Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional 1 H NMR. The far-and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a T m of 76.0 ؎ 0.2°C and no appreciable loss of the secondary structure up to 85°C. This is in contrast to the coincident denaturation of both tertiary and secondary structures with T m values of 74.8 ؎ 0.4°C and 74.3 ؎ 0.7°C, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.

Metastin, also known as KiSS-1, the cognate ligand for the metastin receptor GPR54, is a peptide known to dramatically reduce metastasis in experimental models. Despite this, there is no reported structure for metastin nor any small molecule modulators of metastin function that could be used either clinically or experimentally. Here we report the NMR solution structure of a 13-residue metastin peptide in a membrane-like environment (SDS micelles) and find it to have a relatively stable helix conformation from residues 7 to 13. In assays for metastin receptor binding and calcium flux with receptor-transfected HEK-293 cells, we demonstrate through alanine scanning and amino acid substitutions that the peptide C-terminus shows helix periodicity in an NMR structural model and that Phe9, Arg12, and Phe13 are crucial to the activity of the peptide. These three residues lie on one face of the helix and define a pharmacophore site for metastin. We used these pharmacophore features in small molecule database searches to identify hits with submicromolar affinity for the metastin receptor. We also show here that molecules mimicking key elements of this pharmacophore site bind to the metastin receptor and act as full agonists, albeit with reduced potency compared to that of metastin itself. Together this structure-activity approach may yield pharmacologically useful compounds relevant in defining and modulating metastin receptor function.

Modulation of aberrant cell cycle regulation is a potential therapeutic strategy applicable to a wide range of tumor types. JNJ-7706621 is a novel cell cycle inhibitor that showed potent inhibition of several cyclin-dependent kinases (CDK) and Aurora kinases and selectively blocked proliferation of tumor cells of various origins but was about 10-fold less effective at inhibiting normal human cell growth in vitro. In human cancer cells, treatment with JNJ-7706621 inhibited cell growth independent of p53, retinoblastoma, or P-glycoprotein status; activated apoptosis; and reduced colony formation. At low concentrations, JNJ-7706621 slowed the growth of cells and at higher concentrations induced cytotoxicity. Inhibition of CDK1 kinase activity, altered CDK1 phosphorylation status, and interference with downstream substrates such as retinoblastoma were also shown in human tumor cells following drug treatment. Flow cytometric analysis of DNA content showed that JNJ-7706621 delayed progression through G 1 and arrested the cell cycle at the G 2 -M phase. Additional cellular effects due to inhibition of Aurora kinases included endoreduplication and inhibition of histone H3 phosphorylation. In a human tumor xenograft model, several intermittent dosing schedules were identified that produced significant antitumor activity. There was a direct correlation between total cumulative dose given and antitumor effect regardless of the dosing schedule. These results show the therapeutic potential of this novel cell cycle inhibitor and support clinical evaluation of JNJ-7706621. (Cancer Res 2005; 65(19): 9038-46)

A series of 1-acyl-1H-[1,2,4]triazole-3,5-diamine analogues were synthesized as cyclin-dependent kinase (CDK) inhibitors. These compounds showed potent and selective CDK1 and CDK2 inhibitory activities and inhibited in vitro cellular proliferation in various human tumor cells. Representative compound 3b demonstrated in vivo efficacy in a human melanoma A375 xenograft model in nude mice.