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

Abstract

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110g) playing key roles in immune signalling. p110g 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 PI3Kg kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

“…The putative binding epitopes are indicated in the source data. Multiple nanobodies caused decreased exchange in the helical domain of p110g, which has been observed for previous p110g antibody and small molecule binding partners (Gangadhara et al, 2019;Rathinaswamy et al, 2021;Shymanets et al, 2015) due to the propagation of allosteric changes to the helical domain.…”

“…This allowed us to identify and characterize p110g-binding nanobodies that activate lipid kinase activity, block activation by Ras, and a p101-binding nanobody that disrupts GPCR activation. Nanobodies were identified that stabilized flexible regions/domains, allowing for high-resolution cryo-EM studies (details described in a separate study, Rathinaswamy et al, 2021). Overall, this work provides an HDX-MS-enabled flow path for the rapid characterization of nanobodies for structural and biochemical studies (Figure 1).…”

“…The map was further refined using non-uniform refinement, yielding a map with an overall resolution of 3.36Å . The full details of the p110g-p101 structure for this map is described in a separate manuscript (Rathinaswamy et al, 2021).…”

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

“…The putative binding epitopes are indicated in the source data. Multiple nanobodies caused decreased exchange in the helical domain of p110 γ , which has been observed for previous p110 γ antibody and small molecule binding partners (Gangadhara et al, 2019; Rathinaswamy et al, 2021; Shymanets et al, 2015) due to the propagation of allosteric changes to the helical domain. Of the nanobodies studied by HDX, we were able to identity putative epitopes for the majority (Fig.…”

Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

“…Whether these mutants function within the tumor or the surrounding immune environment remains to be confirmed. Activating oncogenic mutants in the regulatory motif of the kinase domain of p110γ (R1021C) have been identified ( 62 ), with bi-allelic inactivating mutations involving the same site (R1021P, R982 frameshift) causing primary immunodeficiencies ( 63 ). We found oncogenic mutations clustered at ABD and p101 interfaces in PIK3CG.…”