PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells.

The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta (PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not. In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice coincides with upregulation of PIK3CG expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals instructing commitment to the ASC fate.

Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα.

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinact and reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kß-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.

A class of highly selective inhibitors bind to an active state of PI3Kγ.

We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kß. On the basis of X-ray crystallography, hydrogen-deuterium exchange-mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.

Targeting Phosphoinositide 3-Kinase - Five Decades of Chemical Space Exploration.

Phosphoinositide 3-kinase (PI3K) plays a key role in a plethora of physiologic processes and controls cell growth, metabolism, immunity, cardiovascular and neurological function, and more. The discovery of wort-mannin as the first potent PI3K inhibitor (PI3Ki) in the 1990s provided rapid identification of PI3K-dependent processes, which drove the discovery of the PI3K/protein kinase B (PKB/Akt)/target of rapamycin (mTOR) pathway. Genetic mouse models and first PI3K isoform-specific inhibitors pinpointed putative therapeutic applications. The recognition of PI3K as target for cancer therapy drove subsequently drug development. Here we provide a brief journey through the emerging roles of PI3K to the development of preclinical and clinical PI3Ki candidates.

Lipid signalling in disease.

Signalling lipids such as eicosanoids, phosphoinositides, sphingolipids and fatty acids control important cellular processes, including cell proliferation, apoptosis, metabolism and migration. Extracellular signals from cytokines, growth factors and nutrients control the activity of a key set of lipid-modifying enzymes: phospholipases, prostaglandin synthase, 5-lipoxygenase, phosphoinositide 3-kinase, sphingosine kinase and sphingomyelinase. These enzymes and their downstream targets constitute a complex lipid signalling network with multiple nodes of interaction and cross-regulation. Imbalances in this network contribute to the pathogenesis of human disease. Although the function of a particular signalling lipid is traditionally studied in isolation, this review attempts a more integrated overview of the key role of these signalling lipids in inflammation, cancer and metabolic disease, and discusses emerging strategies for therapeutic intervention.

Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110γ.

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining ß-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in ß-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes ß-adrenergic receptor density and improves contractility in failing hearts.

PKCß phosphorylates PI3Kγ to activate it and release it from GPCR control.

All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCß phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca²âº- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCß-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca²âº and PKCß. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.

C-C motif chemokine CCL3 and canonical neutrophil attractants promote neutrophil extravasation through common and distinct mechanisms.

Initial observations suggested that C-C motif chemokines exclusively mediate chemotaxis of mononuclear cells. In addition, recent studies also implicated these chemotactic cytokines in the recruitment of neutrophils. The underlying mechanisms remained largely unknown. Using in vivo microscopy on the mouse cremaster muscle, intravascular adherence and subsequent paracellular transmigration of neutrophils elicited by the chemokine (C-C motif) ligand 3 (CCL3, synonym MIP-1α) were significantly diminished in mice with a deficiency of the chemokine (C-C motif) receptor 1 (Ccr1(-/-)) or 5 (Ccr5(-/-)). Using cell-transfer techniques, neutrophil responses required leukocyte CCR1 and nonleukocyte CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of G protein-receptor coupling and PI3Kγ-dependent signaling, while neutrophil recruitment induced by the canonical neutrophil attractants chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) or the lipid mediator platetelet-activating factor (PAF) was only partially reduced. Moreover, Ab blockade of ß(2) integrins, of α(4) integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly attenuated CCL3-, CXCL1-, or PAF-elicited intravascular adherence and paracellular transmigration of neutrophils. These data indicate that the C-C motif chemokine CCL3 and canonical neutrophil attractants exhibit both common and distinct mechanisms for the regulation of intravascular adherence and transmigration of neutrophils.

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis.

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

PI3Kγ within a nonhematopoietic cell type negatively regulates diet-induced thermogenesis and promotes obesity and insulin resistance.

Obesity is associated with a chronic low-grade inflammation, and specific antiinflammatory interventions may be beneficial for the treatment of type 2 diabetes and other obesity-related diseases. The lipid kinase PI3Kγ is a central proinflammatory signal transducer that plays a major role in leukocyte chemotaxis, mast cell degranulation, and endothelial cell activation. It was also reported that PI3Kγ activity within hematopoietic cells plays an important role in obesity-induced inflammation and insulin resistance. Here, we show that protection from insulin resistance, metabolic inflammation, and fatty liver in mice lacking functional PI3Kγ is largely consequent to their leaner phenotype. We also show that this phenotype is largely based on decreased fat gain, despite normal caloric intake, consequent to increased energy expenditure. Furthermore, our data show that PI3Kγ action on diet-induced obesity depends on PI3Kγ activity within a nonhematopoietic compartment, where it promotes energetic efficiency for fat mass gain. We also show that metabolic modulation by PI3Kγ depends on its lipid kinase activity and might involve kinase-independent signaling. Thus, PI3Kγ is an unexpected but promising drug target for the treatment of obesity and its complications.

Transient targeting of phosphoinositide 3-kinase acts as a roadblock in mast cells' route to allergy.

