Improvement of metabolic state in an animal model of nutrition-dependent type 2 diabetes following treatment with S 23521, a new glucagon-like peptide 1 (GLP-1) analogue.

Glucagon-like peptide 1 (GLP-1) analogues are considered potential drugs for type 2 diabetes. We studied the effect of a novel GLP-1 analogue, S 23521 ([a8-des R36] GLP-1-[7-37]-NH2), on the metabolic state and beta-cell function, proliferation and survival in the Psammomys obesus model of diet-induced type 2 diabetes. Animals with marked hyperglycaemia after 6 days of high-energy diet were given twice-daily s.c. injection of 100 microg/kg S 23521 for 15 days. Food intake was significantly decreased in S 23251-treated P. obesus; however, there was no significant difference in body weight from controls. Progressive worsening of hyperglycaemia was noted in controls, as opposed to maintenance of pre-treatment glucose levels in the S 23521 group. Prevention of diabetes progression was associated with reduced mortality. In addition, the treated group had higher serum insulin, insulinogenic index and leptin, whereas plasma triglyceride and non-esterified fatty acid levels were decreased. S 23521 had pronounced effect on pancreatic insulin, which was 5-fold higher than the markedly depleted insulin reserve of control animals. Immunohistochemical analysis showed islet degranulation with disrupted morphology in untreated animals, whereas islets from S 23521-treated animals appeared intact and filled with insulin; beta-cell apoptosis was approximately 70% reduced, without a change in beta-cell proliferation. S 23521 treatment resulted in a 2-fold increase in relative beta-cell volume. Overall, S 23521 prevented the progression of diabetes in P. obesus with marked improvement of the metabolic profile, including increased pancreatic insulin reserve, beta-cell viability and mass. These effects are probably due to actions of S 23521 both directly on islets and via reduced food intake, and emphasize the feasibility of preventing blood glucose deterioration over time in type 2 diabetes.

Peptide dynamic fingerprints: a tool for investigating the role of conformational flexibility for GLP-1 analogs affinity.

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide implicated in short-term appetite regulation. Its analogs are presumed to be potential drugs against obesity and non-insulin dependent diabetes mellitus (NIDDM or type 2 diabetes). This study examined how the dynamic fingerprints can be used for establishing dynamics-activity relationships in a series of peptides for which the mechanism of action is unknown and in which mutations can cause an increase or decrease in biological activity. The 3D autocorrelation method was used to generate maps of both active and inactive analogs. As the active conformation of GLP-1 is not yet clearly defined, the dynamic fingerprints of peptides in an aqueous environment were compared to explain the high affinity of the peptide for its receptor. The suggestion that the peptide could bind to the receptor in a folded conformation has been examined. In the case of the GLP-1 analogs, it was shown that the folding tendency cannot be directly related to affinity values and the results do not favor a folded active conformation model of GLP-1.

Heterocyclization of functionalized vinylic derivatives of imidazo.

Heterocyclization of functionalized vinylic derivatives of imidazo[1,2-a]pyridines was explored experimentally and theoretically using semiempirical AM1 and ab initio methods. A range of functionalized vinylic derivatives (azido, amino, and carbodiimide groups) were prepared for conversion into pyrroloazaindoles 19-22, imidazo[1,x]-, (x = 5, 6, 7, 8), [2,6]-, and [2,7]naphthyridines 28-30, 35-38 by thermal reaction. In the case of vinylic groups in the 5 position, peri annulation also was observed. The experimental and theoretical data are compared and discussed.

A genetic algorithm for the automated generation of small organic molecules: drug design using an evolutionary algorithm.

Rational drug design involves finding solutions to large combinatorial problems for which an exhaustive search is impractical. Genetic algorithms provide a novel tool for the investigation of such problems. These are a class of algorithms that mimic some of the major characteristics of Darwinian evolution. LEA has been designed in order to conceive novel small organic molecules which satisfy quantitative structure-activity relationship based rules (fitness). The fitness consists of a sum of constraints that are range properties. The algorithm takes an initial set of fragments and iteratively improves them by means of crossover and mutation operators that are related to those involved in Darwinian evolution. The basis of the algorithm, its implementation and parameterization, are described together with an application in de novo molecular design of new retinoids. The results may be promising for chemical synthesis and show that this tool may find extensive applications in de novo drug design projects.

