Pascale Gaillard

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.

Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats

Myocardial ischemia/reperfusion is associated with inflammation, apoptosis and necrosis. During this process, c‐jun N‐terminal kinase is activated in cardiac myocytes resulting in apoptosis. This study investigates the effects of AS601245, a nonpeptide ATP competitive JNK inhibitor, on infarct size caused by myocardial ischemia/reperfusion in anaesthetized rats. The left descending coronary artery of anaesthetized rats was occluded for 30 min and then reperfused for 3 h. AS601245 was administered 5 min before the end of the ischemia period as an i.v. bolus (1.5, 4.5 or 15 mg kg−1 i.v.) followed by continuous i.v. infusion (18, 55 and 183 μg kg−1 min−1, respectively) during reperfusion. Controls received saline only. 3‐Aminobenzamide, a poly(ADP‐ribose) polymerase inhibitor, was used as reference compound at 10 mg kg−1 i.v. bolus plus 0.17 mg kg−1 min−1 continuous infusion. AS601245 significantly reduced infarct size at 4.5 mg kg−1 (−44%; P<0.001) and 15 mg kg−1 i.v. (−40.3%; P<0.001) similarly to 3‐aminobenzamide (−44.2%; P<0.001). This protective effect was obtained without affecting hemodinamics or reducing ST‐segment displacement. The beneficial effects on infarct size correlated well with the reduction of c‐jun phosphorylation (−85%; P<0.001 versus control) and of TUNEL‐positive cells (−82.1%; P<0.001) in post‐ischemic cardiomyocytes. No change in the phosphorylation state of p38 MAPK and ERK in post‐ischemic heart was observed in the presence of AS601245 in comparison to the vehicle‐treated group. These results demonstrate that blocking the JNK pathway may represent a novel therapeutic approach for treating myocardial ischemia/reperfusion‐induced cardiomyocyte death.

Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia.

c‐Jun N‐terminal kinase (JNK), a member of the mitogen‐activated protein kinase family, is activated in response to a number of extracellular stimuli, including inflammatory cytokines, UV irradiation and ischaemia. A large body of evidence supports a role for JNK signalling in stress‐induced apoptosis. It has been hypothesized that JNK may contribute to the apoptotic response by regulating the intrinsic cell death pathway involving the mitochondria. Here, we examined the role of the JNK signalling pathway in hippocampal CA1 apoptotic neurones following transient ischaemia in gerbils. We showed early activation of death receptor‐dependent apoptosis (caspase‐8 activation 2 days after ischaemia) and a biphasic activation of caspase‐3 and caspase‐9 after ischaemia. Activation of the mitochondrial pathway, as measured by cytochrome c release, appeared as a late event (5–7 days after ischaemia). AS601245, a novel JNK inhibitor, antagonized activation of both pathways and significantly protected CA1 neurones from cell death. Our results suggest a key role of JNK in the control of death receptor and mitochondrial‐dependent apoptosis after transient ischaemia.

AS601245, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduces axon/dendrite damage and cognitive deficits after global cerebral ischaemia in gerbils

Based on their proven ability, in animal models of stroke, to reduce damage to brain grey matter, many drugs have been tested in clinical trials but without success. Failure to save axons from injury and to protect functional outcome has been proposed as the major reason for this lack of success. We have previously demonstrated in two rodent models of cerebral ischaemia, that AS601245 (1,3‐benzothiazol‐2‐yl (2‐{[2‐(3‐pyridinyl) ethyl] amino}‐4 pyrimidinyl) acetonitrile), an inhibitor of the c‐Jun NH2‐terminal kinase (JNK), has neuroprotective properties. The aim of the present study was to further investigate if AS601245 in addition to its ability to protect neurons also could protect neurites and preserve memory after cerebral ischaemia, in gerbils.

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.