BACKGROUND: Tissue mast cell numbers are dynamically regulated by recruitment of progenitors from the vasculature. It is unclear whether progenitors are recruited during allergic sensitization and whether recruitment promotes allergic responses. OBJECTIVE: We sought to (1) determine the effect of mast cell recruitment on acute allergic responses and (2) to define the role of phosphoinositide 3-kinase (PI3K) isoforms in sequential steps to allergic responses. METHODS: Gene-targeted mice for PI3Kγ or PI3Kδ or mice treated with isoform-specific PI3K inhibitors (a novel PI3Kγ-specific inhibitor [NVS-PI3-4] and the PI3Kδ inhibitor IC87114) were used to monitor IgE-mediated mast cell recruitment, migration, adhesion by means of intravital microscopy, degranulation, TNF-α release, and subsequent endothelial cell activation in vivo or in bone marrow-derived mast cells. RESULTS: Functional PI3Kγ, but not PI3Kδ, was crucial for mast cell accumulation in IgE-challenged skin, TNF-α release from IgE/antigen-stimulated mast cells, and mast cell/endothelial interactions and chemotaxis. PI3Kγ-deficient bone marrow-derived mast cells did not adhere to the endothelium in TNF-α-treated cremaster muscle, whereas PI3Kδ was not required. Depletion of TNF-α blocked IgE-induced mast cell recruitment, which links tissue mast cell-derived cytokine release to endothelial activation and mast cell recruitment. Interference with mast cell recruitment protected against anaphylaxis and was superior to blockage of tissue mast cell degranulation. CONCLUSIONS: Interference with mast cell recruitment to exacerbated tissues provides a novel strategy to alleviate allergic reactions and surpassed attenuation of tissue mast cell degranulation. This results in prolonged drug action and allows for reduction of drug doses required to block anaphylaxis, an important feature for drugs targeting inflammatory disease in general.

Cell-permeant and photocleavable chemical inducer of dimerization.

Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360 nm cleaves the methyl-6-nitroveratryl core of MeNV-HaXS. MeNV-HaXS covalently links HaloTag- and SNAP-tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV-HaXS-mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV-HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV-HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi-targeted cargo and the assessment of nuclear import kinetics.

Correction: A high affinity pan-PI3K binding module supports selective targeted protein degradation of PI3Kα.

[This corrects the article DOI: 10.1039/D3SC04629J.].

A high affinity pan-PI3K binding module supports selective targeted protein degradation of PI3Kα.

Class I phosphoinositide 3-kinases (PI3Ks) control cellular growth, but are also essential in insulin signaling and glucose homeostasis. Pan-PI3K inhibitors thus generate substantial adverse effects, a reality that has plagued drug development against this target class. We present here evidence that a high affinity binding module with the capacity to target all class I PI3K isoforms can facilitate selective degradation of the most frequently mutated class I isoform, PI3Kα, when incorporated into a cereblon-targeted (CRBN) degrader. A systematic proteomics study guided the fine tuning of molecular features to optimize degrader selectivity and potency. Our work resulted in the creation of WJ112-14, a PI3Kα-specific nanomolar degrader that should serve as an important research tool for studying PI3K biology. Given the toxicities observed in the clinic with unselective PI3Kα inhibitors, the results here offer a new approach toward selectively targeting this frequently mutated oncogenic driver.

A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress.

Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S1P-triggered signaling events. S1P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.

Mal connects TLR2 to PI3Kinase activation and phagocyte polarization.

The recognition of bacterial lipoproteins by toll-like receptor (TLR) 2 is pivotal for inflammation initiation and control in many bacterial infections. TLR2-dependent signalling is currently believed to essentially require both adaptor proteins MyD88 (myeloid differentiation primary response gene 88) and Mal/TIRAP (MyD88-adapter-like/TIR-domain-containing adaptor protein). TLR2-dependent, but MyD88-independent responses have not been described yet. We report here on a novel-signalling pathway downstream of TLR2, which does not adhere to the established model. On stimulation of the TLR2/6 heterodimer with diacylated bacterial lipoproteins, Mal directly interacts with the regulatory subunit of phosphoinositide 3-kinase (PI3K), p85alpha, in an inducible fashion. The Mal-p85alpha interaction drives PI3K-dependent phosphorylation of Akt, phosphatidylinositol(3,4,5)P3 (PIP(3)) generation and macrophage polarization. MyD88 is not essential for PI3K activation and Akt phosphorylation; however, cooperates with Mal for PIP(3) formation and accumulation at the leading edge. In contrast to TLR2/6, TLR2/1 does not require Mal or MyD88 for Akt phosphorylation. Hence, Mal specifically connects TLR2/6 to PI3K activation, PIP(3) generation and macrophage polarization.

Genetic ablation of PI3Kγ results in defective IL-17RA signalling in T lymphocytes and increased IL-17 levels.

The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato-poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro-inflammatory cytokine IL-17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL-17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL-17 were not due to an aberrant expansion of IL-17-producing cells. Furthermore, we also identified an increase in IL-17RA expression on PI3Kγ(-/-) CD4(+) T cells, yet these cells exhibited impaired PI3K-dependent signalling in response to IL-17A, and subsequent NF-κB phosphorylation. In vivo, instillation of recombinant IL-17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL-17 was also reduced in PI3Kγ(-/-) lungs. These data demonstrate PI3Kγ-dependent signalling downstream of IL-17RA, which plays a pivotal role in regulating IL-17 production in T cells.

PI3Ks-drug targets in inflammation and cancer.

Phosphoinositide 3-kinases (PI3Ks) control cell growth, proliferation, cell survival, metabolic activity, vesicular trafficking, degranulation, and migration. Through these processes, PI3Ks modulate vital physiology. When over-activated in disease, PI3K promotes tumor growth, angiogenesis, metastasis or excessive immune cell activation in inflammation, allergy and autoimmunity. This chapter will introduce molecular activation and signaling of PI3Ks, and connections to target of rapamycin (TOR) and PI3K-related protein kinases (PIKKs). The focus will be on class I PI3Ks, and extend into current developments to exploit mechanistic knowledge for therapy.