PI3K-δ and PI3K-γ Inhibition by IPI-145 Abrogates Immune Responses and Suppresses Activity in Autoimmune and Inflammatory Disease Models

SummaryPhosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and in vivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and T cell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored the therapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multiple inflammatory, autoimmune, and hematologic diseases

Membrane recognition and targeting by lipid-binding domains

Modular domains that recognize and target intracellular membranes play a critical role in the assembly, localization, and function of signaling and trafficking complexes in eukaryotic cells. Large domain families, including PH, FYVE, PX, PHD, and C2 domains, combine specific, nonspecific, and multivalent interactions to achieve selective membrane targeting. Despite structural and functional diversity, general features of lipid recognition are evident in the various membrane-targeting mechanisms

Specificity and membrane partitioning of Grsp1 signaling complexes with Grp1 family Arf exchange factors

The Arf exchange factor Grp1 selectively binds phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P(3)], which is required for recruitment to the plasma membrane in stimulated cells. The mechanisms for phosphoinositide recognition by the PH domain, catalysis of nucleotide exchange by the Sec7 domain, and autoinhibition by elements proximal to the PH domain are well-characterized. The N-terminal heptad repeats in Grp1 have also been shown to mediate homodimerization in vitro as well as heteromeric interactions with heptad repeats in the FERM domain-containing protein Grsp1 both in vitro and in cells [Klarlund, J. K., et al. (2001) J. Biol. Chem. 276, 40065-40070]. Here, we have characterized the oligomeric state of Grsp1 and Grp1 family proteins (Grp1, ARNO, and Cytohesin-1) as well as the oligomeric state, stoichiometry, and specificity of Grsp1 complexes with Grp1, ARNO, and Cytohesin-1. At low micromolar concentrations, Grp1 and ARNO are homodimeric whereas Cytohesin-1 and Grsp1 are monomeric. When mixed with Grsp1, Grp1 homodimers and Cytohesin-1 monomers spontaneously re-equilibrate to form heterodimers, whereas approximately 50% of ARNO remains homodimeric under the same conditions. Fluorescence resonance energy transfer experiments suggest that the Grsp1 heterodimers with Grp1 and Cytohesin-1 adopt a largely antiparallel orientation. Finally, formation of Grsp1-Grp1 heterodimers does not substantially influence the binding of Grp1 to the headgroups of PtdIns(3,4,5)P(3) or PtdIns(4,5)P(2), nor does it influence partitioning with liposomes containing PtdIns(3,4,5)P(3), PtdIns(4,5)P(2), and/or phosphatidylserine

Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors

Arf GTPases regulate membrane trafficking and actin dynamics. Grp1, ARNO, and Cytohesin-1 comprise a family of phosphoinositide-dependent Arf GTPase exchange factors with a Sec7-pleckstrin homology (PH) domain tandem. Here, we report that the exchange activity of the Sec7 domain is potently autoinhibited by conserved elements proximal to the PH domain. The crystal structure of the Grp1 Sec7-PH tandem reveals a pseudosubstrate mechanism of autoinhibition in which the linker region between domains and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete activation. Critical determinants of autoinhibition also contribute to insulin-stimulated plasma membrane recruitment. Autoinhibition can be largely reversed by binding of active Arf6 to Grp1 and by phosphorylation of tandem PKC sites in Cytohesin-1. These observations suggest that Grp1 family GEFs are autoregulated by mechanisms that depend on plasma membrane recruitment for activation

The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation.

The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110δ catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in PIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110δ inhibitor that has been FDA approved to treat certain B cell malignancies. p110δ inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110δ inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110γ isoform has partially overlapping roles with p110δ in B cell development, but little is known about its role in B cell function. We found that the p110γ inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110δ/γ inhibitor, IPI-443, had comparable effects to p110δ inhibition alone. These findings show that p110δ is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110δ function in immune cells

The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation.

The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110δ catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in PIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110δ inhibitor that has been FDA approved to treat certain B cell malignancies. p110δ inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110δ inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110γ isoform has partially overlapping roles with p110δ in B cell development, but little is known about its role in B cell function. We found that the p110γ inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110δ/γ inhibitor, IPI-443, had comparable effects to p110δ inhibition alone. These findings show that p110δ is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110δ function in immune cells

Regulation of T cell alloimmunity by PI3Kγ and PI3Kδ

Phosphatidylinositol-3-kinases (PI3K) γ and δ are preferentially enriched in leukocytes, and defects in these signaling pathways have been shown to impair T cell activation. The effects of PI3Kγ and PI3Kδ on alloimmunity remain underexplored. Here, we show that both PI3Kγ −/− and PI3Kδ D910A/D910A mice receiving heart allografts have suppression of alloreactive T effector cells and delayed acute rejection. However, PI3Kδ mutation also dampens regulatory T cells (Treg). After treatment with low dose CTLA4-Ig, PI3Kγ −/−, but not PI3Κδ D910A/D910A, recipients exhibit indefinite prolongation of heart allograft survival. PI3Kδ D910A/D910A Tregs have increased apoptosis and impaired survival. Selective inhibition of PI3Kγ and PI3Kδ (using PI3Kδ and dual PI3Kγδ chemical inhibitors) shows that PI3Kγ inhibition compensates for the negative effect of PI3Kδ inhibition on long-term allograft survival. These data serve as a basis for future PI3K-based immune therapies for transplantation

Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)‑γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate

Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (<b>26</b> or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors