19 December, 2022 at 15:00, Dr. Erhan Keles from Stanford University is going to be at SABITALKS. The event will take place as hybrid. You can attend the event in person in the SABITA seminar hall.
Location: Istanbul Medipol University Kavacik Nourth Side: https://goo.gl/maps/JDDjygVtFLWiPiMJA
*Zoom participation link will be active at the event time.
*Participants from outside SABITA must fill in the participation form.
Erhan Keles, PhD
Phosphoinositide 3-kinase (PI3K) signaling has key roles in the regulation of cellular processes such as cell growth, proliferation, and metabolism1. Constitutive activation of PI3K in tumors is frequent and drives cancer progression2. Considering the contribution of aberrant PI3K signaling in cancer progression, pharmacological intervention strategies to inhibit PI3K-driven malformations have been broadly explored as a therapeutic target, but many pan-PI3K inhibitors displayed a low response rate in clinical trials mainly due to on target metabolic side effects3. Acute pan-PI3K inhibition triggers a rapid increase in blood glucose and insulin level since PI3K and PI3K isoforms have redundant roles in insulin signaling in the hepatocyte, and acute inhibition of the both isoforms impairs glucose homeostasis4. Given that, isoform-selective PI3K inhibition may alleviate hyperglycemia and hyperinsulinemia. However, the selectivity of the claimed PI3K-specific drugs is currently limited at their physiologically effective concentrations.
Herein, this project aimed to develop a rational drug design approach to increase target selectivity of a pan-PI3K inhibitory scaffold, PQR5146, by its covalent attachment to an isoform-specific non-conserved nucleophilic amino acid side chain, Cys862 in PI3K. PQR514 reversible scaffold was derivatized to attach an electrophilic moiety (warhead) after adjusted improvements in warhead stability and warhead intrinsic chemical reactivity. The warhead proximity and its orientation to the covalent anchor site were optimized through our “active volume scanning” strategy to promote isoform-selective covalent attachment on the target site.
In order to validate the target using the active volume scanning strategy, a highly reactive warhead was utilized to scan the dynamic protein space and generate “reactive hits” in terms of successful covalent labeling of the target7. Our novel covalent inhibitors already containing drug-like warheads were metabolically stable and outperformed CNX13518, the only reported PI3K-selective covalent inhibitor, in terms of biochemical and cellular potency, physicochemical properties, and metabolic stability. Concerning “drug-likeness” of the currently available PI3K covalent inhibitors, the only covalent PI3K inhibitor in clinical trials was a pan-PI3K covalent inhibitor, PX866, which had been tested in clinical trials for 15 years but failed due to poor clinical outcome.9-12 Although there are many reversible class I PI3K inhibitors that have been already tested in the clinics, the isoform-selective covalent inhibition strategy is not thoroughly exploited in order to improve the isoform-selectivity profile of the currently available reversible PI3K inhibitors. Therefore, there is a need to develop isoform selective, highly potent, and metabolically stable drug-like covalent PI3K inhibitors not only to treat PI3K-driven malignancies but also to deconvolute class I PI3K signaling activities in cells in order to unravel redundant and non-redundant functions of PI3K isoforms.
Our covalent inhibition strategy based on a covalent PI3K/non-covalent (reversible) pan-PI3K inhibition approach could allow novel scenarios to reversibly target class I PI3Ks (PI3K, PI3K, PI3K), while only PI3K isoform is irreversibly inhibited for a prolonged period of time. Even tumors with loss of PTEN can be transiently targeted, while PI3K inactivation will persist for a prolonged period of time after systemic elimination of the drug. This mode of action is more suitable for intermittent dosing suggested by a clinical trial with PQR309 (Bimiralisib)13. In intermittent treatment regimen, PQR309 maintained the suppression of tumor growth with lessened on-target metabolic side effects in rodents13 and patients [NCT02249429, NCT03740100]. Therefore, a covalent inhibition strategy may introduce an improved therapeutic window. Through a structure-activity relationship (SAR) study, highly potent, metabolically stable, and drug-like PI3K-selective covalent inhibitors were developed as a tool to fine-tune pharmacology in PI3K inhibitor cancer therapy. Optimization of linker length and warhead proximity toward the nucleophilic Cys862 side chain in PI3K promoted increased covalent bond formation efficiency up to a two-order of magnitude without modifying the electrophilicity (or intrinsic reactivity) of warheads. Rigorous cellular characterizations pinpointed low nanomolar potency in inhibition of PI3K downstream activity in cancer cells and prolonged inhibitory activity after drug washout. Moreover, Nano Bioluminescence Resonance Energy Transfer (NanoBRET) experiments exploiting PI3K Cys862Ser genetic point mutation confirmed the involvement of Cys862 in drug action in intact HEK293 cells. In agreement with this, X-ray crystal structures of PI3K in complex with our novel covalent inhibitors validated the covalent modification of Cys862 in PI3K. Our lead compounds outperformed the rapidly metabolized CNX1351, which is the only reported PI3K irreversible inhibitor8. Moreover, our inhibitors exhibited excellent cellular activity with a superior physicochemical profile compared to CNX1351. Our results represent a step towards an increased local and temporal control of PI3K inhibition, and our rational covalent inhibitor design strategy paves the way to a more efficient targeting of a broader panel of cysteines in the human kinome.
In addition to the development of novel PI3K targeting pharmacological probes, a dual pan-PI3K/mTOR-selective inhibitor (PQR530)14 and an mTOR-selective inhibitor (PQR626)15 were developed to deconvolute PI3K and mTOR signaling and to evaluate novel treatment modalities against epileptic seizures occurring due to loss of tuberous sclerosis complex (TSC) function. TSC2 (tuberin) together with its binding partner TSC1 (hamartin) have key functions to integrate multiple inputs from PI3K, ERK, Wnt, and energy signals through the attenuation of mTORC1 activity16,17. Given that, TSC1 and TSC2 function as tumor suppressors, and genetic mutations disrupting TSC function cause a malformation called tuberous sclerosis complex (TSC) disease, which is manifested by the formation of cysts and benign tumors in vital organs such as brain18 and kidney19. Targeting mTOR in the treatment of epileptic seizures using blood-brain barrier (BBB) permeable, orally bioavailable, and mTOR-selective drug-like small molecule inhibitor, PQR626, reduced the loss of TSC1-caused mortality in a TSC1GFAPCKO mouse model and did not induce metabolic side effects including hyperglycemia and hyperinsulinemia15. mTOR-selective/PI3K-sparing inhibition strategy with PQR626 introduced certain advantages over dual mTOR/pan-PI3K inhibition strategy with PQR530 in order to circumvent on target metabolic side effects of pan-PI3K inhibition.
Standford University
Dr. Erhan Keles graduated from Montana State University Bachelor of Science in Bioengineering in 2014. He received his master’s degree in Bioengineering from Washington State University. He completed his PhD in Genetics at University of Basel as Doctor of Philosophy (emphasis on Chemical Genetics). Between 2012-2014, he worked as a research assistant at Montana State University, Department of Chemical Engineering and Bioengineering. During this period, he worked as a research assistant at Istanbul Technical University, Nano/Micro ElectroMechanical Systems Laboratories for 3 months. He interned at Molecular Neurotherapy and Imaging Laboratory at Harvard Stem Cell Institute. From 2014 to 2016, he worked as a graduate research assistant in Chemical Engineering and Bioengineering, at Washington State University. Between 2016-2021, he worked as a pre-doctoral researcher at University of Basel. As of October 2021, he is a postdoc at Stanford Cancer Institute, Stanford University.