Agostino Casapullo

Dactylolide, a New Cytotoxic Macrolide from the Vanuatu SpongeDactylospongia sp.

Dactylolide (1), a new cytotoxic 20-membered macrolide, was isolated from a marine sponge of the genus Dactylospongia collected off the coast of the Vanuatu islands. It co-occurred with other known bioactive macrolides: latrunculin A (2), laulimalide (3), isolaulimalide (4) and with the anthelminthic mycothiazole (5). The structure of 1, which is a minor metabolite, was elucidated by spectroscopy (mainly by 1D/2D NMR and MS techniques). It showed cytotoxic activity against the L1210 and SK-OV-3 tumor cell lines (63% and 40% inhibition at 3.2 μg/mL).

Fixed or Invertible Calixarene-Based Directional Shuttles

The first examples of rotaxanes based on calixarenes threaded by dialkylammonium ions, which also represent the first examples of calixarene-based molecular shuttles, are reported. The base/acid treatment demonstrated that these systems act as molecular shuttles, which move between three sites on the axle. When small OMe groups are appended at the calix[6]arene lower rim an unprecedented inversion of its shuttling direction is observed, which occurs through a cone-to-cone inversion of the macrocycle.

Scalaradial, a Dialdehyde-Containing Marine Metabolite That Causes an Unexpected Noncovalent PLA2 Inactivation

Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti‐inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A2 (sPLA2). Scalaradial, a 1,4‐dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti‐inflammatory profile, both in vitro and in vivo, through selective sPLA2 inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on‐going research on anti‐PLA2 natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA2 inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA2, a model sPLA2 that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA2 inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand–enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti‐inflammatory compounds that target sPLA2 enzymes.

The Binding Mode of Petrosaspongiolide M to the Human Group IIA Phospholipase A2: Exploring the Role of Covalent and Noncovalent Interactions in the Inhibition Process

We report an analysis of the mechanism of human group IIA secretory phospholipase A(2) (sPLA(2)-IIA) inhibition by the natural anti-inflammatory sesterterpene petrosaspongiolide M (PM). The amphiphilic PM, a gamma-hydroxybutenolide marine terpenoid, selectively reacts with the sPLA(2)-IIA Lys67 residue, located near the enzyme-membrane interfacial binding surface, and covalently modifies the enzyme through imine formation. Furthermore, PM is able to target the active site of sPLA(2)-IIA through several van der Waals/electrostatic complementarities. The two events cannot co-occur on a single PLA(2) molecule, so they may contribute separately to enzyme inhibiton. A more intriguing hypothesis suggests a double interaction of PM with two enzyme molecules, one of them covalently modified and the other contacting the inhibitor through its active site. We have explored the occurrence of this unusual binding mode leading to PM-induced PLA(2) supramolecular complexes. These insights could suggest new PLA(2)-inhibition-based therapeutic strategies.

Plakilactones from the marine sponge Plakinastrella mamillaris. Discovery of a new class of marine ligands of peroxisome proliferator-activated receptor γ.

In this paper we report the isolation and the molecular characterization of a new class of PPARγ ligands from the marine environment. Biochemical characterization of a library of 13 oxygenated polyketides isolated from the marine sponge Plakinastrella mamillaris allowed the discovery of gracilioether B and plakilactone C as selective PPARγ ligands in transactivation assays. Both agents covalently bind to the PPARγ ligand binding domain through a Michael addition reaction involving a protein cysteine residue and the α,β-unsaturated ketone in their side chains. Additionally, gracilioether C is a noncovalent agonist for PPARγ, and methyl esters 1 and 2 are noncovalent antagonists. Structural requirements for the interaction of these agents within the PPARγ ligand binding domain were obtained by docking analysis. Gracilioether B and plakilactone C regulate the expression of PPARγ-dependent genes in the liver and inhibit the generation of inflammatory mediators by macrophages.

Molecular Basis of Phospholipase A2 Inhibition by Petrosaspongiolide M

Petrosaspongiolide M (PM) is an anti‐inflammatory marine metabolite that displays a potent inhibitory activity toward group II and III secretory phospholipase A2 (PLA2) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA2 and γ‐hydroxybutenolide‐containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA2 has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM–PLA2 adduct is consistent with the formation of a Schiff base by reaction of a PLA2 amino group with the hemiacetal function (masked aldehyde) at the C‐25 atom of the PM γ‐hydroxybutenolide ring. Proteolysis of the modified PLA2 by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA2 α‐amino terminal group of the Ile‐1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA2 was also monitored and results showed that even with excess inhibitor, the prevalent product is a 1:1 (inhibitor:enzyme) adduct, although a 2:1 adduct is present as a minor component. The 2:1 adduct was also characterized, which showed that the second site of reaction is located at the ε‐amino group of the Lys‐85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA2 binding site were obtained for manoalide, a paradigm for irreversible PLA2 inhibitors, which suggests that the present results may be considered of more general interest within the field of anti‐inflammatory sesterterpenes that contain the γ‐hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA2 binding site through AFFINITY calculations. The model provides an interesting insight into the PM–PLA2 inhibition process and may prove useful in the design of new anti‐inflammatory agents that target PLA2 secretory enzymes.

Makaluvamine P, a new cytotoxic pyrroloiminoquinone from Zyzzya cf. fuliginosa.

A new pyrroloiminoquinone alkaloid (1) belonging to the makaluvamine family has been isolated from the sponge Zyzzya cf. fuliginosa collected in the waters off the Vanuatu Islands. The compound, designated makulavamine P, was characterized on the basis of its spectral data and displayed cytoxicity in the microM range on KB cells and antioxidant activity.

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.

Isolation and NMR characterization of rosacelose, a novel sulfated polysaccharide from the sponge Mixylla rosacea.

Rosacelose, a new anti-HIV polysaccharide composed of glucose and fucose sulfate, has been isolated from an aqueous extract of the marine sponge Mixylla rosacea. Extensive use of 1H and 13C multidimensional NMR spectroscopy, combined with chemical analysis were used to establish a linear polysaccharide structure composed mainly of 4,6-disulfated 3-O-glycosylated alpha-D-glucopyranosyl and 2,4-disulfated 3-O-glycosylated alpha-L-fucopyranosyl residues (in a 3:1 molar ratio).

Chemical Proteomics Reveals Heat Shock Protein 60 To Be the Main Cellular Target of the Marine Bioactive Sesterterpene Suvanine

Marine bioactive compounds are potential drug leads because of their diverse pharmacological effects against human diseases. The identification of their cellular targets is crucial for a rational approach to their application in medicinal chemistry. Thus, we have analyzed the cell interactome of suvanine, a sulfated tricyclic terpenoid of marine origin endowed with an interesting anti‐inflammatory activity, by application of a chemical proteomic approach. Heat Shock Protein 60, a chaperone involved in the inflammatory response, is the main cellular target of suvanine, which is also able to interfere with protein chaperone activity, giving evidence for its anti‐inflammatory properties.