A Hybrid Indoloquinolizidine Peptide as Allosteric Modulator of Dopamine D1 Receptors □S

The indoloquinolizidine-peptide 28 [(3S,12bR)-N-((S)-1-((S)-1-((S)2-carbamoylpyrrolidin-1-yl)-3-(4-fluorophenyl)-1-oxopropan-2ylamino)-4-cyclohexyl-1-oxobutan-2-yl)-1,2,3,4,6,7,12, 12b-octahydroindolo[2,3-a]quinolizine-3-carboxamide], a transindoloquinolizidine-peptide hybrid obtained by a combinatorial approach, behaved as an orthosteric ligand of all dopamine D2like receptors (D2, D3, and D4) and dopamine D5 receptors, but as a negative allosteric modulator of agonist and antagonist binding to striatal dopamine D1 receptors. Indoloquinolizidine-peptide 28 induced a concentration-dependent hyperbolic increase in the antagonist apparent equilibrium dissociation constant values and altered the dissociation kinetics of dopamine D1 receptor antagonists. The negative allosteric modulation was also found when agonist binding to D1 receptors was assayed. Indoloquinolizidinepeptide 28 was a weak ago-allosteric modulator but markedly led to a decreased potency without decreasing the maximum partial/ full agonist-mediated effect on cAMP levels. Compounds able to decrease the potency while preserving the efficacy of D1 receptor agonists are promising for exploration in psychotic pathologies. G-protein-coupled receptors (GPCRs) represent a high percentage of the current market for therapeutic agents and remain a primary focus of many biomedical research and pharmaceutical drug discovery programs. Much attention is focused in the identification and study of molecules that act as orthosteric ligands at a given GPCR to elicit a pharmacological effect. These compounds compete with the endogenous ligand(s) and thus preclude simultaneous occupation of the receptor by the two molecules. In addition to orthosteric sites, many GPCRs have been found to possess allosteric binding sites that are structurally distinct from the orthosteric sites (Christopoulos, 2002; May et al., 2007; Bridges and Lindsley, 2008). Allosteric sites may be less conserved across subtypes than orthosteric sites, providing a means for true selectivity (Bridges and Lindsley, 2008). One characteristic feature of This work was supported by the Spanish Ministerio de Ciencia y Tecnología [Grants SAF2008-00146, SAF2009-07276] (to E.I.C. and R.F., respectively); and Fundació La Marató de TV3 [Grant 060110 ] (to E.I.C.). A.S. and M.V. contributed equally to this work. 1 Current affiliation: Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Helios, Singapore. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.158824. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: GPCR, G-protein-coupled receptor; IP28, indoloquinolizidine-peptide 28, (3S,12bR)-N-((S)-1-((S)-1-((S)-2-carbamoylpyrrolidin-1-yl)3-(4-fluorophenyl)-1-oxopropan-2-ylamino)-4-cyclohexyl-1-oxobutan-2-yl)-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizine-3-carboxamide (C40H51FN6O4); SCH 23390, R-( )-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SKF 38393, 2,3,4,5-tetrahydro-7,8dihydroxy-1-phenyl-1H-3-benzazepine; SKF 81297, ( )-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide; A77636, (1R,3S)-3-(1 -adamantyl)-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran; SKF 83566, 8-bromo-2,3,4,5-tetrahydro-3-methyl-5phenyl-1H-3-benza zepin-7-ol hydrobromide; YM-09151–2, cis-5-chloro-2-methoxy-4(methylamine)-N-[2-methyl-1-(phenylmethyl)-3-pyrrolidinyl]benzamide; HBSS, Hank’s balanced salt solution; FRT, Flp recombination target; HEK, human embryonic kidney. 0022-3565/10/3323-876–885$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 3 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 158824/3564671 JPET 332:876–885, 2010 Printed in U.S.A. 876 http://jpet.aspetjournals.org/content/suppl/2009/12/21/jpet.109.158824.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 4, 2017 jpet.asjournals.org D ow nladed from the allosteric interaction is that the receptor is able to simultaneously bind an orthosteric and an allosteric ligand, introducing complexity into pharmacological responses by modifying the affinity or the signal imparted by orthosteric ligand (May et al., 2007). The ability of allosteric modulators to fine tune pharmacological responses has sparked interest in their potential applications in both clinical and basic science settings (Bridges and Lindsley, 2008; Conn et al., 2009). This interest is more relevant in the case of neurotransmitter receptor targets, where synaptic neurotransmission occurs in extremely complex circuits implicated in many neurological functions. An allosteric modulator will preserve the physiological relevance of receptor