Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα

Abstract

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 ( k chem ), rate of covalent bond formation and proximity ( k inact and reaction space volume V r ), 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 k chem , k inact , K i , and V r , 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.

Construction of Covalent Bisubstrate Inhibitor of Protein Kinase Reacting with Cysteine Residue at Substrate-Binding Site

“…However, the results suggest that the low reactivity toward PKAcα stems from the non-optimal positioning of the FKM warhead in respect to Cys199 of PKAcα and it is not related to the insufficient electrophilicity of FMK warhead which, on the contrary, is highly reactive. Highly reactive warhead could have advantages in the first stage of inhibitor development, but it should be replaced with less reactive electrophile in the course of optimization of the structure of the inhibitor to increase the selectivity of the chemical reaction …”

“…Highly reactive warhead could have advantages in the first stage of inhibitor development, but it should be replaced with less reactive electrophile in the course of optimization of the structure of the inhibitor to increase the selectivity of the chemical reaction. 47 Finally, to confirm that the covalent modification of PKAcα occurs at Cys199 residue, LC-MS/MS analysis of trypsinated compound 4-PKAcα adduct was carried out. The peptide fragment of PKAcα incorporating modified Cys199 (TWTLC 199 GTPEYLAPEIILSK) with mass difference corresponding to the attached compound 4 (MW + 1235 Da) was detected that localized the modification unambiguously to Cys199.…”

Mailuoshutong pill for varicocele-associated male infertility—Phytochemical characterisation and multitarget mechanism

“…Hypoxia-inducible factor-1alpha (HIF1α) is crucial in varicocele-induced oxidative stress and apoptosis, and we found that PI3K/Akt, mTOR, and HIF-1 signaling pathways play an important role in HIF1α expression by enrichment analysis and review of related literature (Babaei et al, 2021), and by constructing the hub targets PPI network, it was found that HIF1α upstream related targets PIK3CA, AKT1, MTOR may be potential core targets for MLST in treating varicocele-associated male infertility. The protein crystal complexes (7R9V, 3O96, 3JBZ) were selected as docking targets for PIK3CA, AKT1, and MTOR, respectively, and the original ligands (2Q7, IQO, ADP) were used as positive controls, respectively (Wu et al, 2010;Lau et al, 2016;Borsari et al, 2022). The initial conformation and re-docking results of the original ligands were shown in Supplementary Table S2.…”

Proteome-Wide Profiling of the Covalent-Druggable Cysteines with a Structure-Based Deep Graph Learning Network

“…First, the noncovalent fragment selectively recognizes and binds to its target by favorable geometric and energetic complementarity. In the meantime, the warhead on the inhibitor is placed in an appropriate position relative to the nucleophilic residue around the pocket, which promotes the occurrence of the covalent-bond formation in the second step [13,14]. Theoretically, the amino acids with nucleophilic groups in the side chains, such as cysteine [9,15], serine [16,17], lysine [18][19][20], and threonine [21], have the potential to react with covalent inhibitors.…”