Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype.

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BACKGROUND AND PURPOSEThe aim of this study was to characterize the functional impact of KCNQ1-encoded voltage-dependent potassium channels (Kv7.1) in the vasculature. EXPERIMENTAL APPROACHMesenteric arteries, intrapulmonary arteries and thoracic aortae were isolated from adult rats. Kv7.1 channel expression was established by fluorescence immunocytochemistry. Wire myography determined functionality of these channels in response to selective blockers and activators. Xenopus oocytes expressing Kv7.1 channels were used to assess the effectiveness of selective Kv7.1 channel blockers. KEY RESULTSKv7.1 channels were identified in arterial myocytes by immunocytochemistry. Kv7.1 blockers HMR1556, L-768,673 (10 mM) and JNJ39490282 (JNJ282; 1 mM) had no contractile effects in arteries, whereas the pan-Kv7 channel blocker linopirdine (10 mM) evoked robust contractions. Application of two compounds purported to activate Kv7.1 channels, L-364 373 (R-L3) and mefenamic acid, relaxed mesenteric arteries preconstricted by methoxamine. These responses were reversed by HMR1556 or L-768,673 but not JNJ282. Similar effects were observed in the thoracic aorta and intrapulmonary arteries. CONCLUSIONS AND IMPLICATIONSIn contrast to previous assumptions, Kv7.1 channels expressed in arterial myocytes are functional ion channels. Although these channels do not appear to contribute to resting vascular tone, Kv7.1 activators were effective vasorelaxants. Abbreviations

As a continuation of our earlier study (J. Med. Chem. 1992, 35, 4334-4343) we conformationally restricted the sigma-receptor ligand 2-(1-pyrrolidinyl)-N-[2-(3,4-dichlorophenyl)ethyl]-N-methylethylamine (1) by incorporating it into a series of homologous piperazines 3-9 and homopiperazines 10 and 11, diazabicyclononanes and decanes, bridgehead bicyclooctanes and nonanes as well as other miscellaneous compounds. sigma-Receptor binding affinities were obtained using [3H](+)-pentazocine in guinea pig brain membrane sigma 1 sites. The studies suggest that the nitrogen lone pair orientation found in the piperazines affords the strongest binding interaction. Other nitrogen lone pair orientations or compounds representing unlikely staggered conformations of 1 [ as in 4-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[3.2.2]nonane (16)] show very weak sigma interaction. Comparison of the binding data of different N-substituted homologues of 1 with those of the 1-[2-(3,4-dichlorophenyl)ethyl]-4-alkylpiperazines suggests that the two nitrogen atoms of 1 are working in opposition to one another in terms of their sensitivity to steric bulk. The high binding affinity of the 1,4-diazabicyclo[4.3.0]nonanes 12 suggests that these may approximate the methyl and pyrrolidine ring conformations found in 1 when it is bound to the sigma receptor. Compound 12 exhibited a 4-fold enantioselectivity favoring (+)-12. The synthesis of 6,7-dichloro-2-[[2-(1-pyrrolidinyl)ethyl]amino]tetralin (19) and its desmethyl derivative 20 permitted constraint of the 3,4-dichlorophenyl and N-methyl moieties of 1 into a gauche orientation. The binding data suggests that this conformation in 1 favors strong binding interaction at sigma-receptors. sigma-Receptor Ki's ranged from 0.55 nM for 1-[2-(3,4-dichlorophenyl)ethyl]-4-n-butylpiperazine (7) to 654 nM for 16. Overall comparison of the results indicate that 1 is subject to considerable conformational freedom and suggests that the sigma receptor is not subject to rigid stereochemical restraints with 1. These results add to our earlier study where we restrained 1 using simple monocyclic heterocycles.

