PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?

Dysregulated signal transduction in innate and adaptive immune cells is known to be associated with the development of various autoimmune and inflammatory diseases. Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases (PI3Ks) generate lipid-based second messengers that control an array of intracellular signalling pathways that are known to have important roles in leukocytes. In light of the recent progress in the development of selective PI3K inhibitors, and the beneficial effects of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis and other immune-mediated diseases.     

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.     

Targeting phosphoinositide 3-kinase: moving towards therapy

Phosphoinositide 3-kinases (PI3K) orchestrate cell responses including mitogenic signaling, cell survival and growth, metabolic control, vesicular trafficking, degranulation, cytoskeletal rearrangement and migration. Deregulation of the PI3K pathway occurs by activating mutations in growth factor receptors or the PIK3CA locus coding for PI3Kalpha, by loss of function of the lipid phosphatase and tensin homolog deleted in chromosome ten (PTEN/MMAC/TEP1), by the up-regulation of protein kinase B (PKB/Akt), or the impairment of the tuberous sclerosis complex (TSC1/2). All these events are linked to growth and proliferation, and have thus prompted a significant interest in the pharmaceutical targeting of the PI3K pathway in cancer. Genetic targeting of PI3Kgamma (p110gamma) and PI3Kdelta (p110delta) in mice has underlined a central role of these PI3K isoforms in inflammation and allergy, as they modulate chemotaxis of leukocytes and degranulation in mast cells. Proof-of-concept molecules selective for PI3Kgamma have already successfully alleviated disease progress in murine models of rheumatoid arthritis and lupus erythematosus. As targeting PI3K moves forward to therapy of chronic, non-fatal disease, safety concerns for PI3K inhibitors increase. Many of the present inhibitor series interfere with target of rapamycin (TOR), DNA-dependent protein kinase (DNA-PK(cs)) and activity of the ataxia telangiectasia mutated gene product (ATM). Here we review the current disease-relevant knowledge for isoform-specific PI3K function in the above mentioned diseases, and review the progress of >400 recent patents covering pharmaceutical targeting of PI3K. Currently, several drugs targeting the PI3K pathway have entered clinical trials (phase I) for solid tumors and suppression of tissue damage after myocardial infarction (phases I,II).     

PI3K inhibition in inflammation: Toward tailored therapies for specific diseases

In the past decade, the availability of genetically modified animals has enabled the discovery of interesting roles for phosphatidylinositol 3‐kinase‐γ (PI3Kγ) and ‐δ (PI3Kδ) in different cell types orchestrating innate and adaptive immune responses. Therefore, these PI3K isoforms appear to be attractive drug targets for the treatment of diseases caused by unrestrained immune reactions. Currently, pharmacological targeting of PI3Kγ and/or PI3Kδ represents one of the most promising challenges for companies interested in the development of novel safe treatments for inflammatory diseases. In this review we provide a general outline of PI3Kγ‐ and PI3Kδ‐specific functions in distinct subsets of inflammatory cells. We also discuss the therapeutic impact of novel compounds targeting PI3Kγ, PI3Kδ or both, in mouse models of autoimmune disorders (systemic lupus erythematosus (SLE) and rheumatoid arthritis), respiratory diseases (allergic asthma and chronic obstructive pulmonary disease) and cardiovascular dysfunctions (atherosclerosis and myocardial infarction).     

Phosphoinositide 3-kinases gamma and delta, linkers of coordinate C5a receptor-Fcgamma receptor activation and immune complex-induced inflammation

Fcgamma receptors (FcgammaR) and the C5a receptor (C5aR) are key effectors of the acute inflammatory response to IgG immune complexes (IC). Their coordinated activation is critical in IC-induced diseases, although the significance of combined signaling by these two different receptor classes in tissue injury is unclear. Here we used the mouse model of the passive reverse lung Arthus reaction to define their requirements for distinct phosphoinositide 3-kinase (PI3K) activities in vivo. We show that genetic deletion of class IB PI3Kgamma abrogates C5aR signaling that is crucial for FcgammaR-mediated activation of lung macrophages. Thus, in PI3Kgamma(-/-) mice, IgG IC-induced FcgammaR regulation, cytokine release, and neutrophil recruitment were blunted. Notably, however, C5a production occurred normally in PI3Kgamma(-/-) mice but was impaired in PI3Kdelta(-/-) mice. Consequently, class IA PI3Kdelta deficiency caused resistance to acute IC lung injury. These results demonstrate that PI3Kgamma and PI3Kdelta coordinate the inflammatory effects of C5aR and FcgammaR and define PI3Kdelta as a novel and essential element of FcgammaR signaling in the generation of C5a in IC disease.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Discovery of N-{5-[3-(3-hydroxypiperidin-1-yl)-1,2,4-oxadiazol-5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (TASP0415914) as an orally potent phosphoinositide 3-kinase γ inhibitor for the treatment of inflammatory diseases

