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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.

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.

The development of cell-based assays for cellular receptors, ion channels, and transporters requires the delivery and expression of transgenes. Viral-mediated gene delivery is a particularly attractive approach for this purpose because of its efficiency and potential to deliver genes to a wide variety of cell types. Recombinant baculoviruses, long used to deliver genes to insect cells for overexpression, also effectively transfer genes to mammalian cells. The only required modification to the virus for this purpose is the addition of transgene expression cassettes controlled by mammalian cell-active promoters. These so-called BacMam viruses are useful for developing mammalian cell-based assays for investigating the function of recombinant proteins and for assessing the action of pharmacological modulators of their function. The use of such viruses is gaining popularity because of the ease of optimizing assay conditions, the ability to deliver multiple gene products, and of their flexibility in terms of host cells and levels of transgene expression. BacMam-mediated assays may be used for studying a wide variety of target proteins and assay technologies. Described in this unit is an example of BacMam-mediated gene delivery to configure a cell-based assay for pharmacological assessment of a G protein-coupled receptor. A protocol is also provided describing the use of a GFP-expressing BacMam to assess the susceptibility of new cell lines to transduction by the virus.

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).

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) = 8.6 +/- 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 (Z)-(4-bromophenyl){1'-[(2,4-dimethyl-1-oxido-3-pyridinyl)carbonyl]-4'-methyl-1,4'-bipiperidin-4-yl}methanone O-ethyloxime (Sch-C; SCH 351125), 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R)-2-(methyloxy)-1-[4-(trifluoromethyl)phenyl]ethyl}-1-piperazinyl)-1-piperidinyl]carbonyl}pyrimidine (Sch-D; SCH 417,690), 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenyl-propyl)cyclohexanecarboxamide (UK-427,857), and N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclo-hepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride (TAK779) blocked the binding of both chemokines (125)I-MIP-1alpha (also known as (125)I-CCL3, (125)I-LD78) and (125)I-RANTES ((125)I-CCL5), 873140 was an ineffectual antagonist of (125)I-RANTES (regulated on activation normal T cell expressed and secreted) binding (but did block binding of (125)I-MIP-1alpha). Furthermore, 873140 blocked the calcium response effects of CCR5 activation by CCL5 (RANTES) (as did the other antagonists), indicating a unique divergence of blockade of function and binding with this antagonist. The antagonism of CCR5 by 873140 is saturable and probe-dependent, consistent with an allosteric mechanism of action. The blockade of CCR5 by 873140 was extremely persistent with a rate constant for reversal of0.004 h(-) (1) (t(1/2)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.