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  • Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

eLife (2021-03-05)
Manoj K Rathinaswamy, Zied Gaieb, Kaelin D Fleming, Chiara Borsari, Noah J Harris, Brandon E Moeller, Matthias P Wymann, Rommie E Amaro, John E Burke
ABSTRACT

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanosine 5′-[γ-thio]triphosphate tetralithium salt, ≥90% (contains < 10% GDP, HPLC), powder
Sigma-Aldrich
L-α-Phosphatidylethanolamine from egg yolk, Type III, 10 mg/mL in chloroform, ≥97%, solution
Sigma-Aldrich
Deuterium oxide, 99.9 atom % D
Sigma-Aldrich
PI 3-Kγ Inhibitor II, The PI 3-Kγ Inhibitor II, also referenced under CAS 648449-76-7, controls the biological activity of PI 3-Kγ. This small molecule/inhibitor is primarily used for Phosphorylation & Dephosphorylation applications.
Supelco
Cholesterol solution, certified reference material, 10 mg/mL in chloroform
Sigma-Aldrich
Sphingomyelin, from chicken egg yolk, ≥95%