Class I phosphoinositide 3-kinases (PI3Ks), particularly PI3Kγ, have become attractive drug targets for inflammatory and autoimmune disorders such as rheumatoid arthritis. Herein, we describe the synthesis and the structure–activity relationships (SAR) of a series of 2-amino-5-oxadiazolyl thiazoles, culminating in the identification of 8j (TASP0415914), an orally potent inhibitor of phosphoinositide 3-kinase γ (PI3Kγ). TASP0415914 demonstrated good potency in a cell-based assay and, furthermore, exhibited in vivo efficacy in a collagen induced arthritis (CIA) model in mice after oral administration.     

PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.     

Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kgamma and PI3Kdelta for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kgamma(-/-) or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kgamma(-/-) or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kdelta inhibitor, IC87114, or a specific PI3Kgamma inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kgamma inhibitor or in PI3Kgamma(-/-) mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kdelta in PI3Kgamma(-/-) mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kgamma(-/-) mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kgamma(-/-) mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kgamma. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kgamma and PI3Kdelta for neutrophil recruitment induced by different chemoattractants in vivo.     

Discovery of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent and selective PI3Kδ inhibitors

As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model.     

Artesunate Abolishes Germinal Center B Cells and Inhibits Autoimmune Arthritis

The antimalarial drug artemisinin and its derivatives exhibit potent immunosuppressive activity in several autoimmune disease models, however the mechanisms are not well-understood. This study was designed to investigate the therapeutic effects and the underlying mechanisms of the artemisinin analog artesunate using the K/BxN mouse model of rheumatoid arthritis. The well-studied disease mechanisms of K/BxN model allowed us to pinpoint the effect of artesunate on disease. Artesunate treatment prevented arthritis development in young K/BxN mice by inhibiting germinal center (GC) formation and production of autoantibodies. In adult K/BxN mice with established arthritis, artesunate diminished GC B cells in a few days. However, artesunate did not affect the follicular helper T cells (Tfh). In contrast to the spontaneous K/BxN model, artesunate treatment exerted minor influence on K/BxN serum transfer induced arthritis suggesting that artesunate has minimal effect on inflammatory responses downstream of antibody production. Finally, we showed that artesunate preferentially inhibits proliferating GC B cells. These results identify GC B cells as a target of artesunate and provide a new rationale for using artemisinin analogues to treat autoimmune diseases mediated by autoantibodies.     

Selective role of PI3K delta in neutrophil inflammatory responses

Although members of the class I phosphoinositide 3-kinases (PI3Ks) have been implicated in neutrophil inflammatory responses, the contribution of the individual PI3K isoforms in neutrophil activation has not been tractable with the non-selective inhibitors, LY294002 and wortmannin. We have developed a novel series of PI3K inhibitors that is selective for PI3K delta, an isoform expressed predominantly in hematopoietic cells. In addition to being selective between members of class I PI3Ks, representatives of these inhibitors such as IC980033 and IC87114 did not inhibit any protein kinases tested. Utilizing these inhibitors we report here a novel role for PI3K delta in neutrophil activation. Inhibition of PI3K delta with IC980033 and IC87114 blocked both fMLP- and TNF1 alpha-induced neutrophil superoxide generation and elastase exocytosis. The PI3K delta inhibitor IC87114 also blocked TNF1 alpha-stimulated elastase exocytosis from neutrophils in a mouse model of inflammation. To our knowledge, this is the first in vivo efficacy demonstration of a PI3K delta inhibitor in an animal model. Inhibition of PI3K delta, however, had no effect on in vitro neutrophil bactericidal activity and Fc gamma R-stimulated superoxide generation. Thus, PI3K delta plays an essential role in certain signaling pathways of neutrophil activation and appears to be an attractive target for the development of an anti-inflammatory therapeutic.     

