Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies.

Rational design of apoptosis signal-regulating kinase 1 inhibitors: discovering novel structural scaffold.

Increased activity of apoptosis signal-regulating kinase 1 (ASK1) is associated with a number of human disorders and the inhibitors of ASK1 may become important compounds for pharmaceutical application. Here we report novel ASK1 inhibitor scaffold, namely 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one, that has been identified using virtual screening and biochemical tests. A series of derivatives has been synthesized and evaluated in vitro towards human protein kinase ASK1. It was revealed that the most active compounds 4-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid and 6-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)hexanoic acid inhibit ASK1 with IC50 of 0.2 µM. Structure-activity relationships of 33 derivatives of 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one have been studied and binding mode of this chemical class has been predicted.

Identification of 3H-naphtho[1,2,3-de]quinoline-2,7-diones as inhibitors of apoptosis signal-regulating kinase 1 (ASK1).

Apoptosis signal-regulating kinase 1 (ASK1) has recently emerged as an attractive therapeutic target for the treatment of cardiac and neurodegenerative disorders. The selective inhibitors of ASK1 may become important compounds for the development of clinical agents. We have identified the ASK1 inhibitor among 3H-naphtho[1,2,3-de]quinoline-2,7-diones using receptor-based virtual screening. In vitro kinase assay revealed that ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline-1-carboxylate (NQDI-1) inhibited ASK1 with a K(i) of 500 nM. The competitive character of inhibition is demonstrated in Lineweaver-Burk plots. In our preliminary selectivity study this compound exhibited strong specific inhibitory activity toward ASK1.

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis.

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.