Carlo De Micheli

synthesis of new Δ2-isoxazoline derivatives and their pharmacological characterization as β-adrenergic receptor antagonists

Abstract A series of Δ 2 -isoxazoline derivatives structurally related to Broxaterol 1 and Falintolol 3 has been prepared and evaluated for their binding affinity to β 1 - and β 2 -adrenergic receptors. Among the tested compounds only the 3-isopropenyl anti derivative 4d is as active as the reference compounds. An electron-releasing group, probably operating through a π – π interaction, in the 3-position of the isoxazoline nucleus greatly enhances the affinity of the compounds. Conversely, the closest analogs of Broxaterol (3-bromo Δ 2 -isoxazolines 4a and 5a ) are at least one order of magnitude less active than the model compound 1 . Throughout the series of derivatives the anti stereoisomers are invariably more active than their syn counterparts.

Nitrile oxides in medicinal chemistry-2. synthesis of the two enantiomers of dihydromuscimol

Abstract The cycloaddition of bromonitrile oxide to monosubstituted olefins has a high regioselectivity yielding 3-bromo-5-substituted isoxazolines contaminated by minor amounts (4-9%) of the 4-substituted isomer. The adducts of bromonitrile oxide to allyl a3lcohol and N-protected allylamine were employed as key intermediates in the preparation of racemic dihydromuscimol (DHM). The synthesis of (R)-(-)- and (S)-(+)-DHM was accomplished by using the two diastereomers obtained by the cycloaddition of bromonitrile oxide to (S)-(+)-isopropylidene-3-buten-1,2-diol. The enantiomeric excesses of (R)-(-)- and (S)-(+)-DHM, determined by capillary GLC on the appropriate precursors, were 98.8 and >99.0 %. A spectroscopic survey of the tautomerism of 3-hydroxyisoxazolines indicates the predominant or exclusive occurrence of the NH form

Drug discovery targeting amino acid racemases.

Drug Discovery Targeting Amino Acid Racemases Paola Conti, Lucia Tamborini, Andrea Pinto, Arnaud Blondel, Paola Minoprio, Andrea Mozzarelli, and Carlo De Micheli* Dipartimento di Scienze Farmaceutiche “P. Pratesi”, via Mangiagalli 25, 20133 Milano, Italy Institut Pasteur, Unit e de Bioinformatique Structurale, CNRS-URA 2185, D epartement de Biologie Structurale et Chimie, 25 rue du Dr. Roux, 75724 Paris, France Institut Pasteur, Laboratoire des Processus Infectieux a Trypanosoma; D epartement d’Infection et Epid emiologie; 25 rue du Dr. Roux, 75724 Paris, France Dipartimento di Biochimica e Biologia Molecolare, via G. P. Usberti 23/A, 43100 Parma, Italy Istituto di Biostrutture e Biosistemi, viale Medaglie d’oro, Roma, Italy

Investigating the Mechanism of Substrate Uptake and Release in the Glutamate Transporter Homologue GltPh through Metadynamics Simulations

A homeostatic concentration of glutamate in the synaptic cleft ensures a correct signal transduction along the neuronal network. An unbalance in this concentration can lead to neuronal death and to severe neurodegenerative diseases such as Alzheimers or Parkinsons. Glutamate transporters play a crucial role in this respect because they are responsible for the reuptake of the neurotransmitter from the synaptic cleft, thus controlling the glutamate concentration. Understanding the molecular mechanism of this transporter can provide the possibility of an exogenous control. Structural studies have shown that this transporter can assume at least three conformations, thus suggesting a pronounced dynamical behavior. However, some intermediate states that lead to the substrate internalization have not been characterized and many aspects of the transporter mechanism still remain unclear. Here, using metadynamics simulations, we investigate the substrate uptake from the synaptic cleft and its release in the intracellular medium. In addition, we focus on the role of ions and substrate during these processes and on the stability of the different conformations assumed by the transporter. The present dynamical results can complement available X-ray data and provide a thorough description of the entire process of substrate uptake, internalization, and release.

Inhibition of Rhodesain as a Novel Therapeutic Modality for Human African Trypanosomiasis

Rhodesain, a cathepsin L-like cysteine protease of T. brucei rhodesiense, is considered a potential target for the treatment of human African trypanosomiasis. Recent findings have confirmed that rhodesain, a lysosomal protease, is essential for parasite survival. Rhodesain is required by T. brucei to cross the blood-brain barrier, degrade host immunoglobulins, and turn over variant surface coat glycoproteins of T. brucei, which impair effective host immune responses. In this Perspective, we discuss the main classes of rhodesain inhibitors, including peptidic, peptidomimetic, and nonpeptidic structures, emphasizing those that have exhibited an optimal match between enzymatic affinity and trypanocidal profile and those for which preclinical investigations are currently in progress.

