Kinji Fuchikami

Discovery of novel and selective IKK-β serine-threonine protein kinase inhibitors. Part 1

IkappaB kinase beta (IKK-beta) is a serine-threonine protein kinase critically involved in the activation of the transcription factor Nuclear Factor kappa B (NF-kappaB) in response to various inflammatory stimuli. We have identified a small molecule inhibitor of IKK-beta. Optimization of the lead compound resulted in improvements in both in vitro and in vivo potency, and provided IKK-beta inhibitors exhibiting potent activity in an acute cytokine release model (LPS-induced TNFalpha).

A selective novel low‐molecular‐weight inhibitor of IκB kinase‐β (IKK‐β) prevents pulmonary inflammation and shows broad anti‐inflammatory activity

1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell‐mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF‐κB). 3 NF‐κB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK‐β. 4 We identified COMPOUND A as a small‐molecule, ATP‐competitive inhibitor selectively targeting IKK‐β kinase activity with a Ki value of 2 nM. 5 COMPOUND A inhibited stress‐induced NF‐κB transactivation, chemokine‐, cytokine‐, and adhesion molecule expression, and T‐ and B‐cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS‐induced TNF‐α in rodents. 7 In mice COMPOUND A inhibited cockroach allergen‐induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed‐type hypersensitivity. 10 These data suggest that IKK‐β inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.

A Versatile High-Throughput Screen for Inhibitors of Lipid Kinase Activity: Development of an Immobilized Phospholipid Plate Assay for Phosphoinositide 3-Kinase γ

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K γ isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on33 P-phosphate transfer from radiolabeled [γ33 P]ATP to phosphatidylinositol immobilized on Maxisorp™ plates. The established assay format for PI3K γ was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K γ determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K γ was observed at pH 7.0. The apparent Km value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3μM (6.0-8.6 μM, 95% confidence interval [CI]). IC50 values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.