Peptides quantitative structure-function relationships: an automated mutation strategy to design peptides and pseudopeptides from substitution matrices.

The process by which analogs in peptide chemistry are currently designed does not include any quantitative basis for amino acid substitutions from pharmacological leads. Here, we show that substitution matrices such as PAM 250 can provide quantitative constraints compatible with biological activity. This article describes its use in a strategy of rational amino acid substitution in peptides and proteins: we have computed a chemically derived matrix equivalent to the well-known PAM 250 matrix, reflecting the natural mutability rates of amino acids in protein evolutions but that can be extended to all the noncoded amino acids. Some of these noncoded amino acids are widely used to mimic secondary structure, to constrain backbone conformation, or to evade protease degradation. An automated sequence mutation (ASM) strategy has been defined to generate mutations within constraints. Application of such a substitution matrix to quantitative structure-function relationship studies will be of use in the design of proteins and peptides destined to become pharmaceutical drugs. In particular, issues such as which functionally conserved substitutions are able to satisfy conformational restrictions, oral bioavailability, or formulation demands can be quantitatively addressed.

Computer-assisted rational design of immunosuppressive compounds.

We describe the rational design of immunosuppressive peptides without relying on information regarding their receptors or mechanisms of action. The design strategy uses a variety of topological and shape descriptors in combination with an analysis of molecular dynamics trajectories for the identification of potential drug candidates. This strategy was applied to the development of immunosuppressive peptides with enhanced potency. The lead compounds were peptides, derived from the heavy chain of HLA class I, that modulate immune responses in vitro and in vivo. In particular, a peptide derived from HLA-B2702, amino acids 75-84 (2702.75-84) prolonged skin and heart allograft survival in mice. The biological activity of the rationally designed peptides was tested in a heterotopic mouse heart allograft model. The molecule predicted to be most potent displayed an immunosuppressive activity approximately 100 times higher than the lead compound.

Solution structure of the antimicrobial peptide ranalexin and a study of its interaction with perdeuterated dodecylphosphocholine micelles.

Ranalexin, a 20-residue peptide isolated from the skin of the bullfrog Rana catesbeiana displays antimicrobial activity. This peptide contains two cysteine residues in positions 14 and 20 linked by a disulphide bridge. Ranalexin was chemically synthesised and close antimicrobial activities were measured for the reduced and oxidised forms. The solution structure of ranalexin was determined by using circular dichroism, proton NMR spectroscopy and molecular modelling techniques. The reduced and oxidised forms of ranalexin are mainly unstructured in water but display an alpha-helical structure spanning residues 8-15 and 8-17, respectively, in a trifluoroethanol/water mixture (3:7, by vol.). Ranalexin was found to interact with micelles of dodecylphosphocholine and to adopt a similar helical structure. Moreover, slow-exchanging amide protons located on the same side of the helix suggest that the hydrophobic face of the helix lies on the micelle surface. Hydrophobic residues of the poorly structured N-terminal part which are important for the biological activity are also involved in the interaction with micelles. Taken together, the results suggest that the disulphide bond does not strongly affect either the conformation or the antimicrobial activity of ranalexin.

Oligomerization of protegrin-1 in the presence of DPC micelles. A proton high-resolution NMR study.

Protegrins are members of a family of five Cys-rich naturally occurring cationic antimicrobial peptides. The NMR solution structure of protegrin-1 (PG-1) has been previously determined as a monomeric beta-hairpin both in water and in dimethylsulfoxide solution. Protegrins are bactericidal peptides but their mechanism of action is still unknown. In order to investigate the structural basis of their cytotoxicity, we studied the effect of lipid micelles on the structure of PG-1. The NMR study reported in the present work indicates that PG-1 adopts a dimeric structure when it binds to dodecylphosphocholine micelles. Moreover, the amide proton exchange study suggests the possibility of an association between several dimers.

Rational choice of molecular dynamics simulation parameters through the use of the three-dimensional autocorrelation method: application to calmodulin flexibility study.

