Synthesis and pharmacological evaluation of a selected library of new potential anti-inflammatory agents bearing the gamma-hydroxybutenolide scaffold: a new class of inhibitors of prostanoid production through the selective modulation of microsomal prostaglandin E synthase-1 expression.

As a part of our drug discovery effort, recently we clarified the molecular basis of phospholipase A2 (PLA2) inactivation by petrosaspongiolide M (PM), an interesting metabolite belonging to a marine sesterterpene family, containing in its structural architecture a gamma-hydroxybutenolide moiety and showing potent anti-inflammatory activity. In the attempt to expand structural diversity as well as to simplify crucial synthetic features of the parent compound, we decided to develop a selected library based on the densely functionalized gamma-hydroxybutenolide scaffold. The synthesized products were tested for their ability to inhibit PLA2 enzymes as well as to modulate the expression of inducible cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1), two key enzymes highly involved in the inflammatory event, in order to discover new promising anti-inflammatory agents with better pharmacological profiles. This led us to the discovery of a promising inhibitor (4e) of prostanoid production acting by in vitro and in vivo selective modulation of microsomal prostaglandin E synthase 1 expression.

Copper-binding activity of Trefoil factor 1 (TFF1): a new perspective in the study of the multifunctional roles of TFFs.

Trefoil factors (TFFs) are gastrointestinal peptides playing an essential role in the epithelial restitution. Among the three known TFF peptides, TFF1 is characterized by three disulfide bonds producing a compact globular structure and an extended and disordered tail formed by amino- and carboxy-termini. The presence of a cysteine surrounded by several negatively charged residues in this region of the protein, highly conserved in different species, suggests the possible formation of a metal-binding site. Affinity chromatography and mass spectrometric analyses allowed us to demonstrate a selective binding affinity of TFF1 for copper. The binding induces conformational changes in the tertiary structure as demonstrated by circular dichroism experiments, while limited proteolysis revealed an altered access to the cleavage sites in the amino- and carboxy-termini. The results of this study reveal a new property of TFF1 and suggest that copper could influence its biological activities by interfering with the dimerization of the peptide and/or the interaction with mucins or putative TFF receptors.

Chemistry and biology of anti-inflammatory marine natural products: molecules interfering with cyclooxygenase, NF-kappaB and other unidentified targets.

The majority of the anti-inflammatory drugs routinely used nowadays are COX (cyclo-oxygenase) inhibitors. The important role of this enzyme, once known as prostaglandin synthase, in inflammation came a consequence of the discovery by the Nobel prize winner John Vane with his path-breaking discovery that aspirin and similar drugs exert their action by blocking the biosynthesis of the prostaglandin group of lipid mediators. (John R. Vane, Nobel Lecture, December 8, 1982 and references cited therein) In the last five years it has become clear that there are two such enzymes involved. One of the "cyclo-oxygenases", called COX1 is responsible for making prostaglandins, which among other things, protect the stomach and kidney from damage. It is now clear that inhibition of COX1 accounts for the unwanted side effects of aspirin-like drugs such as gastric irritation and renal damage. The other enzyme, COX2, is induced by inflammatory stimuli and it is prostaglandins made by this enzyme that contribute to the inflammation in diseases such as rheumatoid arthritis. However, concerning inflammation-related targets, one should not limit the interest to COX and PLA2 enzymes. In recent years, it has steadily become more clear, that modulation in the expression of genes underlies most cellular responses, and inflammation is certainly not an exception in this sense. It does not come as surprise that molecules showing ability to interfere with factors involved in the modulation of genes expression, such as NF-kB, have also to be considered potential anti-inflammatory agents. Also in this respect, marine natural products (MNP) have brought a collection of novel molecular entities displaying ability to target COX1/COX2, NF-kappaB or acting through molecular mechanisms yet-to-be-discovered. Following, the marine natural products accounted for within this review will be grouped on the basis of their bio-molecular targets. Chemical synthesis of particular relevant molecules will be also discussed, especially in those cases where the natural products can be considered as lead compounds for the development of simplified derivatives or analogues of potential pharmaceutical interest.

