Marco De Amici

Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity

Selective modulation of cell function by G protein‐coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype‐selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine‐like orthosteric activator with M2‐selective bis(ammonio)alkane‐type allosteric fragments. Radioligand binding in wild‐type and mutant receptors supplemented by receptor docking simulations proved M2 selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid‐induced dynamic mass redistribution in CHO‐hM2 cells disclosed pathway‐specific signaling. Selective receptor activation (M2>M1>M3) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.— Antony, J., Kellershohn, K., Mohr‐Andrä, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Tränkle, C., Kostenis, E., Christopoulos, A., Höltje, H.‐D., Barocelli, E., De Amici, M., Holzgrabe, U., Mohr, K. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J. 23, 442–450 (2009)

Rational design of dualsteric GPCR ligands: quests and promise

Dualsteric ligands represent a novel mode of targeting G protein‐coupled receptors (GPCRs). These compounds attach simultaneously to both, the orthosteric transmitter binding site and an additional allosteric binding area of a receptor protein. This approach allows the exploitation of favourable characteristics of the orthosteric and the allosteric site by a single ligand molecule. The orthosteric interaction provides high affinity binding and activation of receptors. The allosteric interaction yields receptor subtype‐selectivity and, in addition, may modulate both, efficacy and intracellular signalling pathway activation. Insight into the spatial arrangement of the orthosteric and the allosteric site is far advanced in the muscarinic acetylcholine receptor, and the design of dualsteric muscarinic agonists has now been accomplished. Using the muscarinic receptor as a paradigm, this review summarizes the way from suggestive evidence for an orthosteric/allosteric overlap binding to the rational design and experimental validation of dualsteric ligands. As allosteric interactions are increasingly described for GPCRs and as insight into the spatial geometry of ligand/GPCR‐complexes is growing impressively, the rational design of dualsteric drugs is a promising new approach to achieve fine‐tuned GPCR‐modulation.

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.

Allosteric ligands for G protein-coupled receptors: A novel strategy with attractive therapeutic opportunities

Allosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter‐bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein‐coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (family A, B, and C receptors). In addition, the behavior at muscarinic receptors of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated.

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

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.

Dynamic ligand binding dictates partial agonism at a G protein–coupled receptor

We present a new concept of partial agonism at G protein-coupled receptors. We demonstrate the coexistence of two functionally distinct populations of the muscarinic M2 receptor stabilized by one dynamic ligand, which binds in two opposite orientations. The ratio of orientations determines the cellular response. Our concept allows predicting and virtually titrating ligand efficacy, which opens unprecedented opportunities for the design of drugs with graded activation of the biological system.

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

Epiboxidine and novel-related analogues : A convenient synthetic approach and estimation of their affinity at neuronal nicotinic acetylcholine receptor subtypes

Racemic exo-epiboxidine 3, endo-epiboxidine 6, and the two unsaturated epiboxidine-related derivatives 7 and 8 were efficiently prepared taking advantage of a palladium-catalyzed Stille coupling as the key step in the reaction sequence. The target compounds were assayed for their binding affinity at neuronal alpha4beta2 and alpha7 nicotinic acetylcholine receptors. Epiboxidine 3 behaved as a high affinity alpha4beta2 ligand (K(i)=0.4 nM) and, interestingly, evidenced a relevant affinity also for the alpha7 subtype (K(i)=6 nM). Derivative 7, the closest analogue of 3 in this group, bound with lower affinity at both receptor subtypes (K(i)=50 nM for alpha4beta2 and K(i)=1.6 microM for alpha7) evidenced a gain in the alpha4beta2 versus alpha7 selectivity when compared with the model compound.

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.