Some Examples Of Gpcr Allosteric Modulators 361 Small Molecule Allosteric Modulators

Clearly, the most obvious potential for exploiting allosteric sites on GPCRs is with respect to the discovery of novel small molecule chemotypes, which traditionally are seen as the most suitable agents amenable for drug development. In recent years, small molecule allosteric modulators have been discovered to act at all three major families of GPCRs (see Table 3.1 for some representative examples). While many of these have remained pharmacological tools, they have highlighted the applicability of allosteric modulation to GPCR- targeted

TABLE 3.1 Representative Allosteric Modulators of GPCRs

Family A

Adenosine A1 Adenosine A2A Adenosine A3 Adrenoceptor a1 Adrenoceptor a2A, a2B Adrenoceptor a2D Adrenoceptor p2

PD 81,723; LUF 5484 Amilorides

VU5455; VU8504; DU124183

Amilorides; benzodiazepines; conopeptide r-TIA

Amilorides

Agmatine

Zinc

TABLE 3.1 (Continued)

Cannabinoid CBi

Org 27569; Org 27759; Org 29647; PSNCBAM-1;

RTI-371

Chemokine CXCR1

Reparixin; SCH 527123; AZ Cmpds. A & B

Chemokine CXCR2

Reparixin; SCH 527123; SB 656933; AZ Cmpds.

A & B

Chemokine CXCR3

IP-10; I-TAC

Chemokine CXCR4

RSVM, ASLW; prichosanthin; plerixafor

Chemokine CCRi

BX-471; CP-481715; UCB35625

Chemokine CCR3

UCB35625; TAK779;

Chemokine CCR5

Trichosanthin; AK602; AK530; TAKK220; TAK779;

SCH 351125; ancriviroc; vicriviroc; aplaviroc;

maraviroc

Dopamine D1

Zinc

Dopamine D2

Amilorides; zinc; L-prolyl-L-leucylglycinamide

Endothelin ETA

Aspirin, sodium salicylate

FFA2 (GPR43)

Phenylacetamides 1 and 2

Follicle stimulating hormone

BMS compounds 2-7

GnRh receptor

Furan-1

Growth hormone secretagogue

L-692,429; GH-releasing peptide 6

Luteinizing hormone

Org 41841; [3H]Org 43553

Muscarinic M1-M5

Gallamine, alcuronium, brucine, W84, C7/3-phth;

WIN 62,577; AC-42; thiochrome; MT7; MT3;

staurosporine; tacrine; McN-A-343; LY2033298

Neurokinin NK1

Heparin

Opioid |i, S

Cannabidiol

Purine P2Y1

2,2'-pyridylsatogen tosylate

Serotonin 5HT1B/1D

5HT moduline

Serotonin 5HT2A, 5HT7

Oleamide

Serotonin 5HT2C

Oleamide; PNU-69176E

Family B

CRF1 receptor

Antalarmin; NBI 35965; DMP696; NBI 27914

CGRP receptor

BIBN4096BS

Glucagon

Bay27 - 9955; L - 168,049

GLP1 receptor

NN compounds 1-6; T-0632

Family C

Calcium sensing receptor

Fendeline; Cinacalcet; NPS 467; NPS 568; L-amino

acids; NPS 2143; Calhex 231

GABAB

CGP7930; CGP13501; GS39783

Glutamate mGluR1

(-)-CPCCOEt; Ro 67-7476, Ro 01-6128; BAY36-

7620; [3H]R214127; NPS 2390; EM-TBPC;

cis-64a; JNJ 16259685

Glutamate mGluR2

LY 487379; Biphenyl - indanone A; LY- 181837;

Ro 67 - 6221

Glutamate mGluR4

SIB - 1893, MPEP; ( - ) - PHCCC; VU0155041;

VU0080421

Glutamate mGluR5

MPEP; MTEP; DFB, DmeoB, DCB; CPPHA;

CDPPB; ADX-47273

Glutamate mGluR7

AMN082; MMPIP

drug discovery. The following sections briefly discuss some exemplar models of modulators from each of the main GPCR families.

