Thomas Rückle

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kγ (encoded by Pik3cg). We show that Pik3cg−/− mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kγ as a therapeutic target. We also describe that oral treatment with a PI3Kγ inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg−/− mice. Our results identify selective PI3Kγ inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

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) γ, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kγ may be a useful target in the treatment of chronic inflammation.

Phosphoinositide 3-kinase p110beta activity : key role in metabolism and mammary gland cancer but not development

The phosphoinositide 3-kinase p110β subunit has noncatalytic functions; its catalytic activity is pertinent to both diabetes and cancer. Unveiling p110β Phosphatidylinositide 3-kinase (PI3K) signaling has been implicated in the response to insulin and various growth factors. However, the specific role of the β isoform of the PI3K catalytic subunit (p110β) has been unclear. Analysis of mouse mutants carrying a catalytically inactive form of p110β reveals that it possesses noncatalytic as well as catalytic functions. Moreover, its catalytic activity is involved in sustaining the response to insulin signaling and in mediating forms of breast cancer associated with oncogenic epidermal growth factor signaling. The phosphoinositide 3-kinase (PI3K) pathway crucially controls metabolism and cell growth. Although different PI3K catalytic subunits are known to play distinct roles, the specific in vivo function of p110β (the product of the PIK3CB gene) is not clear. Here, we show that mouse mutants expressing a catalytically inactive PIK3CBK805R mutant survived to adulthood but showed growth retardation and developed mild insulin resistance with age. Pharmacological and genetic analyses of p110β function revealed that p110β catalytic activity is required for PI3K signaling downstream of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors as well as to sustain long-term insulin signaling. In addition, PIK3CBK805R mice were protected in a model of ERBB2-driven tumor development. These findings indicate an unexpected role for p110β catalytic activity in diabetes and cancer, opening potential avenues for therapeutic intervention.

PI3Kγ inhibition: towards an 'aspirin of the 21st century'?

Class IB phosphatidylinositol 3-kinase p110γ (PI3Kγ) has gained increasing attention as a promising drug target for the treatment of inflammatory disease. Extensive target-validation data are available, which are derived from studies using both pharmacological and genetic tools. More recent findings have uncovered further therapeutic applications for PI3Kγ inhibitors, opening up potentially huge opportunities for these drugs. Several companies have been pursuing small-molecule PI3Kγ inhibitor projects, but none of them has progressed to the clinic yet. Here, we discuss the insights gained so far and the main challenges that are emerging on the path to developing PI3Kγ inhibitors for the treatment of human disease.

The p110[delta] structure: mechanisms for selectivity and potency of new PI(3)K inhibitors

Deregulation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in numerous pathologies like cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small molecule and ATP-competitive PI3K inhibitors with a wide range of selectivities have entered clinical development. In order to understand mechanisms underlying isoform selectivity of these inhibitors, we developed a novel expression strategy that enabled us to determine the first crystal structure of the catalytic subunit of the class IA PI3K p110δ. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI3K inhibitors reveal that selectivity towards p110δ can be achieved by exploiting its conformational flexibility and the sequence diversity of active-site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110δ with greatly improved potencies.

Isoform-Specific Functions of Phosphoinositide 3-Kinases: p110δ but Not p110γ Promotes Optimal Allergic Responses In Vivo

The leukocyte-enriched p110γ and p110δ isoforms of PI3K have been shown to control in vitro degranulation of mast cells induced by cross-linking of the high affinity receptor of IgE (FcεRI). However, the relative contribution of these PI3K isoforms in IgE-dependent allergic responses in vivo is controversial. A side-by-side comparative analysis of the role of p110γ and p110δ in mast cell function, using genetic approaches and newly developed isoform-selective pharmacologic inhibitors, confirms that both PI3K isoforms play an important role in FcεRI-activated mast cell degranulation in vitro. In vivo, however, only p110δ was found to be required for optimal IgE/Ag-dependent hypersensitivity responses in mice. These observations identify p110δ as a key therapeutic target among PI3K isoforms for allergy- and mast cell-related diseases.

Genetic and pharmacological targeting of phosphoinositide 3-kinase-gamma reduces atherosclerosis and favors plaque stability by modulating inflammatory processes.

Background— The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a notable quest for novel and innovative drugs for the treatment of atherosclerosis. The lipid kinase phosphoinositide 3-kinase-&ggr; (PI3K&ggr;) is thought to be a key player in various inflammatory, autoimmune, and allergic processes. These properties and the expression of PI3K&ggr; in the cardiovascular system suggest that PI3K&ggr; plays a role in atherosclerosis. Methods and Results— Here, we demonstrate that a specific PI3K&ggr; inhibitor (AS605240) is effective in murine models of established atherosclerosis. Intraperitoneal administration of AS605240 (10 mg/kg daily) significantly decreased early atherosclerotic lesions in apolipoprotein E–deficient mice and attenuated advanced atherosclerosis in low-density lipoprotein receptor–deficient mice. Furthermore, PI3K&ggr; levels were elevated in both human and murine atherosclerotic lesions. Comparison of low-density lipoprotein receptor–deficient mice transplanted with wild-type or PI3K&ggr;-deficient bone marrow demonstrated that functional PI3K&ggr; in the hematopoietic lineage is required for atherosclerotic progression. Alleviation of atherosclerosis by targeting of PI3K&ggr; activity was accompanied by decreased macrophage and T-cell infiltration, as well as increased plaque stabilization. Conclusions— These data identify PI3K&ggr; as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment.

BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity

Bone morphogenetic proteins (BMPs) are potent regulators of several cellular events. We report that exposure of C2C12 cells to BMP2 leads to an increase in cell migration and a rapid rearrangement of the actin filaments into cortical protrusions. These effects required independent and parallel activation of the Cdc42 small GTPase and the α-isoform of the phosphoinositide 3-kinase (PI3Kα), because ectopic expression of a dominant-negative form of Cdc42 or distinct pharmacological PI3K inhibitors abrogated these responses. Furthermore, we demonstrate that BMP2 activates different group I and group II PAK isoforms as well as LIMK1 with similar kinetics to Cdc42 or PI3K activation. BMP2 activation of PAK and LIMK1, measured by either kinase activity or with antibodies raised against phosphorylated residues at their activation loops, were abolished by blocking PI3K-signaling pathways. Together, these findings suggest that Cdc42 and PI3K signals emanating from BMP receptors are involved in specific regulation of actin assembly and cell migration.

Regulation of the severity of neuroinflammation and demyelination by TLR-ASK1-p38 pathway

Apoptosis signal‐regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen‐activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll‐like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1‐p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE‐induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR‐ASK1‐p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.

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 PI3Kγ and PI3Kδ for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kγ−/− 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 PI3Kγ−/− or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kδ inhibitor, IC87114, or a specific PI3Kγ inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kγ inhibitor or in PI3Kγ−/− mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kδ in PI3Kγ−/− mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kγ−/− mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kγ−/− mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kγ. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kγ and PI3Kδ for neutrophil recruitment induced by different chemoattractants in vivo.