Chemistry and biology of chromatin remodeling agents: state of art and future perspectives of HDAC inhibitors.

Chromatin remodeling is a fundamental phenomenon in the life of eukaryotic cells, bearing implications to numerous physiological and pathological phenomena. This review outlines the chemistry of natural and synthetic agents endowed with the ability to interfere with such biological function, with a particular emphasis on histone deacetylase (HDAC) inhibitors. Other aspects covered in this article comprise structure activity relationships (SAR) and modes of action at molecular level, including the description of crystal structures of enzyme-inhibitor complexes.

Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M.

Proinflammatory mediators, namely eicosanoids, reactive oxygen and nitrogen species and cytokines, are clearly involved in the pathogenesis of intestinal bowel disease. bolinaquinone (BQ) and petrosaspongiolide M (PT), two marine products with potent anti-inflammatory action, have been shown to control the production of mediators in acute and chronic inflammatory processes. Hence, we have tested here the hypothesis that BQ and PT could ameliorate inflammation and oxidative stress parameters in 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis in Balb/c mice. BQ and PT were given orally in doses of 10 or 20mg/kg/day. Treatment of the animals with BQ or PT at the highest dose significantly protected against TNBS-induced inflammation, as assessed by a reduced colonic weight/length ratio and histological scoring. Neutrophilic infiltration, interleukin (IL)-1beta and prostaglandin E(2) (PGE(2)) levels, as well as cyclooxygenase-2 (COX-2) protein expression were inhibited by both compounds. Colonic nitrite and nitrate levels and protein expression of inducible nitric oxide synthase (iNOS) were also lower in the treated groups in comparison to the TNBS control. BQ and PT reduced nitrotyrosine immunodetection and colonic superoxide anion production. Neither compound inhibited the expression of the protective protein heme oxygenase-1 (HO-1), although they reduced the extension of apoptosis. Our study also indicated that PT could interfere with the translocation of p65 into the nucleus, a key step in nuclear factor-kappaB (NF-kappaB) activation. Altogether, the results suggest that BQ and PT can have potential protective actions in intestinal inflammatory diseases.

Advances in the universal NMR database approach. 2'-substituted taxanes as probes for an improved protocol of diastereomeric differentiation.

[structure: see text] The configuration of the alpha-substituted alpha-hydroxy-beta-aminoester moiety in a series of 2'-substituted taxanes was analyzed according to the recently proposed Universal NMR Database (UDB) approach. A critical analysis of the results showed that modifications regarding chemical shift adjustment (so as to render the shifts virtually connectivity independent) were necessary to get consistent stereoassignments in this set of compounds. On this basis, a modified UDB-based strategy, especially tailored to the configurational assignment of densely substituted diastereomeric fragments, is proposed.

Quantum mechanical calculations of conformationally relevant 1H and 13C NMR chemical shifts of calixarene systems.

[graphs: see text] QM GIAO calculations of 13C and 1H chemical shift values of the ArCH2Ar group have been performed, using the hybrid DFT functional MPW1PW91 and the 6-31G(d,p) basis set, on some representative calixarenes and on a series of simplified calixarene models allowing derivation of chemical shift surfaces versus phi and chi dihedral angles. A good reproduction of experimental data was obtained. The applicability of chemical shift surfaces in the study of calixarene conformational features is illustrated.

Inhibition of the NF-kappaB signaling pathway mediates the anti-inflammatory effects of petrosaspongiolide M.

