Activation of seven-transmembrane (7TM) receptors by agonists does not always lead to uniform activation of all signaling pathways mediated by a given receptor. Relative to other ligands, many agonists are "biased" toward producing subsets of receptor behaviors. A hallmark of such "functional selectivity" is cell type dependence; this poses a particular problem for the profiling of agonists in whole cell test systems removed from the therapeutic one(s). Such response-specific cell-based variability makes it difficult to guide medicinal chemistry efforts aimed at identifying and optimizing therapeutically meaningful agonist bias. For this reason, we present a scale, based on the Black and Leff operational model, that contains the key elements required to describe 7TM agonism, namely, affinity (K(A) (-1)) for the receptor and efficacy (τ) in activating a particular signaling pathway. Utilizing a "transduction coefficient" term, log(τ/K(A)), this scale can statistically evaluate selective agonist effects in a manner that can theoretically inform structure-activity studies and/or drug candidate selection matrices. The bias of four chemokines for CCR5-mediated inositol phosphate production versus internalization is quantified to illustrate the practical application of this method. The independence of this method with respect to receptor density and the calculation of statistical estimates of confidence of differences are specifically discussed.

4-{[4-({(3R)-1-Butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140) is a potent noncompetitive allosteric antagonist of the CCR5 receptor (pK B ϭ 8.6 Ϯ 0.07; 95% CI, 8.5 to 8.8) with concomitantly potent antiviral effects for HIV-1. In this article, the receptor-based mechanism of action of 873140 is compared with four other noncompetitive allosteric antagonists of CCR5. Although 857), and N, with an allosteric mechanism of action. The blockade of CCR5 by 873140 was extremely persistent with a rate constant for reversal of Ͻ0.004 h Ϫ1 (t 1/2 Ͼ 136 h). Coadministration studies of 873140 with the four other allosteric antagonists yielded data that are consistent with the notion that all five of these antagonists bind to a common allosteric site on the CCR5 receptor. Although these ligands may have a common binding site, they do not exert the same allosteric effect on the receptor, as indicated by their differential effects on the binding of 125 I-RANTES. This idea is discussed in terms of using these drugs sequentially to overcome HIV viral resistance in the clinic.With the discovery that the R5 strain of HIV uses the chemokine C CCR5 receptor for cell infection (Alkhatib et al., 1996;Choe et al., 1996;Doranz et al., 1996; Dragic et al., 1996;Deng et al., 1997;Shieh et al., 1998;Zhang and Moore, 1999) has come the opportunity for a completely new approach to preventing HIV infection: blockade of CCR5 receptor interaction with the viral coat protein gp120. Subsequent reports of potent antagonists of CCR5-mediated HIV entry (Baba et al., 1999;Finke et al., 2001;Strizki et al., 2001;Kazmierski et al., 2003; Demarest et al., 2004a,b, Maeda et al., 2004 have validated this approach and have possibly opened a new era of AIDS therapy. There are data to support the notion that an allosteric mechanism is involved in the antagonism of HIV by low molecular weight antagonists of CCR5 (Kazmierski et al., 2002). The large size of the proteins involved in HIV fusion (i.e., CCR5 and gp120) and the fact that mutational studies indicate that numerous regions of both CCR5 (Atchison et al., 1996;Rucker et al., 1996;Doms and Peiper, 1997;Doranz et al., 1997;Picard et al., 1997 ABBREVIATIONS: MIP-1␣, macrophage inflammatory protein 1-alpha (standard nomenclature CCL3, also known as LD78); CHO, Chinese hamster ovary; SPA, scintillation proximity assay; DMSO, dimethyl sulfoxide; RT, room temperature; HEK, human embryonic kidney; FLIPR, fluorometric imaging plate reader; RANTES, regulated on activation, normal T cell expressed and secreted (standard nomenclature for this chemokine is CCL5); Sch-C (SCH 351125), (Z)-(4-bromophenyl

Insurmountable antagonism (maximal response to the agonist depressed) can result from a temporal inequilibrium involving a slow offset orthosteric antagonist or be the result of an allosteric modulation of the receptor. The former mechanism is operative when the antagonist, agonist, and receptors cannot come to proper equilibrium during the time allotted for collection of agonist response (hemi-equilibrium conditions). Allosteric effects (changes in the conformation of the receptor through binding of the allosteric modulator to a separate site) can preclude the agonist-induced production of response, leading to depression of maximal responses. In these cases, the effects on receptor affinity can be observed as well. The first premise of this article is that system-independent estimates of insurmountable antagonist potency can be made with no prior knowledge of molecular mechanism through the use of pA 2 (Ϫlog molar concentration of antagonist producing a 2-fold shift of the concentration response curve) measurements The relationship between the pA 2 and antagonist pK B (Ϫlog equilibrium dissociation constant of the antagonist-receptor complex) is described; the former is an extremely close approximation of the latter in most cases. The second premise is that specially designed experiments are required to differentiate orthosteric versus allosteric mechanisms; simply fitting of data to orthosteric or allosteric theoretical models can lead to ambiguous results. A strategy to determine whether the observed antagonism is orthosteric (agonist and antagonist competing for the same binding site on the receptor) or allosteric in nature is described that involves the detection of the hallmarks of allosteric response, namely saturation and probe dependence of effect.Two major considerations in a drug discovery program for antagonists are the need for 1) system-independent estimates of potency and 2) knowledge of the molecular mechanism of action. The former enables systematic study of structure and activity and subsequent optimization of activity, whereas the latter allows prediction of the properties of the antagonist in the therapeutic situation. The major premise of this study is that knowledge of the mechanism of action of insurmountable antagonists is not required for the systemindependent measure of antagonist potency. In fact, verisimilitude of data to specific theoretical models is an unreliable way to determine mechanism of action (vide infra). It will be proposed that specifically designed experiments are required to do so.By definition, antagonists interfere with the ability of agonists to produce pharmacological response. The way they express this interference varies but generally involves changing the location parameter (EC 50 ; molar concentration proArticle, publication date, and citation information can be found at

We describe robust chemical approaches toward putative CCR5 scaffolds designed in our laboratories. Evaluation of analogues in the (125)I-[MIP-1beta] binding and Ba-L-HOS antiviral assays resulted in the discovery of 64 and 68 in the 4,4-disubstitited piperidine class H, both potent CCR5 ligands (pIC 50 = 8.30 and 9.00, respectively) and HIV-1 inhibitors (pIC 50 = 7.80 and 7.84, respectively, in Ba-L-HOS assay). In addition, 64 and 68 were bioavailable in rodents, establishing them as lead molecules for further optimization toward CCR5 clinical candidates.

We recently described ( J. Med. Chem. 2008 , 51 , 6538 - 6546 ) a novel class of CCR5 antagonists with strong anti-HIV potency. Herein, we detail SAR converting leads 1 and 2 to druglike molecules. The pivotal structural motif enabling this transition was the secondary sulfonamide substituent. Further fine-tuning of the substituent pattern in the sulfonamide paved the way to enhancing potency and bioavailability and minimizing hERG inhibition, resulting in discovery of clinical compound 122 (GSK163929).

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