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ABSTRACT: The c-jun N-terminal kinase 3 (JNK3) is a promising drug target for the treatment of neurological disorders. Here we report a direct ELISA including the optimization of a nonradioactive immunosorbent JNK3 activity assay to determine inhibitory potency of small-molecule inhibitors. Based on our previous JNK3 assay and our recently optimized p38α mitogen activated protein kinase (MAPK) protocol for monitoring the phosphorylation of activating-transcription factor 2 (ATF-2), we present a rapid and straightforward alternative to conventional radioactive and indirect ELISA kinase assays. To validate the assay with the optimized assay conditions we used reference compounds and achieved well comparable IC(50) results to published data. The use of a linked monoclonal antibody increased the specificity and the sensitivity of the assay, reducing the required antibody concentration by approximately 100-fold. The novel protocol is an accurate, easy-to-handle and robust screening assay for JNK3 and the assay performance was reduced from 7.5 to 3h.
Journal of pharmaceutical and biomedical analysis 01/2011; 55(1):236-40. · 2.45 Impact Factor
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ABSTRACT: Two series of new compounds with the general formula 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles and their isosteric 2-imino derivatives were synthesized by the multicomponent reaction of the appropriate acetophenone, aromatic aldehyde, ammonium acetate, and malononitrile or ethyl cyanoacetate. The products were obtained with excellent yields. The prepared compounds were evaluated for their in vitro ability to inhibit p38 alpha-MAP kinase. Several compounds showed p38 MAP kinase inhibitory properties with IC(50) as low as 0.07 microM. This is the first time to report compounds with such scaffold as p38 alpha-MAP kinase inhibitors. This asserts the potentiality of multicomponent reactions in drug discovery.
Journal of Combinatorial Chemistry 07/2010; 12(4):559-65. · 3.41 Impact Factor
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ABSTRACT: The need for the development of selective agents, which only inhibit the mainly "harmful" cyclooxygenase-2 (COX-2) while leaving physiological COX-1 mostly unaffected, still remains, especially after the recent issues related to cardiovascular toxicity caused by some COX-2 selective agents. Thus there is still a demand for sensitive and rapid methods to assay for COX-2 selective agents. Among several in vitro testing systems the whole blood assay (WBA) is a well-known method to examine non-steroidal anti-inflammatory drugs (NSAIDs) in view of their potency to inhibit COX activity. This assay has some major advantages over enzyme-based or isolated cell assays. Emergence of artifacts due to cell separation steps is kept to a minimum and substances, even in disproportional high concentrations, can be examined outside the body in a physiological environment resembling most closely the in vivo conditions in living humans, i.e., 37 degrees C, homeostasis, presence of all blood compounds and cell-cell interactions remain intact. While COX-1 human whole blood assays are performed within less than 2 h, for established COX-2 assays one still has to allow for an overnight incubation step before gaining the desired plasma. The aim of the assay described in this chapter is to characterize an optimized human whole blood assay (hWBA). We present a simple, fast and reliable method to examine the capacity of NSAIDs at inhibiting COX-2 activity that can be applied for rapid and routine screening purposes.
Methods in molecular biology (Clifton, N.J.) 01/2010; 644:91-116.
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ABSTRACT: p38 MAP kinase has received considerable interest in the pharmaceutical industry and remains a valid and interesting target for the treatment of inflammation. To discover novel p38 inhibitors, we applied the ligand-based virtual screening technique, FieldScreen, to 1.2 million commercially available compounds. Fifty-eight diverse compounds were selected for biological analysis, using molecular field similarity to known inhibitors, while explicitly removing any structure that shared a scaffold with previously reported p38 inhibitors. Of these, 11 (19%) showed >or=20% inhibition of p38 at 10 microM. We chose to prepare analogues of two distinct chemical series resulting in a potential lead compound with pIC(50) of 6.4. Modeling of SAR using FieldAlign, a ligand alignment protocol, was used to rationalize the SAR of the series of thiadiazole based inhibitors.
Journal of Medicinal Chemistry 06/2009; 52(14):4200-9. · 4.80 Impact Factor
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ABSTRACT: In light of emerging interest in the relevance of c-Jun NH2-terminal protein kinase 3 (JNK3) as a promising drug target, we describe here an advanced non-radioactive immunosorbent JNK3 activity assay that is applicable for routine screening of small molecule ATP-competitive enzyme inhibitors. We modified and established a JNK3/ATF-2 protocol based on our previously described p38 MAPK method [1] for a substrate-bound non-radioactive procedure that represents a convenient alternative to conventional radioactive protein kinase assays. The objective of the present study was to validate these conditions by using the reference compounds SP600125 and SB203580 to achieve comparable IC(50) results to published data. Furthermore, an IC(50) for staurosporine was determined. The protocol we describe here represents an accessible and robust screening assay for JNK3 inhibitors.
Combinatorial Chemistry & High Throughput Screening 10/2006; 9(8):613-8. · 1.78 Impact Factor