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Isoxazoline derivatives. Part VI. Regioselectivity in the 1,3-dipolar cycloaddition of nitrile oxides to αβ-unsaturated ketones

1,3-Cycloadditions of nitrile oxides to αβ-unsaturated ketones have been studied. In most cases a mixture of the two regioisomeric 3-substituted 4- and 5-acyl-Δ2-isoxazolines was obtained. The individual compounds were characterised by chemical and/or spectroscopic methods. Effects of steric and electronic factors on the cycloadditions are discussed.

Engineering of α-conotoxin MII-derived peptides with increased selectivity for native α6β2* nicotinic acetylcholine receptors

α6β2∗ Nicotinic acetylcholine receptors are expressed in selected central nervous system areas, where they are involved in striatal dopamine (DA) release and its behavioral consequences, and other still uncharacterized brain activities. α6β2∗ receptors are selectively blocked by the α‐conotoxins MII and PIA, which bear a characteristic N‐ terminal amino acid tail [arginine (R), aspartic acid (D), and proline (P)]. We synthesized a group of PIA‐related peptides in which R1 was mutated or the RDP motif gradually removed. Binding and striatal DA release assays of native rat α6β2∗ receptors showed that the RDP sequence, and particularly residue R1, is essential for the activity of PIA. On the basis of molecular modeling analyses, we synthesized a hybrid peptide (RDP‐MII) that had increased potency (7‐fold) and affinity (13‐fold) for α6β2∗ receptors but not for the very similar α3β2∗ subtype. As docking studies also suggested that E11 of MII might be a key residue engendering α6β2∗ vs. α3β2∗ selectivity, we prepared MII[E11R] and RDP‐MII[E11R] peptides. Their affinity and potency for native α6β2∗ receptors were similar to those of their parent analogues, whereas, for the oocyte expressed rat α3β2∗ subtype, they showed a 31‐ and 14‐fold lower affinity and 21‐ and 3.5‐fold lower potency. Thus, MII[E11R] and RDP‐MII[E11R] are potent antagonists showing a degree of α6β2∗ vs. α3β2∗ selectivity in vivo.—Pucci, L., Grazioso, G., Dallanoce, C., Rizzi, L., De Micheli, C., Clementi, F., Bertrand, S., Bertrand, D., Longhi, R., De Amici, M., Gotti, C. Engineering of α‐conotoxin MII‐derived peptides with increased selectivity for native α6β2* nicotinic acetylcholine receptors. FASEB J. 25, 3775–3789 (2011). www.fasebj.org

Synthesis and in vitro/in vivo Evaluation of the Antitrypanosomal Activity of 3-Bromoacivicin, a Potent CTP Synthetase Inhibitor

The first convenient synthesis of enantiomerically pure (αS,5S)‐α‐amino‐3‐bromo‐4,5‐dihydroisoxazol‐5‐yl acetic acid (3‐bromoacivicin) is described. We demonstrate that 3‐bromoacivicin is a CTP synthetase inhibitor three times as potent as its 3‐chloro analogue, the natural antibiotic acivicin. Because CTP synthetase was suggested to be a potential drug target in African trypanosomes, the in vitro/in vivo antitrypanosomal activity of 3‐bromoacivicin was assessed in comparison with acivicin. Beyond expectation, we observed a 12‐fold enhancement in the in vitro antitrypanosomal activity, while toxicity against mammalian cells remained unaffected. Despite its good in vitro activity and selectivity, 3‐bromoacivicin proved to be trypanostatic and failed to completely eradicate the infection when tested in vivo at its maximum tolerable dose.

Alpha7 Nicotinic Acetylcholine Receptor Agonists: Prediction of Their Binding Affinity Through a Molecular Mechanics Poisson-Boltzmann Surface Area Approach

A group of agonists for the α7 neuronal nicotinic acetylcholine receptors (nAChRs) was investigated, and their free energies of binding ΔGbind were calculated by applying the molecular mechanics Poisson–Boltzmann surface area (MM‐PBSA) approach. This method, based on molecular dynamics simulations of fully solvated protein–ligand complexes, allowed us to estimate the contribution of both polar and nonpolar terms as well as the entropy to the overall free energy of binding. The calculated results were in a good agreement with the experimentally determined ΔGbind values, thereby pointing to the MM‐PBSA protocol as a valuable computational tool for the rational design of specific agents targeting the neuronal α7 nAChR subtypes.