We examined the effects of several adjustable parameters for use in molecular dynamics simulations of proteins using both standard criteria (radius of gyration, root mean square deviation from starting coordinates, molecular mechanics energy) and a new description of protein conformations by 3-D autocorrelation vectors (3-D ACV). We chose calmodulin (CaM) as a protein model and analysed 23 simulations using different combinations of the four molecular dynamics parameters studied, such as the dielectric constant (epsilon), the heating phase time (H), the thermal bath coupling time (zeta T) and the time step size (delta t). The correctness of the various trajectories generated with different parameter sets was evaluated through geometric analysis and use of a knowledge-based profile method. It is shown that 3-D ACV combined with multivariate statistical analysis provides a convenient way to describe and compare molecular dynamics simulations and constitutes a valuable complementary tool to standard methods. Using these methods, comparison of the various simulations performed on CaM indicated that the best in vacuo parameter set was epsilon = 1 x r, H = 15 ps, zeta T = 0.1 ps and delta t = 1 fs in fairly good agreement with previous less extensive comparisons of molecular dynamics trajectories.

Synthesis and solution structure of the antimicrobial peptide protegrin-1.

Protegrins are members of a family of five Cys-rich, cationic antimicrobial peptides recently isolated from porcine cells. We have synthesised an 18-amino-acid peptide that corresponds to protegrin-1. After Cys oxidation, the peptide has bactericidal activity against gram-positive and gram-negative bacteria, similar to that described for the natural peptide. The solution structure of protegrin-1 was investigated by means of 1H-NMR spectroscopy in water and in (CD3)2SO, with distance-geometry and simulated-annealing calculations. The C6-C15 and C8-C13 disulfide pattern was determined on the basis of NMR-derived constraints. These two parallel disulfide bridges stabilised a beta-sheet structure which comprised two antiparallel strands (residues 5-9 and 12-16) linked by a distorted beta-turn (residues 9-12). The N-terminus and C-terminus were essentially disordered. The distribution of hydrophobic and hydrophilic residues at the peptide surface was found to be a structural feature shared with tachyplesin-1, a related peptide which displays cytolytic activity, and, to a lesser extent, with mammalian defensins. These findings led us to assume that the distribution pattern could be required for the cytolytic activity of these peptides.

Change in membrane permeability induced by protegrin 1: implication of disulphide bridges for pore formation.

Protegrin 1 (PG-1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG-1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.

Variable mapping of structure-activity relationships: application to 17-spirolactone derivatives with mineralocorticoid activity.

Fifty-four steroid homologs, belonging to the series of 17-spirolactones, were modelled by molecular and quantum mechanics. We studied the affinity of these compounds for the cytosolic mineralocorticoid receptor by way of various parameters describing each structure and its molecular properties. After the failure of a classic preliminary QSAR study, demonstrating the nonlinear relationships between affinity and structural descriptors, we constructed a model allowing us to predict the affinity of new compounds. Our method is based on simple graphic tools coupled to a cluster significance analysis. A complementary study of the activity relating the prediction of the antagonist/agonist character of 37 high-affinity compounds was also carried out using the same methodology. The principal electronic and structural characteristics leading to a selective activity were revealed.

Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry.

The binding of Ca2+ and Mg2+ to four calmodulins (SynCaM 1, SynCaM 8, SynCaM 12A, and SynCaM 18A) has been studied by ESI-MS. The mass spectra were recorded by dissolving the apoproteins in methanol/water (20/80, v/v) containing 1 mM CaCl2 or 1 mM MgCl2 and the pH adjusted to 6.0 with ammonia. The carrier solvent was methanol/water (20/80, v/v). In the case of Ca2+ complexation, ESI-MS reveals the presence of three kinds of sites: the first of high affinity corresponding to those determined using flow and equilibrium dialysis techniques and two others with lower affinities. These results clearly confirm the conclusion of Milos et al. [Milos, M., Comte, M., Schaer, J. J., & Cox, J. A. (1989) J. Inorg. Biochem. 36, 11-25] that there should exist between four and six auxiliary sites for Ca2+. Concerning the complexation of magnesium, the four proteins are able to bind two Mg2+ almost certainly on auxiliary cationic sites.

Use of TSAR as a new tool to analyze the molecular dynamics trajectories of proteins.

There is a lack of tools to analyze simulations of protein molecular dynamics quantitatively. Our aim is to use calmodulin, a prototypical calcium-binding protein, to describe a strategy and some tools for extracting relevant information from dynamics calculations. Our main conclusions are as follows: Autocorrelation vectors may be used to represent a 3D conformation in an n-dimensional space, where n is variable (n < or = 20-30). On such a transformation, classic statistical tools (PCA, clustering, etc.) may be used to differentiate or characterize dynamics trajectories quantitatively. TSAR, an integrated package used for quantitative structure-activity relationships, is well suited (after minor modifications) for such a purpose. Finally, this type of strategy is able to point out the effects of the solvent screening parameters of the Amber software on the dynamics trajectories of calmodulin.