The V600E missense mutation in B-Raf kinase leads to an anomalous regulation of the MAPK pathway, uncontrolled cell proliferation, and initiation of tumorigenesis. While the ATP-competitive B-Raf inhibitors block the MAPK pathway in B-Raf mutant cells, they induce conformational changes to wild-type B-Raf kinase domain leading to heterodimerization with C-Raf causing a paradoxical hyperactivation of MAPK pathway. A new class of inhibitors (paradox breakers) has been developed that inhibit B-Raf(V600E) activity without agonistically affecting the MAPK pathway in wild-type B-Raf cells. In this study, we explore the structural, conformational, and cellular effects on the B-Raf kinase domain upon binding of paradox breakers and inducers. Our results indicate that a subtle structural difference between paradox inducers and breakers leads to significant conformational differences when complexed with B-Raf. This study provides a novel insight into the activation of B-Raf by ATP-competitive inhibitors and can aid in the design of more potent and selective inhibitors without agonistic function.

Abstract. There is a growing number of biologically active peptides which have potential for the development of new therapeutics. However, native peptides are only rarely directly usable as drugs, due to inherent limitations which include rapid proteolysis and metabolism, poor transport properties, rapid excretion by the liver and kidneys, and low oral activity. Furthermore, peptides are often aselective in their actions owing to their flexible structure. In efforts to address these limitations, peptides are modified into mimetics with specific physical, chemical and biological characteristics. These so-called peptidomimetics are derived from peptides by partly or completely removing the amide bonds while retaining essential amino acid side chains in a defined, spatial relationship. They can be developed in a rational design cycle which starts either from a lead compound obtained in a screening programme or from the parent peptide. In the latter approach, which is the focus of this overview, first the smallest, active sequence is found, and the significance of each amino acid for the biological activity is determined. In the following steps the bioactive conformation is defined by the introduction of local and global constraints. In the final step the essential amino acid side chains are positioned onto a scaffold, preferably a small, polyfunctional ring of defined stereochemistry, using structural information obtained in earlier steps. Five examples of the application of the peptidomimetic design cycle in medicinal chemistry are described here, illustrating the progression from native peptides to therapeutically useful drugs.

that they are a major source of energy. TGs are synthesized from glycerol and three FA molecules; each FA is attached via an ester bond to hydroxyl groups of the glycerol backbone. Like many neutral lipids, TGs contain FA molecules with varying chain lengths; the most common are 16, 18, or 20 carbons. The two major biosynthetic pathways of TG are the glycerol-3-phosphate pathway, which exists primarily in liver and adipose tissues, and the monoacylglycerol pathway, which exists predominately in the intestine. The fi nal step of the glycerol-3-phosphate biosynthetic pathway can be catalyzed by either diacylglycerol acyltransferase 1 (DGAT1) or DGAT2 ( 1, 2 ). Although DGAT1 and DGAT2 both convert diacylglycerol (DAG) to TG, they do not share similarity in either their nucleotide or amino acid sequences.It has been reported that knockout mice lacking DGAT1 (DGAT1 Ϫ / Ϫ ) do not display obvious changes in TG metabolism in the liver ( 3 ). In contrast, knockout mice lacking DGAT2 (DGAT2 Ϫ / Ϫ ) display severely reduced TG content in the liver ( 4 ). Furthermore, studies have shown that suppression of DGAT2 with antisense oligonucleotides reduced hepatic TG content in rodents ( 5, 6 ) producing reversed diet-induced hepatic steatosis and insulin resistance in rats ( 5 ). These results suggest that DGAT1 and DGAT2 function differently in TG biosynthesis. The fi nding that multiple enzymes catalyze the synthesis of TG from DAG presents an opportunity to modulate one catalytic mechanism of this biochemical reaction. This type of modulation may produce therapeutic results without the potential adverse side effects that might occur if TG synthesis were completely inhibited in all tissues. By specifically inhibiting the activity of DGAT1 or DGAT2, compounds that inhibit the conversion of DAG to TG will be useful in lowering absorption and circulating concentrations of TG.Abstract Acyl -CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGATexpressing cells with stable isotope-labeled [

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.