Therapeutic effect of a poly(ADP-ribose) polymerase-1 inhibitor on experimental arthritis by downregulating inflammation and Th1 response

Poly(ADP-ribose) polymerase-1 (PARP-1) synthesizes and transfers ADP ribose polymers to target proteins, and regulates DNA repair and genomic integrity maintenance. PARP-1 also plays a crucial role in the progression of the inflammatory response, and its inhibition confers protection in several models of inflammatory disorders. Here, we investigate the impact of a selective PARP-1 inhibitor in experimental arthritis. PARP-1 inhibition with 5-aminoisoquinolinone (AIQ) significantly reduces incidence and severity of established collagen-induced arthritis, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of AIQ is associated with a striking reduction of the two deleterious components of the disease, i.e. the Th1-driven autoimmune and inflammatory responses. AIQ downregulates the production of various inflammatory cytokines and chemokines, decreases the antigen-specific Th1-cell expansion, and induces the production of the anti-inflammatory cytokine IL-10. Our results provide evidence of the contribution of PARP-1 to the progression of arthritis and identify this protein as a potential therapeutic target for the treatment of rheumatoid arthritis.     

Design and synthesis of phenethyl benzo[1,4]oxazine-3-ones as potent inhibitors of PI3Kinasegamma

The Type 1 PI3Kinases comprise a family of enzymes, which primarily phosphorylate PIP2 to give the second messenger PIP3, a key player in many intracellular signaling processes [Science, 2002, 296, 1655; Trends Pharmacol. Sci.2003, 24, 366]. Of the four type 1 PI3Ks, the gamma-isoform, which is expressed almost exclusively in leukocytes [Curr. Biol., 1997, 7, R470], is of particular interest with respect to its role in inflammatory diseases such as rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD) [Mol. Med. Today, 2000, 6, 347]. Investigation of a series of 4,6-disubstituted-4H-benzo[1,4]oxazin-3-ones has led to the identification of single-digit nanomolar inhibitors of PI3Kgamma, several of which had good cell based activity and were shown to be active in vivo in an aspectic peritonitis model of inflammatory cell migration.     

Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.     

Better understanding of phosphoinositide 3-kinase (PI3K) pathways in vasculature: Towards precision therapy targeting angiogenesis and tumor blood supply

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kβ, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg^–/–/Pik3cd^–/–) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 μM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10 μM) and PI-103 (1 μM), but not by the PI3Kβ isoform inhibitor TGX 221 (100 nM). Pik3cg^–/–/Pik3cd^–/–-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α₁-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.     

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.     

Successful treatment of animal models of rheumatoid arthritis with small-molecule cyclin-dependent kinase inhibitors

Intraarticular gene transfer of cyclin-dependent kinase (CDK) inhibitors to suppress synovial cell cycling has shown efficacy in treating animal models of rheumatoid arthritis. Endogenous CDK inhibitors also modulate immune function via a CDK-independent pathway. Accordingly, systemic administration of small molecules that inhibit CDK may or may not ameliorate arthritis. To address this issue, alvocidib (flavopiridol), known to be tolerated clinically for treating cancers, and a newly synthesized CDK4/6-selective inhibitor were tested for antiarthritic effects. In vitro, they inhibited proliferation of human and mouse synovial fibroblasts without inducing apoptosis. In vivo, treatment of collagen-induced arthritis mice with alvocidib suppressed synovial hyperplasia and joint destruction, whereas serum concentrations of anti-collagen type II (CII) Abs and proliferative responses to CII were maintained. Treatment was effective even when therapeutically administered. Treated mice developed arthritis after termination of treatment. Thus, immune responses to CII were unimpaired. The same treatment ameliorated arthritis induced by K/BxN serum transfer to lymphocyte-deficient mice. Similarly, the CDK4/6-selective inhibitor suppressed collagen-induced arthritis. Both small-molecule CDK inhibitors were effective in treating animal models of rheumatoid arthritis not by suppressing lymphocyte function. Thus, the two small-molecule CDK inhibitors ameliorated arthritis models in a distinctive way, compared with other immunosuppressive drugs.