Anti-CLK4 monoclonal antibody Made to order

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimmagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT 116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT1 16 cells with VAN at minimally toxic concentralions (0.5-2.5 mM) reduced the spontaneous HPRT mutant fraction (MR, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5 mu M yielded a 13-56% reduction of the spontaneous MR Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimmagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip((R))) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116 + chr3) and deficient (HCT 116) cells at concentrations that were antimutagenic in HCT 116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations. (c) 2006 Elsevier B.V. All rights reserved.

The cdc2-like kinases (CLKs) are an evolutionarily conserved group of dual specificity kinases belonging to the CMGC (cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK) and CDK-like kinases). The CLK family consists of four isoforms namely CLK1, CLK2, CLK3 and CLK4. The human CLK1 encoded protein comprises 454 amino acids and the catalytic domain of CLK1 exhibits the typical protein kinase fold. CLK1 has been shown to autophosphorylate on serine, threonine and tyrosine residues and phosphorylate exogenous substrates on serine and threonine residues. CLK1 plays an important role in the regulation of RNA splicing through phosphorylation of members of the serine and arginine-rich (SR) family of splicing factors. CLK1 is involved in the pathophysiology of Alzheimer's disease by phosphorylating the serine residue in SR proteins. Nuclear speckles of the nucleoplasm contain the stored form of SR proteins and are moderately responsible for the choice of splicing sites during pre-mRNA splicing. Hence, the inhibition of CLK1 can be used as a therapeutic strategy for Alzheimer's disease. Many natural and synthetic molecules are reported to possess CLK1 inhibitory activity. Some specific examples are Marine alkaloid Leucettamine B and KH-CB19. Leucettamine B is a potent inhibitor of CLK1 (15 nM), Dyrk1A (40 nM), and Dyrk2 (35 nM) and a moderate inhibitor of CLK3 (4.5 M) whereas KH-CB19 is a highly specific and potent inhibitor of the CLK1/CLK4. X-ray crystallographic studies have revealed the binding mode of marine sponge metabolite hymenialdisine and a dichloroindolyl enamino nitrile (KH-CB19) to CLK1. This review focuses on the role of CLKs in the pathophysiology of Alzheimer's disease and therapeutic potential of targeting CLK1 in Alzheimer's disease drug discovery and development. In addition, the recent developments in drug discovery efforts targeting human CLK1 are also highlighted.