signaling while modulating the potency of the endogenous neurotransmitter (Conn et al., 2009). Allosteric sites have been described for dopamine receptors (Schetz, 2005). Dopamine receptors are grouped into two classes: D1-like receptors, which include D1 and D5 receptors, and D2-like receptors, which include D2, D3, and D4 receptors (Neve et al., 2004). As for many GPCRs (see Ferré et al., 2009 for review), there is evidence that dimerization/oligomerization of D1 receptors (Ng et al., 1994; George et al., 1998) is important for membrane expression (Kong et al., 2006). In addition, dopamine D2 receptors form higher-order oligomers at physiological expression levels with dimers, the minimal repetitive structural unit (Guo et al., 2008; Han et al., 2009). An allosteric site associated with the D2 dopamine receptor is recognized by amiloride and analogs of this diuretic drug, such as benzamil and methylisobutylamiloride (Hoare et al., 2000). These compounds also decrease antagonist binding to D1, D3, and D4 dopamine receptors (Hoare et al., 2000). L-Proline-L-leucine-L-glycine, an endogenous hypothalamic factor that inhibits the release of melanocyte-stimulating hormone from the anterior pituitary, is another dopamine receptor allosteric modulator enhancing agonist binding to D2 and D4 receptors (Verma et al., 2005). The allosteric modulation of dopamine receptors by ions has been described extensively (Schetz, 2005). At millimolar concentrations, sodium decreases agonist binding but increases antagonist binding to D2 dopamine receptors (Ericksen et al., 2009). Zinc ion allosterically modulates dopamine receptors (Schetz and Sibley, 2001), and it may be therapeutically relevant in antipsychotic drug treatments (Schetz, 2005). Because dopamine is an important neurotransmitter involved in the regulation of several biological functions, including locomotor activity, emotion, cognition, and neuroendocrine secretion, and because striatum receives the densest dopamine innervations and contains the highest density of dopamine receptors in the brain (Gerfen, 2004), dopamine receptors are targets in the pathophysiology of Parkinson’s disease and schizophrenia (Andersen and Nielsen, 1991; Wu et al., 2005). We have recently developed a library of indoloquinolizidine-peptide hybrids as multiple ligands for different dopamine receptor subtypes by a combinatorial approach that combines the solution-phase synthesis of two indolo[2,3a]quinolizidine scaffolds with solid-phase peptide chemistry (Vendrell et al., 2009). Some trans-indoloquinolizidine-peptide hybrids were selected showing an affinity (KD) in the low-micromolar range for both families of dopamine receptors (Vendrell et al., 2009). Here, the functional characterization of one of these compounds, the indoloquinolizidinepeptide 28 (IP28), on binding to dopamine D1-like and D2-like receptors is described. By means of kinetic assays and competition experiments in radioligand binding, it has been demonstrated that the IP28 behaved as an orthosteric ligand of dopamine D2, D3, D4, and D5 receptors but as an allosteric modulator of the D1 dopamine receptors. IP28 decreased the affinity of both agonist and antagonist binding to the receptor and, at the same time, behaved as a D1 receptor partial agonist. IP28, which was then the first described ago-allosteric modulator of D1 dopamine receptors, decreased receptor potency, whereas it preserved agonist-induced maximal cAMP production. This type of compound may be relevant to treat some psychotic pathologies. Materials and Methods Cell Transfection and Generation of an Inducible Cell Line Expressing the Human Dopamine D1 Receptor. Human embryonic kidney 293 (HEK-393) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units ml 1 penicillin, 100 g ml 1 streptomycin, 2 mM L-glutamine, and 100 g ml 1 sodium pyruvate (all from Invitrogen, Paisley, UK), at 37°C in a humidified atmosphere of 5% CO2. HEK-293 cells were grown to 60% confluence and transfected by the polyethylenimine (Sigma-Aldrich Chemie, Steinheim, Germany) method with 5 g of cDNA corresponding to human dopamine D3, D4.4, or D5 receptors. Cells were incubated (4 h) with the corresponding cDNA together with polyethylenimine (5.47 mM in nitrogen residues) and 150 mM NaCl in a serum-starved medium. After 4 h, the medium was changed to a fresh complete culture medium. Forty-eight hours after transfection, cells were washed twice in quick succession in Hank’s balanced salt solution (HBSS) with 10 mM glucose, detached, and resuspended in the same buffer containing 1 mM EDTA. Human dopamine receptor expression was tested by binding to membranes