Petrosaspongiolide M (PT) is a potent secretory phospholipase A(2) inhibitor and anti-inflammatory agent. This marine metabolite reduced the production of nitrite, prostaglandin E(2), and tumor necrosis factor-alpha in the mouse air pouch injected with zymosan. These effects were also observed in mouse peritoneal macrophages stimulated with zymosan. Inhibition of these inflammatory mediators was related to reductions in inducible nitric oxide synthase, cyclo-oxygenase-2, and tumor necrosis factor-alpha expression. Since nuclear factor-kappaB (NF-kappaB) appears to play a central role in the transcriptional regulation of these proteins by macrophages, we investigated the effects of PT on this transcription factor. We found that PT was a potent inhibitor of the NF-kappaB pathway since at 1 microM it strongly decreased NF-kappaB-DNA binding in response to zymosan, in mouse peritoneal macrophages. Our study also indicated that PT could interfere with a key step in NF-kappaB activation, the phosphorylation of IkappaBalpha, resulting in inhibition of IkappaBalpha degradation. The control of a wide range of mediators by PT suggests a potentially wide therapeutic spectrum for this marine metabolite in inflammatory conditions.

Quantum mechanical calculations of NMR J coupling values in the determination of relative configuration in organic compounds.

[structure: see text] An approach relying on quantum mechanical calculations of proton-proton and proton-carbon J coupling values is proposed as a tool for assigning the relative configuration on chiral organic compounds. The method is suitable for carbon frameworks containing several adjacent stereogenic centers and may allow significant advances in the extensive use of spin-spin couplings in structural elucidation.

Extension of the J-based configuration analysis to multiple conformer equilibria: an application to sapinofuranone A.

[structure: see text] A new strategy that extends the application of the J-based configuration analysis to systems characterized by multiple conformer equilibria is described and applied to sapinofuranone A (1), a phytotoxic molecule produced by three strains of Sphaeropsis sapinea. This method, based on a combination of computational techniques and NMR spectroscopy, uses ab initio calculations to predict a set of theoretical homo- and heteronuclear J values which can be compared against experimental NMR data.

Cycloamphilectenes, a new type of potent marine diterpenes: inhibition of nitric oxide production in murine macrophages.

The inhibitory effect of a series of 6 cycloamphilectenes, novel marine diterpenes based on amphilectene skeletons and isolated from the Vanuatu sponge Axinella sp., on NO, PGE(2) and TNFalpha production in murine peritoneal macrophages was studied. These compounds reduced potently nitric oxide production in a concentration-dependent manner with IC(50) values in the submicromolar range (0.1-4.3 microM). Studies on intact cells and Western blot analysis showed that the more potent cycloamphilectenes reduced the expression of inducible nitric oxide synthase without affecting cyclo-oxygenase-2 expression. Among them cycloamphilectene 2, the unique compound bearing an exocyclic methylene group, was able to reduce NO production without affecting TNFalpha release. Cycloamphilectene 2, which is an inhibitor of the nuclear factor-kB pathway, exhibited topical anti-inflammatory activity.

Petrosaspongiolide M reduces morphine withdrawal in vitro.

The bee venom phospholipase A(2) (PLA(2)) inhibitory activity of petrosaspongiolide M (PM), a marine metabolite displaying a potent anti-inflammatory activity and able to covalently bind and block group II and III secretory PLA(2) enzymes, has been investigated by mass spectrometry and molecular modeling. The model reveals interesting insight on the PM-PLA(2) inhibition process and may prove useful in the design of new anti-inflammatory agents targeting PLA(2) secretory enzymes. In this paper, the effect of PM has been investigated on opiate withdrawal in an in vitro model. After a 4 min in vitro exposure to morphine a strong contracture of guinea pig isolated ileum was observed after the addition of naloxone. PM treatment 1 x 10(-8), 5 x 10(-8), 1 x 10(-7) M was able to reduce morphine withdrawal. These results suggest that PM effect in this in vitro model of opiate withdrawal may be due to extracellular type II PLA(2) inhibition.

Design and synthesis of cyclopeptide analogues of the potent histone deacetylase inhibitor FR235222.

Various structurally modified analogues of FR235222 (1), a natural tetrapeptide inhibitor of mammalian histone deacetylases, were prepared in a convergent approach. The design of the compounds was aimed to investigate the effect of structural modifications of the tetrapeptide core involved in enzyme binding in order to overcome some synthetic difficulties connected with the natural product 1. The modifications introduced could also help identify key structural features involved in the mechanism of action of these compounds. The prepared molecules were subjected to in vitro pharmacological tests, and their potency was tested on cultured cells. Two of the components of the array were found to be more potent than the parent compound 1 and almost as efficient as trichostatin A (TSA). These results demonstrate that it is possible to synthesize highly active cyclic tetrapeptides using commercially available amino acids (with the exception of 2-amino-8-oxodecanoic acid, Ahoda). The nature of the residue in the second position of the cyclic peptide and the stereochemistry of the Ahoda tail are important for the inhibitory activity of this class of cyclic tetrapeptide analogues.

