Characterization of Novel PI3Kδ Inhibitors as Potential Therapeutics for SLE and Lupus Nephritis in Pre-Clinical Studies.

SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.

Leukocyte transmigration is modulated by chemokine-mediated PI3Kgamma-dependent phosphorylation of vimentin.

Phosphoinositide 3-kinase gamma (PI3Kgamma) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine-stimulated WT or PI3Kgamma-null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kgamma signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kgamma or the kinase activity of PI3Kgamma (PI3Kgamma(KD/KD)), phosphorylation of vimentin was reduced. PI3Kgamma-null macrophages displayed impaired chemokine-driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N-terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kgamma-null macrophages with a vimentin mutant mimicking serine phosphorylation of N-terminal residues rescued the transendothelial migration defect. These results define vimentin N-terminal phosphorylation and fiber reorganization as a target of chemokine-dependent PI3Kgamma signaling in leukocytes.

Inactivation of PI3Kgamma and PI3Kdelta distorts T-cell development and causes multiple organ inflammation.

Mice lacking both the p110gamma and p110delta isoforms display severe impairment of thymocyte development. Here, we show that this phenotype is recapitulated in p110gamma-/-/p110delta(D910A/D910A) (p110gamma(KO)delta(D910A)) mice where the p110delta isoform has been inactivated by a point mutation. Moreover, we have examined the pathological consequences of the p110gammadelta deficiency, which include profound T-cell lymphopenia, T-cell and eosinophil infiltration of mucosal organs, elevated IgE levels, and a skewing toward Th2 immune responses. Using small-molecule selective inhibitors, we demonstrated that in mature T cells, p110delta, but not p110gamma, controls Th1 and Th2 cytokine secretion. Thus, the pathology in the p110gammadelta-deficient mice is likely to be secondary to a developmental block in the thymus that leads to lymphopenia-associated inflammatory responses.

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages.

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.