Family A GPCRs The mAChR family is arguably one of the most well-characterized Family A GPCRs that possess at least one (and likely two) small molecule allosteric binding site(s). Indeed, the full spectrum of allosteric ligand phenotypes have been described for this family, from prototypical allosteric inhibitors and enhancers, such as gallamine and alcuronium, to allosteric agonists, such as LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b] pyridine-2-carboxylic acid cyclopropylamide) and bitopic ligands, such as McN-A-343 [23, 27, 38, 39] . Examples of the entire range (and complexities) of allosteric behaviors are also evident for the muscarinic receptor family, including positive and inverse agonism through the allosteric site, probe dependence of allosteric interactions, subtype selectivity driven through coop-erativity rather than affinity, and differential allosteric modulation of efficacy and affinity of the same orthosteric ligand [40]. In this regard, the muscarinic receptors have been widely utilized as a model system in the development of assay techniques and application of theoretical models, such as the ATCM and the operational model of allosterism, to experimental data. In addition, the location of the allosteric site of muscarinic receptors utilized by prototypical allosteric modulators has been well characterized via numerous structure-function studies identifying the importance of regions such as the second extracellular loop and the top of transmembrane (TM) domain 7 for allosteric drug interaction.

From a therapeutic viewpoint, the M1 and M4 subtypes have more recently been identified as desirable targets for allosteric modulation. Recent drug discovery efforts have uncovered highly functionally selective M1 mAChR agonists, such as AC - 42 (4 - n - butyl - 1 - [4 - 2(2 - methylphenyl) -4-oxo-1-butyl]-piperidine) [41], 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl) propyl]-3,3-dihydro-2(1 H)-quinolinone), AC-260584 (4-[3-(butylpiperidin-1-yl)propyl]-7-fluoro-4H-benzo[1,4]oxasin-3-one) [42, 43], and TBPB ((1-(1'-2-methylbenzyl)-1,4'-bipiperin-4-yl)-1Hbenzo[d]imidazole-1(3H)-one) [44], which have been suggested to mediate at least some of their effects via allosteric mechanisms. For example, the mode of action of AC-42 has been characterized using a combination of mutagenesis, signaling, binding, and dissociation kinetic assays, which have suggested that this compound operates at a distinct location to the orthosteric site [41-43, 45] . N-desmethlyclozapine is another putative allosteric agonist identified for the M1 mAChR [46]. A truly M1 selective allosteric agonist could be used to treat cognitive function and behavioral symptoms of Alzheimer's disease, with an allosteric mode of action overcoming the lack of selectivity and subsequent dose- l imiting side effects associated with orthosteric agonists. For the M4 mAChR subtype, recent studies have reported M4-selective positive allosteric modulators, LY2033298 and VU10010 (3-amino-N-(4-chlorobenzyl)-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide) [39, 47] . with the exciting potential of providing a novel approach to treating schizophrenia. Mechanistic studies of these ligands indicated that they exert their effects at the M4 mAChR by increasing the affinity of ACh and its coupling to G proteins. Interestingly, LY2033298 has also been shown to restore the function of ACh at a mutant muscarinic receptor with dramatically impaired orthosteric site functionality [48], highlighting the potential to use allosteric ligands to rescue function of receptors with impaired orthosteric pockets and/or signaling.

The adenosine family of GPCRs is another noteworthy example where many studies have focused on the potential for selective allosteric modulators. Indeed, the A1 adenosine receptor subtype was the first GPCR for which positive allosteric modulators were reported [49]. A1 allosteric enhancers have been pursued as an avenue for drug development in the treatment of a number of pathologies including neurological, cardiac, sleep, immune, and inflammatory disorders as well as cancer. Initial modulators were generated from an aroylthiophene chemical scaffold, which, although showing promise, exhibited factors that made it unattractive for further development [50]. Better compounds have since been developed from 6-aryl-8H-indeno[1,2-d]thiazol-2-ylamines, including IDTA-3x [51]. In addition, VUF5455 (4-methoxy-N-[7-methyl-3-(2-pyridinyl)-1-isoquinolinyl] benzamide) and DU124183 (2-cyclopentyl-4-phenylamino-1H-imidazo[4,5-c] quinoline) have been developed as selective, nanomolar potency, enhancers for the adenosine A3 receptor [52, 53]. All these compounds, however, appear to exhibit a mixed orthosteric/allosteric binding mode, which may have an impact on their use therapeutically.

