Positive Allosteric Modulator of the Human 5-HT_2CReceptor

Abstract

The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.

Exploration of Synthetic Approaches and Pharmacological Evaluation of PNU-69176E and Its Stereoisomer as 5-HT2C Receptor Allosteric Modulators

“…This profile for compounds 1 and 2 in 5-HT 2C R-CHO cells was distinguished Multiple allosteric modulators of G-protein-coupled receptors have been developed and predicted to have robust effects in a variety of CNS disorders. Our preliminary data with the lead compound 1 demonstrate our ability to detect positive, and perhaps negative, allosteric activity (Figure 4) selectively at the 5-HT 2C R versus the highly homologous 5-HT 2A R. In our hands, compound 1 produced the anticipated characteristics based upon a previous study 5 which identified positive allosteric modulation by PNU-69176E in the presence of 5-HT at concentrations less than 10 μM and negative allosteric modulation at higher concentrations. These investigators also detected intrinsic activation of GTPγS binding and inositol 1,4,5-triphosphate (IP 3 ) release/[ 3 H]IP accumulation by PNU-69176E in the absence of 5-HT; in contrast, we did not detect intrinsic agonist activity for compound 1 in the 5-HT 2C Rinduced Ca i 2+ release assay ( Figure 5).…”

“…Such differences may be attributable to the choice of expression system and the protein expression level for the 5-HT 2C R. In the present studies, we employed a stably transfected CHO cell line (∼250 fmol/mg protein) which expresses vastly lower levels of the 5-HT 2C R protein relative to the stably transfected HEK293 cell line (∼45 pmol/mg protein) used in the previous report. 5 These technical aspects highlight the nuances that have hampered GPCR allosteric modulator drug discovery in the past, but also present new prospects for preclinical lead discovery. 14 Compound 1 potently enhanced 5-HT-induced Ca i 2+ release in the 5-HT 2C R-CHO cells with modest efficacy.…”

“…4 P N U -6 9 1 7 6 E [ ( 2 S , 4 R ) -N -( ( 1 S , 2 S ) -2 -c h l o r o -1 -((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(methylthio)tetrahydro-2H-pyran-2-yl)propyl)-4-undecylpiperidine-2-carboxamide (1); Figure 1)] is the only reported 5-HT 2C R selective positive allosteric modulator, which was identified via screening of a chemical library of Pharmacia (now Pfizer). 5 Structurally, 1 consists of two moieties, a piperidinyl ring with a long alkyl chain (undecyl) and a polar moiety including the α-Dgalactopyranoside, and can be viewed as an analogue of pirlimycin with a long alkyl chain (Figure 1). Although the preparation of pirlimycin and its analogues is available in the literature, 6 to our knowledge neither the synthesis nor the chemical characterization of 1 and its diastereomer 2 have been reported.…”

Central Serotonin2C Receptor: From Physiology to Pathology

“…Recently, it has been shown that the affinity and efficacy of 5-HT for 5-HT 2C receptors can be potentiated also via allosteric modulation by amphipathic lipid metabolites [106]. Im and colleagues hypothesized that this modulation could also occur in nature and represent another route of constitutive activation of the 5-HT 2C receptors [106].…”

“…Furthermore, it has been shown that the typical antipsychotic haloperidol despite its absence of affinity for the 5-HT 2C receptors increases the level of their constitutive activity [102], and this, most likely, through an action at the level of common pathways [105]. Recently, it has been shown that the affinity and efficacy of 5-HT for 5-HT 2C receptors can be potentiated also via allosteric modulation by amphipathic lipid metabolites [106]. Im and colleagues hypothesized that this modulation could also occur in nature and represent another route of constitutive activation of the 5-HT 2C receptors [106].…”