Nevertheless, we employed this mutation to generate a p110δ-dependent mechanistic cellular model for PI3Ki assessment. In contrast, the similar E1021K substitution in p110δ occurs as a rare germline mutation that causes activated PI3K delta syndrome (APDS) 10. In this regard, the high frequency of activating point mutations in p110α, e.g., H1047R, in solid tumors points to an important targetable oncogenic potential 8, 9. The four class I PI3K catalytic subunit isoforms have distinct though overlapping roles in pivotal physiological cell functions as well as in oncogenic signaling 7. Among these, the serine threonine kinase AKT serves as an important signaling hub and AKT phosphorylation is commonly used to monitor PI3K activity in cells. By interaction of their pleckstrin-homology (PH) domains with PIP3, downstream mediators are recruited to the plasma membrane, where they are activated. PI3Ks catalyze the phosphorylation of the membrane constituent phosphoinositide-(4,5)-biphosphate (PIP2) to yield phosphoinositide-(3,4,5)-triphosphate (PIP3) 2. Among targeted agents, PI3Ki therefore currently plays a less prominent role in the treatment of CLL compared to BTK inhibitors and BH3 mimetics 6. Idelalisib, the first clinically applied PI3Ki, is an effective treatment of chronic lymphocytic leukemia (CLL) 3, but limited by immune-related adverse events and resistance 4, 5. Thus, disrupting the activity of p110δ for these indications resulted in the first clinical approvals of PI3K inhibitors (PI3Ki) 2. The treatment of B lymphoid malignancies has been revolutionized by targeting kinases involved in B cell receptor (BCR) signaling, which include the phosphoinositide-3-kinase (PI3K) isoform δ 1. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability.

Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes.

Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development.

Bh3 electron domain geometry