Novel PI3K and mTOR selective inhibitors to deconvolute PI3K signaling
PI3K sinyal iletim yolağının ayrıştırılmasına yönelik yeni PI3K ve mTOR selektif inhibitörleri
- Tez No: 797550
- Danışmanlar: PROF. DR. MATTHİAS P. WYMANN
- Tez Türü: Doktora
- Konular: Onkoloji, Genetik, Moleküler Tıp, Oncology, Genetics, Molecular Medicine
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2021
- Dil: İngilizce
- Üniversite: Unıversıty Of Basel
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Biyokimya ve Moleküler Biyoloji Ana Bilim Dalı
- Bilim Dalı: İlaç Tasarımı ve Geliştirilmesi Bilim Dalı
- Sayfa Sayısı: Belirtilmemiş.
Özet
Fosfoinositit 3-kinaz (PI3K) sinyalizasyon yolağı hücre büyümesi, proliferasyon ve metabolizma gibi hücresel proseslerin düzenlenmesinde kilit rollere sahiptir. PI3K'nin tümörlerde konstittitif aktivasyonü yaygındır ve kanser ilerlemesine neden our. Anormal PI3K sinyalizasyonunun kanser ilerlemesindeki anormal katkısı dikkate alınarak, PI3K kaynaklı malformasyonu onlemeye yonelik farmakolojik müdahaleler sağaltici hedef olarak geniş bir şekilde araştırılmıştır, fakat çogu pan-PI3K inhibitorleri temel olarak hedef üzerindeki metabolik yan etkiler nedeniyle, klinik denemelerde düşük bir tepki oranı sergilemiştir. Aküt pan-PI3K inhibisyonü; PI3Kalfa ve PI3Kbeta izoformlarinin hepatositteki insülin sinyalizasyonünda gereksiz rollere sahip olmalari ve her iki izoformün aküt inhibisyonünün glikoz homeostazini bozması nedeniyle, kan glikozü ve insülin seviyelerinde hızlı bir artışı tetikler. Buna göre, izoform-selektif PI3Kalfa inhibisyonü hiperglisemi ve hiperinsülinemiyi hafifletebilir. Bununla birlikte, öne sürülen PI3Kalfa'ya ozgü ilaçların selektivitesi fizyolojik olarak etkili konsantrasyonlarında sınırlıdır. Burada, bu proje bir pan-PI3K inhibitor iskelesi PQR514'ün hedef selektivitesini, bunun izoforma-özgü korunumsuz nükleofilik amino asit yan zincirine, PI3Kalfa'daki Cys862'ye eklenmesi yoluyla arttirmak icin, rasyonel bir ilaç tasarımı yaklaşımı geliştirmeyi amaçlamaktadır. PQR514 tersinir iskelesi, savaş başlığı stabilitesindeki ayarlanmış iyileştirmelerden ve savaş başlığı intrinsik kimyasal reaktiviteden sonra bir elektrofilik parça (savaş başlığı) eklemek icin derive edilmiştir. Savaş başlığı proksimitesi ve bunun kovalent bağlanti alanına oryantasyonu, izoform-selektif kovalentin hedef alana eklenmesini teşvik etmek amacıyla,“aktif hacim tarama”stratejimiz optimize edilmiştir.
Özet (Çeviri)
Phosphoinositide 3-kinase (PI3K) signaling has key roles in the regulation of cellular processes such as cell growth, proliferation, and metabolism. Constitutive activation of PI3K in tumors is frequent and drives cancer progression. 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 effects. Acute pan-PI3K inhibition triggers a rapid increase in blood glucose and insulin level since PI3Kalpha and PI3Kbeta isoforms have redundant roles in insulin signaling in the hepatocyte, and acute inhibition of the both isoforms impairs glucose homeostasis. Given that, isoform-selective PI3Kalpha inhibition may alleviate hyperglycemia and hyperinsulinemia. However, the selectivity of the claimed PI3Kalpha-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, PQR514, by its covalent attachment to an isoform-specific non-conserved nucleophilic amino acid side chain, Cys862 in PI3Kalpha. 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 target. If a covalent bond was formed between the warhead and the targeted amino acid side chain in the target protein of interest, further modifications were carried out using the steric modifications to optimize warhead proximity and the nucleophilic attack vector in order to maximize covalent bond formation efficiency. Accordingly, the highly reactive warhead was exchanged with a moderately reactive, drug-like warhead in order to minimize unwanted side reactions and promote its metabolic stability. Our novel covalent inhibitors already containing drug-like warheads were metabolically stable and outperformed CNX13517, the only reported PI3Kalpha-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. Its labile wortmannin core and unstable Schiff-base forming warhead with the targeted amino acid side chains were not optimal for in vivo efficacy. 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 PI3Kalpha-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 PI3Kalpha/non-covalent (reversible) pan-PI3K inhibition approach could allow novel scenarios to reversibly target class I PI3Ks (PI3Kbeta, PI3Kdelta, PI3Kgamma), while only PI3Kalpha isoform is irreversibly inhibited for a prolonged period of time. Even tumors with loss of PTEN can be transiently targeted, while PI3Kalpha 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). In intermittent treatment regimen, PQR309 maintained the suppression of tumor growth with lessened on-target metabolic side effects in rodents14 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 PI3Kalpha-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 PI3Kalpha 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 PI3Kalpha 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 PI3Kalpha in complex with our novel covalent inhibitors validated the covalent modification of Cys862 in PI3Kalpha. Our lead compounds outperformed the rapidly metabolized CNX1351, which is the only reported PI3Kalpha irreversible inhibitor. 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) and an mTOR-selective inhibitor (PQR626) 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 activity. 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 brain and kidney. 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 hyperinsulinemia. 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.
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