Design, Synthesis, and Structure-Activity Relationship Optimization of Pyrazolopyrimidine Amide Inhibitors of Phosphoinositide 3-Kinase γ (PI3Kγ).

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Transcriptional profiling identifies novel regulators of macrophage polarization.

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. Major advances have occurred in understanding the transcriptional, epigenetic, and functional differences in various macrophage subsets by in vitro modeling and gene expression and epigenetic profiling for biomarker discovery. However, there is still no standardized protocol for macrophage polarization largely due to the lack of thorough validation of macrophage phenotypes following polarization. In addition, transcriptional regulation is recognized as a major mechanism governing differential macrophage polarization programs and as such, many genes have been identified to be associated with each macrophage subset. However, the functional role of many of these genes in macrophage polarization is still unknown. Moreover, the role of other regulatory mechanisms, such as DNA methylation, in macrophage polarization remains poorly understood. Here, we employed an optimized model of human M1 and M2 macrophage polarization which we used for large-scale transcriptional and DNA methylation profiling. We were unable to demonstrate a role for DNA methylation in macrophage polarization, as no significant changes were identified. However, we observed significant changes in the transcriptomes of M1 and M2 macrophages. Additionally, we identified numerous novel differentially regulated genes involved in macrophage polarization, including CYBB and DHCR7 which we show as important regulators of M1 and M2 macrophage polarization, respectively. Taken together, our improved in vitro human M1 and M2 macrophage model provides new understandings of the regulation of macrophage polarization and candidate macrophage biomarkers.