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Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5′-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinactand reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes., Journal of the American Chemical Society, 144 (14), ISSN:0002-7863, ISSN:1520-5126

Plasmodium falciparum phosphatidylinositol 4-kinase (PfPI4K) has emerged as a promising new drug target for novel antimalarial therapeutics. In the absence of a reliable high-resolution three-dimensional structure, a homology model of PfPI4K was built as a tool for structure-based drug design. This homology model has been validated against three distinct chemical series of potent inhibitors using docking and energy minimizations to elucidate the interactions crucial for PI4K inhibition and potent antiplasmodium activity. Despite its potential as an antimalarial target, the similarity between PfPI4K and structurally related human kinases poses a risk for human off-target kinase activity and associated toxicity. Comparative docking between PfPI4K and human phosphoinositide kinases (PIKs) presents compelling evidence for the origins of selectivity. This in-depth analysis of the PfPI4K homology model, the binding modes of the inhibitors, and the interactions responsible for selectivity over human kinases provides a powerful template for future optimization of Plasmodium PI4K inhibitors.

The lipid kinases that generate the lipid signalling phosphoinositides have been established as fundamental signalling enzymes that control numerous aspects of how cells respond to their extracellular environment. In addition, they play critical roles in regulating membrane trafficking and lipid transport within the cell. The class I phosphoinositide kinases which generate the critical lipid signal PIP

The lipid kinases that generate the lipid signalling phosphoinositides have been established as fundamental signalling enzymes that control numerous aspects of how cells respond to their extracellular environment. In addition, they play critical roles in regulating membrane trafficking and lipid transport within the cell. The class I phosphoinositide kinases which generate the critical lipid signal PIP3 are hyperactivated in numerous human pathologies including cancer, overgrowth syndromes, and primary immunodeficiencies. The type III phosphatidylinositol 4-kinase beta isoform (PI4KB), which are evolutionarily similar to the class I PI3Ks, have been found to be essential host factors mediating the replication of numerous devastating pathogenic viruses. Finally, targeting the parasite variant of PI4KB has been established as one of the most promising strategies for the development of anti-malarial and anti-cryptosporidium strategies. Therefore, the development of targeted isoform selective inhibitors for these enzymes are of paramount importance. The first generation of PI3K inhibitors have recently been clinically approved for a number of different cancers, highlighting their therapeutic value. This review will examine the history of the class I PI3Ks, and the type III PI4Ks, their relevance to human disease, and the structural basis for their regulation and inhibition by potent and selective inhibitors.

Inhibitors of the phosphatidylinositol 3-kinase (PI3K) - protein kinase B (PKB/Akt) - mechanistic target of rapamycin (mTOR) axis are considered valuable assets in cancer therapy. A considerable effort has been dedicated to the development of drugs targeting class I PI3Ks, which are evaluated in preclinical and clinical studies.[1-5] Here we present a strategy to convert a phase II clinical candidate, a pan-PI3K inhibitor (PQR309, bimiralisib)[5,6], into a highly selective, covalent PI3Kα inhibitor with the aim to minimize off-target and on-target metabolic side effects of PI3K inhibitor cancer therapy. We exploited a rational approach to increase target selectivity by covalently targeting PI3Kα at the non-conserved nucleophilic Cys862. A combination of warhead activity design, proximity screening and an optimized orientation allowed a tight control of reversible inhibitor binding in combination with an isoform-specific covalent reaction. To avoid off-target reactions, all warheads' reactivities were determined and optimize for selectivity and of Cys862 modification. An extensive Structure Activity Relationship (SAR) study was performed and a wide range of linear and restricted rotation linkers were introduced. A comprehensive understanding of the kinetics of irreversible inhibition acquired by kinetic TR-FRET assays and subsequent determination of kchem, kinact and calculated Ki allowed the establishment of a SAR, for compound selection with minimal off-target reactivity and high PI3Kα selectivity. X-ray crystallography and MS-based proteomics validated the covalent modification of Cys862. Our pilot compounds exceed specificity and potency over an experimental dimethyl-substituted enone, CNX-1351.[7] Moreover, our compounds display increased stability in rat liver microsomal assays and outperform the rapidly metabolized CNX-1351. Our strategy to investigate and tune warheads' reactivity represents a major step forward in the rational design of covalent chemical tools, overcoming the serendipity in the discovery of irreversible compounds. Moreover, we provide highly selective chemical tools to dissect PI3K isoform signaling in physiology and disease. A clarification of the role of the different PI3K isoforms in insulin signaling allows to address the challenges in isoform selectivity and to develop PI3K inhibitors showing ideal isoform specificity. [1] Rageot D et. al. J Med Chem. 2019, 62 (13), 6241-6261. [2] Borsari C et. al. ACS Med Chem Lett. 2019, 10 (10), 1473-1479. [3] Marone R et al. Biochim Biophys Acta 2008, 1784 (1), 159-185. [4] Wymann MP and Schneiter R Nat Rev Mol Cell Biol. 2008, 9 (2), 162-176. [5] Beaufils F et. al. J Med Chem. 2017, 60 (17), 7524-7538. [6] Wicki A et. al. Eur J Cancer. 2018, 96, 6-16. [7] Nacht M et. al. J Med Chem. 2013, 56 (3), 712-721. Citation Format: Chiara Borsari, Erhan Keles, Jacob McPhail, Alexander Schäfer, Rohitha Sriramaratnam, Martina De Pascale, Matthias Gstaiger, John Burke, Matthias P. Wymann. Volume scanning, a rational approach to covalent PI3Kα inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1377.