The molecular mechanism of bee venom phospholipase A2 inactivation by bolinaquinone.

The molecular basis of the inactivation of bee venom PLA2 by the marine natural product bolinaquinone (BLQ) was studied by several spectral techniques (CD, fluorescence, and NMR spectroscopy, mass spectrometry), biomimetic reactions, and molecular modeling. Our data suggest competitive inhibition based on a BLQ-PLA2 noncovalent molecular recognition. However, BLQ is also able to react selectively with Lys133 through conjugate addition followed by a beta elimination. The biological implications of both the covalent and noncovalent molecular events are discussed.

On the role of stereo-electronic effects in tuning the selectivity and rate of DNA alkylation by duocarmycins.

The role of local geometric and stereo-electronic effects in tuning the alkylation of DNA by duocarmycins has been analyzed by an integrated computational tool rooted in the density functional theory and the polarizable continuum model. Our study points out that together with steric accessibility, different electronic delocalisations also contribute to determine the higher reactivity of adenine with respect to guanine. Also the effect of the methyl ester group on the alkylating agent has an electronic origin. Furthermore, deviations from the planarity in the drug structure (conformational catalysis) could be less important than currently accepted since, according to our computations, compounds with strongly different reactivity have nearly constant and very similar out of plane distortions before and after the reaction. Model computations suggest, instead, that specific non covalent interactions could discriminate between different drugs selectively reducing some activation energies with respect to the corresponding processes in solution.

Synthesis of 2-amino-8-oxodecanoic acids (Aodas) present in natural hystone deacetylase inhibitors.

[reaction: see text] Differently substituted 2-amino-8-oxodecanoic acids (Aodas), present in naturally occurring inhibitors of hystone deacetylase (HDAC), have been prepared using a convergent approach. The configuration in position 2 was derived from enantiomerically pure allylglycine or glutamic acid, whereas the stereochemistry of the substituent in position 9 derived from lactic acid or glyceraldehyde derivatives. Starting from allylglycine, (S)-Aodas, protected at the nitrogen as Boc or Fmoc, were obtained in four steps in about 30% overall yield. These products have been used to prepare a simplified analogue of a natural cyclic tetrapeptide HDAC inhibitor by SPPS.

Synthesis, conformational analysis and CB1 binding affinity of hairpin-like anandamide pseudopeptide mimetics.

We have designed, synthesized and evaluated the CB(1) binding affinity of a number of new conformationally restricted lipopeptides (1-17). All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Among them, compounds 1-3 and 8 showed CB(1) affinities in competitive binding assays with K(i) values in the micromolar range (K(i) of AEA = 0.8 microM in the same assay). The remaining pseudopeptides showed little binding to the CB(1) receptor (with K(i) values >or= 50 microM). Conformational analysis on two representative compounds, performed by a combination of NMR studies, restrained molecular dynamics and QM calculations, allowed us to shed light on the structure-activity relationships (SAR), pointing to a correlation between the predominance of the hairpin-like structural motif and the CB(1) binding affinity. In a more general context, the present study may also prove useful in gaining additional insight into the biological relevance of the various AEA conformations.

The inactivation of phospholipase A2 by scalaradial: a biomimetic study by electrospray mass spectrometry.

A biomimetic approach was employed to shed light on the nature of chemical reactions occurring in the covalent inactivation of phospholipase A(2) (PLA(2)) by scalaradial (1), a marine dialdehyde terpenoid endowed with potent anti-inflammatory activity. To this end, a detailed study of the reaction profile between the nitrogenous nucleophile isopropylamine and scalaradial was performed under biologically relevant conditions.