Development of selective inhibitors of phosphatidylinositol 3-kinase C2α.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Phosphoinositide Conversion Inactivates R-RAS and Drives Metastases in Breast Cancer.

Breast cancer is the most prevalent cancer and a major cause of death in women worldwide. Although early diagnosis and therapeutic intervention significantly improve patient survival rate, metastasis still accounts for most deaths. Here it is reported that, in a cohort of more than 2000 patients with breast cancer, overexpression of PI3KC2α occurs in 52% of cases and correlates with high tumor grade as well as increased probability of distant metastatic events, irrespective of the subtype. Mechanistically, it is demonstrated that PI3KC2α synthetizes a pool of PI(3,4)P2 at focal adhesions that lowers their stability and directs breast cancer cell migration, invasion, and metastasis. PI(3,4)P2 locally produced by PI3KC2α at focal adhesions recruits the Ras GTPase activating protein 3 (RASA3), which inactivates R-RAS, leading to increased focal adhesion turnover, migration, and invasion both in vitro and in vivo. Proof-of-concept is eventually provided that inhibiting PI3KC2α or lowering RASA3 activity at focal adhesions significantly reduces the metastatic burden in PI3KC2α-overexpressing breast cancer, thereby suggesting a novel strategy for anti-breast cancer therapy.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.