Phosphatidylinositol-3-kinase (PI3K) activity is aberrant in tumors, and PI3K inhibitors are investigated as cancer therapeutics.[1-5] The major obstacles to the successful implementation of PI3K-targeted cancer therapy are on-target adverse effects on insulin signaling. Isotype selective PI3K inhibitors have been exploited to answer fundamental questions regarding the role of PI3K isoforms in cell biology. However, the availability of claimed isoform-selective PI3Kα inhibitors is limited to BYL719 (Alpelisib)[6] and GDC0032 (Taselisib)[7], which do not maintain PI3Kα selectivity at a concentration required in cellular experimental settings and clinical applications. Highly selective PI3Kα inhibitors are expected to represent ideal tools to elucidate the role of PI3Kα isoform in tumor development and insulin signaling. As the systemic inactivation of PI3Kα is embryonic lethal, genetic approaches are currently limited to organ-specific targeting, and a specific inactivation of PI3Kα in an adult organism has not been achieved up-to-date. Herein, we generate high-quality PI3Kα chemical probes to dissect the role of PI3Kα in cancer and metabolism. We exploit covalent inhibitors, permanently blocking target functions, as a strategy to enhance the ligand binding selectivity for proteins in the same family. The non-conserved nucleophilic amino acid Cys862 in PI3Kα represents a promising target for covalent modifiers. We converted the reversible scaffold of PQR514[5] into irreversible compounds. An extensive Structure Activity Relationship (SAR) study was performed using CNX-1351[8] reacting group and introducing different heteroaliphatic rings in the linker. X-ray crystallography and bottom-up LC-MS/MS based proteomics validated the covalent modification of Cys862. Our pilot chemical probes exceeded in vitro and cellular potency over CNX-1351. The generation of a novel class of covalent PI3Kα-specific inhibitors with improved selectivity and persistency of PI3Kα-inhibition will shed light on the role of PI3Kα in cancer and metabolism. Our results will pave the way for the dissociation of PI3Ki antitumor activity from adverse effects on insulin action. [1] Beaufils F et al. J Med Chem. 2017, 60 (17), 7524-7538. [2] Rageot R et al. J Med Chem. 2019, 62 (13), 6241-6261. [3] Wymann MP et al. Nat Rev Mol Cell Biol. 2008, 9 (2), 62-176. [4] Marone R et al. Biochim Biophys Acta. 2008, 1784 (1), 159-185. [5] Borsari C et al. ACS Med Chem Lett. 2019, 10 (10), 1473-1479. [6] Markham A Drugs 2019, 79 (11), 1249-1253. [7] Zumsteg ZS et al. Clin Cancer Res. 2019, 22 (8), 2009-2019. [8] Nacht M et al. J Med Chem. 2013, 56 (3), 712-721. Citation Format: Martina De Pascale, Chiara Borsari, Erhan Keles, Jacob McPhail, Alexander Schäfer, Rohitha Sriramaratnam, Matthias Gstaiger, John Burke, Matthias Wymann. Development of optimized chemical probes targeting PI3Ka to deconvolute the role of class I PI3Ks isoforms in insulin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 291.

SummaryThe lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi, and is essential for the viral replication of specific enteroviruses. We used hydrogen deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 was also required for proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.Highlightsc10orf76 forms a direct complex with PI4KB, with the interface formed by a disorder-to-order transition in the kinase linker of PI4KBThe c10orf76 binding site of PI4KB can be phosphorylated by PKA, with phosphorylation leading to decreased affinity for c10orf76Complex-disrupting mutants of PI4KB and c10orf76 reveal that PI4KB recruits c10orf76 to the Golgi/TGNDepletion of c10orf76 leads to decreases in both active Arf1 and Golgi PI4P levelsEnteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76-PI4KB complex

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

Type III Phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking, and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles, and may lead to novel anti-viral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design we have designed novel potent and selective (>1000 fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed anti-viral activity against Hepatitis C Virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as anti-viral therapeutics.

Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks. * ACBD3, : acyl-CoA-binding domain containing protein 3; NCS-1, : neuronal calcium sensor 1; NS5A, : non-structural protein 5A; PI4K, : phosphatidylinositol 4 kinase; PI4KIIIα/β, : type III phosphatidylinositol 4 kinase α/β; PI4P, : phosphatidylinositol 4-phosphate; PIP2, : phosphatidylinositol 4,5-bisphosphate; PIP3, : phosphatidylinositol 3,4,5-trisphosphate; TGN, : trans -Golgi network; TTC7, : tetratricopeptide repeat protein 7

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting; Plasmodium; phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant; Plasmodium falciparum; and; Plasmodium vivax; clinical isolates. Excellent; in vitro; antiplasmodial activity translated into high efficacy in; Plasmodium berghei; and humanized; P. falciparum; NOD-; scid IL-2R; γ; null; mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate; in vivo; intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation; Plasmodium; PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches., PDB Code(s): 5C46; 5C4G