Investigating the high affinity and low sequence specificity of calmodulin binding to its targets.

Calmodulin (CaM) is a calcium binding protein that regulates a wide range of enzymes. Recently the structures of a number of complexes between CaM and synthetic target peptides have been determined. The peptides correspond to the CaM-binding domain of skeletal and smooth muscle myosin light-chain kinase (MLCK) and calmodulin-dependent protein kinase II alpha. Comparison of the peptide-free and peptide-bound structures reveals that CaM undergoes a large conformational change when forming a complex, resulting in the formation of a binding surface that provides for an optimal interaction with its target. In this work, the available co-ordinates of the NMR solution structure of CaM-skeletal MLCK peptide are used as a basis upon which several molecular models of binding are built. The detailed features of the protein's peptide binding surface are revealed through two-dimensional topographical projections. Negatively charged margins at the binding surface extremities interact strongly with basic peptide residues separated by nine or ten positions. The binding surface core is hydrophobic and displays a groove with four deep pockets, which can accommodate bulky peptide residues at relative positions 4 and 8 (pocket A), 11 (pocket B), 13 (pocket C), 14 and 17 (pocket D). Therefore, both electrostatic and van der Waals' features contribute to the high affinity binding. A search for alternative peptide placements in the binding tunnel reveals the dominant role of specific electrostatic interactions in the binding energy. Apolar interactions are more permissive, such that the hydrophobic side-chains that line the binding tunnel adapt in order to maintain favourable van der Waals' contacts. The model suggests that the structure can accommodate large peptide translations (up to 5 A) and a reversed peptide binding mode, with a little loss in binding interaction energy. These calculations are compared with available experimental data, providing a structural rationale for the low sequence specificity of the CaM target recognition.

Synthesis of new 11 beta-substituted spirolactone derivatives. Relationship with affinity for mineralocorticoid and glucocorticoid receptors.

Various steroidal 17-spirolactones substituted in the 11 beta-position were synthesized to study the relationship between the nature of the 11 beta-arm and their affinity for cytosolic mineralocorticoid (MR) and glucocorticoid (GR) receptors prepared from adrenalectomized rabbit kidney or liver. One of them, the 11 beta-allenyl-3-oxo-19-nor-17-pregna-4,9-diene-21,17- carbolactone derivative, exhibited the same affinity for MR as aldosterone and a 5-fold higher affinity than mespirenone. Its affinity for GR was found to be relatively low. As suggested by molecular modeling, the marked differences in mineralocorticoid receptor binding affinity could be related to the structural features induced by this 11 beta-allenic substituent.

Synthesis and antibacterial activity of some imidazo[1,2-a]pyrimidine derivatives.

A series of 75 imidazo[1,2-a]pyrimidine derivatives were synthesized. The "in vitro" antibacterial activity of these compounds and their corresponding alpha-bromoketones against a variety of gram (+), gram (-) bacteria and Mycobacterium species is reported. Some of the prepared derivatives exhibited potent antimicrobial activity.

Comparative sensitivity study of Clostridium perfringens towards 16 antibiotics from different classes.

A series of 16 antibiotics from different classes (5-nitro imidazoles, beta-lactams, cyclins, macrolids, chloramphenicol) was examined for their bacteriostatic and bactericidal activities on 45 strains of Clostridium perfringens, with determination of MIC and MBC values. Several techniques of multivariate analysis were used in order to visualize differentiations in sensitivity profiles: Principal component analysis (PCA) and preferentially Correspondence factorial analysis (CFA). This approach revealed the efficacy of antibiotics from both general and family classifications.

[Hydroxyiminoacetone-like reactivators of phosphorylated cholinesterases. Structure-activity relationships via molecular modelling]. / Hydroxyiminocétones réactivatrices de cholinestérases phosphorylees. Approche des relations structure-activite par modelisation moleculaire.

A structure-activity relationships (S.A.R.) is performed on a series of iminocetones acting like reactivators of phosphorylated cholinesterases. The structural and electronic molecular characteristics are computed using a molecular modelling system (Moldesign). A multidimensional statistical analysis point out the role of MEP's which are representative of the whole molecular electronic distribution.