The CC-chemokine receptor 5 (CCR5) is the major coreceptor for the entry of macrophage-tropic (R5) HIV-1 strains into target cells. Posttranslational sulfation of tyrosine residues in the N-terminal tail of CCR5 is critical for high affinity interaction of the receptor with the HIV-1 envelope glycoprotein gp120 in complex with CD4. Here, we focused on defining precisely the sulfation pattern of the N terminus of CCR5 by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a synthetic peptide that corresponds to amino acids 2-18 of the receptor (CCR5 2-18). Analysis of the reaction products was made with a combination of reversed-phase HPLC, proteolytic cleavage, and matrix-assisted laser desorption͞ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We found that CCR5 2-18 is sulfated by both TPST isoenzymes leading to a final product with four sulfotyrosine residues. Sulfates were added stepwise to the peptide producing specific intermediates with one, two, or three sulfotyrosines. The pattern of sulfation in these intermediates suggests that Tyr-14 and Tyr-15 are sulfated first, followed by Tyr-10, and finally Tyr-3. These results represent a detailed analysis of the multiple sulfation reaction of a peptide substrate by TPSTs and provide a structural basis for understanding the role of tyrosine sulfation of CCR5 in HIV-1 coreceptor and chemokine receptor function. The CC-chemokine receptor 5 (CCR5) is a member of the protein superfamily of G protein-coupled receptors (GPCRs) (1-3). High-affinity binding of the CC-chemokines MIP-1␣, MIP-1␤, RANTES (1-3), or MCP-2 (4) to CCR5 induces signaling through G proteins of the G i subfamily (1) and leads to chemotactic responses in CCR5-expressing leukocytes (5).In addition to their physiological function in chemokine signaling, some chemokine receptors are used as coreceptors by HIV-1. Entry of HIV-1 into target cells is mediated by the sequential interaction of the envelope glycoprotein gp120 with CD4 and a chemokine receptor on the cell membrane (6). CCR5 and CXCR4 are the primary HIV-1 coreceptors in vivo (7,8). CCR5, in particular, is the principal coreceptor for macrophage-tropic HIV-1 strains (R5 isolates) that are commonly transmitted between individuals (6, 9). A naturally occurring CCR5 mutant (⌬32) with a deletion in the second extracellular loop results in impaired membrane expression of the receptor and leads to resistance to HIV-1 infection in homozygous individuals (6).Interaction with both types of CCR5 ligands, CC-chemokines and the HIV-1 gp120-CD4 complex, involves the N-terminal domain, as well as other extracellular regions of the receptor (10-13). Within the N-terminal domain, a region rich in tyrosine residues and acidic amino acid residues ( Fig. 1; residues 2-18) was identified as a major determinant of HIV-1 coreceptor function (11,12,(14)(15)(16)(17). Sequence similarities with proteins known to be modified by tyrosine O-sulfation, a posttranslational modification mediated by tyrosylprotein sulfotransfera...

The discovery of vibegron, a potent and selective human β3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical β3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived β3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.

Although the physiologic role of muscarinic receptors in bladder function and the therapeutic efficacy of muscarinic antagonists for the treatment of overactive bladder are well established, the role of β-adrenergic receptors (βARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel βAR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of βAR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full βAR agonist across species, and it dose dependently increased bladder capacity, decreased micturition pressure, and increased bladder compliance in rhesus monkeys. The relaxation effect of vibegron was enhanced when combined with muscarinic antagonists, but differentially influenced by muscarinic receptor subtype selectivity. The effect was greater when vibegron was co-administered with tolterodine, a nonselective antagonist, compared with coadministration with darifenacin, a selective M3 antagonist. Furthermore, a synergistic effect for bladder strip relaxation was observed with the combination of a βAR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel βAR agonist probe, [H]MRL-037, that selectively labels β receptors in both urothelium and detrusor smooth muscle. Vibegron administration caused a dose-dependent increase in circulating glycerol and fatty acid levels in rhesus and rat in vivo, suggesting these circulating lipids can be surrogate biomarkers. The translation of our observation to the clinic has yet to be determined, but the combination of βAR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder.

A series of conformationally restricted acetanilides were synthesized and evaluated as β3-adrenergic receptor agonists (β3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine β3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent β3-AR mediated responses in a rat bladder hyperactivity model.