E. Novellino

Calmodulin-Dependent Kinase II Mediates Vascular Smooth Muscle Cell Proliferation and Is Potentiated by Extracellular Signal Regulated Kinase

Vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling in atherosclerosis and hypertension. Calcium-dependent signaling through calcium/calmodulin-dependent kinase II (CaMKII) and ERK1/2 activation plays an important role in the regulation of VSMC proliferation by agents such as alpha-adrenergic receptor agonists. Nevertheless, how the CaMKII and ERK pathways interact in VSMCs has yet to be characterized. The aim of the present study was to clarify this interaction in response to alpha(1)-adrenergic receptor-mediated VSMC proliferation. We discovered that phenylephrine stimulation resulted in complex formation between CaMKII and ERK in a manner that facilitated phosphorylation of both protein kinases. To assess the effects of CaMKII/ERK association on VSMC proliferation, we inhibited endogenous CaMKII either pharmacologically or by adenoviral-mediated gene transfer of a kinase-inactive CaMKII mutant. Inhibition of CaMKII activation but not CaMKII autonomous activity significantly decreased formation of the CaMKII/ERK complex. On the contrary, the expression of constitutively active CaMKII enhanced VSMC growth and CaMKII/ERK association. In addressing the mechanism of this effect, we found that CaMKII could not directly phosphorylate ERK but instead enhanced Raf1 activation. By contrast, ERK interaction with CaMKII facilitated CaMKII phosphorylation and promoted its nuclear localization. Our results reveal a critical role for CaMKII in VSMC proliferation and imply that CaMKII facilitates assembly of the Raf/MEK/ERK complex and that ERK enhances CaMKII activation and influences its subcellular localization.

A Critical Review of Recent CoMFA Applications

Comparative molecular field analysis (CoMFA) is a technique for determining threedimensional quantitative structure‐activity relationships (3D QSAR). In a standard CoMFA procedure, a bioactive conformation of each compound under study is chosen, and all the structures are superimposed in a manner defined by the supposed mode of interaction with the target macromolecule. Then, the steric and the electrostatic fields of these molecules are calculated with a probe atom, such as sp 3 carbon atom with +1 charge, at regularly spaced (1 or 2 A) points of a three-dimensional grid. Sometimes other fields or physico-chemical parameters are also included. The calculated energy values and other descriptor values are then analyzed with the partial least-squares (PLS) statistical technique. The optimum number of components for the CoMFA model is selected based on the cross-validation test results. The final CoMFA model is derived using the optimum number of components selected. The results are usually displayed as coefficient contour maps. A good CoMFA model should show satisfactory statistical significance, explanatory capability of the variance in the activity of the compounds in the training set and predictive power of the potency of new compounds. This work describes the CoMFA studies published since 1993. Any aspects of the standard CoMFA procedure or the works described in the previous volume [156L] * of this book or those subjects that are extensively discussed in other chapters of this volume are not discussed in any detail. For such subjects, readers are referred to the corresponding chapters in this volume. There are many choices to be considered in a CoMFA analysis: [134L] biological data, selection of compounds and series design, generation of three-dimensional structure and charges of the ligand molecules, conformational analysis and establishment of the bioactive conformation of each molecule, alignment of the molecules, position of the lattice points, choice of force fields and calculation of the interaction energies, statistical analysis of the data and the selection of the final model, display of the results in contour maps and their interpretations, and design and forecasting the activity of unknown compounds. Those studies reported in the last few years can be largely divided into two groups. The first group includes those that studied various aspects of CoMFA procedures to

Conformational studies on a synthetic C-terminal fragment of the α subunit of GS proteins

It has recently been reported that synthetic peptides corresponding to the C-terminal sequence of G alpha, can be used to study the molecular mechanisms of interaction between this protein and G protein coupled receptors (Hamm et al., Science, 1988, Vol. 241, pp. 832-835). A conformational analysis on a 11 amino acids peptide from the G alpha(S) C-terminus, G alpha(S)(384-394) (H-QRMHLRQYELL-OH), was performed by nmr spectroscopy and molecular modeling methods. Two-dimensional nmr spectra, recorded in hexafluoroacetone/water, a mixture with structure stabilizing properties, showed an unusually high number of nuclear Overhauser effects, forming significative pattern to the drawing of a secondary structure. Conformations consistent with experimental NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers corresponding to an alpha-turn and a type III beta-turn involving the last five C-terminal residues. Interestingly, the alpha-turn conformation was found to overlap with good agreement the crystallographic structure of the same fragment in the G alpha(S) protein.

New small molecules, ISA27 and SM13, inhibit tumour growth inducing mitochondrial effects of p53.

Background:p53 is a transcription factor with tumour suppressor properties, which is able to induce mitochondrial apoptosis independently of its transcriptional activity. We recently synthesised two new compounds (ISA27 and SM13), which block p53-MDM2 interaction and induce apoptosis in p53 wild-type (WT) tumour cells. The aim of this study was to verify the effectiveness of these compounds in tumours carrying a mutated form of p53 gene with no transcriptional activity.Methods:In vitro we evaluated the effectiveness of our compounds in cancer cell lines carrying WT, mutated and null p53 gene. In vivo study was performed in Balb/c nude mice and the mitochondrial-dependent apoptotic signalling was evaluated by western blot.Results:Both ISA27 and SM13 reduced cell proliferation and induced apoptosis in vitro in cells carrying either p53 WT or mutated gene, suggesting that its effect is independent from p53 transcriptional activity. On the contrary, SM13 had no effect in a p53 null cell line. In vivo, ISA27 and SM13 induced cancer cell death in a dose-dependent manner through the activation of the mitochondrial-dependent death signalling in p53-mutated cells. In vivo, SM13 reduced tumour growth.Conclusions:Our study proposes SM13 as anticancer compound to use for the treatment of p53-dependent tumours, even in the absence of p53 transcriptional activity.

Design, Synthesis and Conformational Analysis of Human Urotensin II (U-II) Analogues with Lactam Bridge

Urotensin II (U-II), a cyclic neuropeptide with potent vasoconstrictor activity, has been originally isolated from the urophysis of the fish goby. Several structural forms of U-II, showing a disulfide bridge between cysteines, have been reported in different species of fish and amphibian, with variation occurring in the first five to seven N-terminal amino acids, while the C-terminal cyclic hexapeptide sequence is conserved in all species [1,2]. The recent cloning of the U-II precursor in frog and human has demonstrated that the peptide is not restricted to the fish urophysis, but is also expressed in the central nervous system of tetrapods. The identification of a human G protein-coupled receptor homologous to rat GPR14, expressed predominantly in cardiovascular tissue, has demonstrated that U-II is an endogenous ligand of this orphan receptor. Human U-II is an 11 amino acids peptide that retains the cyclohexapeptide sequence typical of this family (GluThrProAspCysPheTrpLysTyrCysVal). The cyclic sequence of U-II is the biologically active fragment, and it has been suggested that the conformation of the disulfide loop might modulate the activity of the molecule [3,4]. U-II is partially homologous and analogous to somatostatin, a peptide whose disulfide bridge is known to be important for activity.

Aromatase Inhibitors: Comparison between a CoMFA Model and the Enzyme Active Site

Aromatase inhibitors are among the most actively studied compounds in the field of antitumour agents, because of their role in the treatment of breast cancer. Aromatase is a cytochrome P 450 isozyme (P 450 XIX), that can be inhibited either competitively or non competitively by various classes of steroidal and non-steroidal compounds.