitro, but also to introduce a completely novel way of reducing platelet activity, possibly much stronger than pharmacological inhibitors.
In summary, the data generated will be useful in guiding the design and development of novel antiplatelet therapies and to potentially counteract thrombosis in patients who do not respond well to the currently available antiplatelet treatment.

Date of Award	29 Sept 2020
Original language	English
Awarding Institution	University of Bristol
Supervisor	Ingeborg Hers (Supervisor) & Alastair W Poole (Supervisor)