With respect to current therapeutic utility of allosteric modulators, the chemokine receptor family is worth highlighting because maraviroc (UK-427,857), an allosteric antagonist of CCR5 receptors, was approved for use by the FDA in 2007 for the treatment of HIV-1 infection [54] . The chemokine receptor family is also of interest in that they possess numerous endogenous ligands and probably multiple allosteric sites, given the recent discovery of at least one class of modulator of the CXCR2 receptor with an intracellular site of action [ 55]. Because of the potential for probe- dependence of allosteric interactions, best practice would be to assess potential allosteric ligands against the full array of endogenous orthosteric ligands for any given chemokine receptor subtype. Indeed, a recent study has shown that a number of metal ion chelator complexes that bind allosterically at the CC- chemokine receptor 1, can simultaneously enhance the binding of one endogenous ligand (CC -chemokine 3), while acting as an antagonist for another (CC-chemokine 5) at the same receptor [56] . Clearly, the phenomenon of probe dependence is an important consideration with respect to the development of allosteric ligands from a drug discovery perspective.

Family B GPCRs Family B GPCRs include a number of peptide hormone GPCRs such as the corticotropin releasing factor (CRF) and glucagon-.ike peptide 1 (GLP1) receptors. Furthermore, this family of GPCRs is character ized by a relatively large N-terminal domain, which comprises most of the orthosteric binding site. Traditionally, this class of receptors has been recalcitrant to drug discovery targeting the diffuse pharmacophore associated with the peptide orthosteric site. However, a number of small molecules have recently emerged that act allosterically, an example of which is found with molecules targeting the CRF-1 receptor. Evidence for the allosteric nature of these molecules has been derived from both pharmacological studies that highlighted behavior inconsistent with the predictions of simple orthosteric competition, and from site- directed mutagenesis studies that demonstrated these compounds bound to residues within the TM regions of the CRF1 receptor rather than the N-terminal domain [57] . To date, the majority of CRF-1 receptor allosteric antagonists are used as pharmacological tools to study anxiety, depression, and irritable bowel syndrome. However, recently a series of tetraazaacenaphthylenes have shown promising results in preclinical studies and a Phase II clinical trial of major depressive disorder [58-60].

Novo Nordisk recently reported the first series of allosteric agonist/modulators, exemplified by compound 2, which specifically targets the GLP-1R [61] . In addition to its own agonist activity, compound 2 was able to increase the affinity of the endogenous ligand for the receptor while not affecting the maximal cyclic adenosine monophosphate (cAMP) response in functional studies. The molecular mechanisms for the effects of compound 2 are, to date, unknown. While there have been relatively few studies on other Family B GPCRs, these findings suggest that the allosteric approach represents a viable path forward for discovering small molecules directed against these receptors.

Family C GPCRs Family C GPCRs have traditionally proved most amenable to allosteric potentiation, and represent the first family of GPCR for which an allosteric modulator was approved and marketed as a novel therapeutic. Cinacalcet (NPS-1493) is a positive allosteric modulator of the calcium-sensing receptor (CaR) and represents an oral calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease [62, 63] . Cinacalcet interacts with the TM regions of the CaR to potentiate the actions of calcium, which binds in the large N-terminal, "Venus flytrap" (VFT) orthosteric binding domain [64] . Interestingly, the removal of the VFT from the CaR converts another positive allosteric modulator, calindol, into an allosteric agonist that exhibits synergy with additional calcium binding sites located within the remaining seven transmembrane (7TM) helical bundle [64]. This highlights not only the interaction between the allosteric and orthosteric sites on these receptors, but also the inherent complexity in some of these systems that must be considered in drug discovery efforts.

Allosteric modulators of mGluRs are also the subject of continued research because of their emerging therapeutic potential for a range of psychiatric and neurological disorders such as pain, anxiety, cognition, Parkinson -s disease, and schizophrenia [65]. Positive allosteric modulators have recently been described for group I (mGluR1 and mGluR5), group II (mGluR2), and group

III (mGluR4) mGluRs. Relative to classical mGluR agonists, these molecules offer improved selectivity, chemical tractability, and may reduce receptor desensitization [65]. Negative allosteric modulators of mGluRs have also been described [66] .

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