Pharmacological Characterization of a Novel Beta 3 Adrenergic Agonist, Vibegron: Evaluation of Antimuscarinic Receptor Selectivity for Combination Therapy for Overactive Bladder.

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 ß3-adrenergic receptors (ß3ARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel ß3AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of ß3AR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full ß3AR 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 ß3AR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel ß3AR agonist probe, [3H]MRL-037, that selectively labels ß3 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 ß3AR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder.

Design of a monomeric 23-residue polypeptide with defined tertiary structure.

Small proteins or protein domains generally require disulfide bridges or metal sites for their stabilization. Here it is shown that the beta beta alpha architecture of zinc fingers can be reproduced in a 23-residue polypeptide in the absence of metal ions. The sequence was obtained through an iterative design process. A key feature of the final design is the incorporation of a type II' beta turn to aid in beta-hairpin formation. Nuclear magnetic resonance analysis reveals that the alpha helix and beta hairpin are held together by a defined hydrophobic core. The availability of this structural template has implications for the development of functional polypeptides.

THE PHYSICAL AND CHEMICAL MICROSTRUCTURE OF THE ACHATINA FULICA EPIPHRAGM.

Microstructural characterization of Achatina fulica Bowdich, 1822 epiphragms and mucus secretions was performed to address two questions: what are the structure and composition of the reinforcing inorganic phase in the epiphragms, and what enables a durable epiphragm to form quickly in comparison to other biomineralized materials? Characterization was performed by a combination of light microscopy (relying on a variety of contrast modes), wet chemical tests, environmental scanning electron microscopy (including the use of energy dispersive X-ray analysis to obtain compositional data), and X-ray diffraction. The morphology of the inorganic phase promotes mechanical interlocking and presents a large surface for binding to the organic matrix. Strong binding occurs between the organic and inorganic phases. The inorganic phase adopts the calcite structure; its composition is Ca(0.912) Mg(0.088) CO(3). Epiphragms can form quickly because pre-grown crystals of the inorganic reinforcing phase are co-deposited with the mucus matrix. Unlike other biomineralized material, the crystals are not solution-grown in situ on an organic template in the final product.

Assessment of chemokine receptor function on monocytes in whole blood: In vitro and ex vivo evaluations of a CCR2 antagonist.

Inhibition of monocyte and macrophage function by targeting chemokine receptors represents an attractive strategy for therapeutic intervention in inflammatory diseases. We describe an assay to assess chemokine receptor function on whole blood monocytes by measuring chemokine stimulated change in cell shape as measured by flow cytometry. The relative potential of the chemokine receptors CCR1, CCR2, CCR5, CX(3)CR1, and CXCR4 to activate monocytes in whole blood was evaluated and compared. Analysis of MCP-1 response for monocytes in blood from numerous donors revealed that the assay method had excellent intra-donor reproducibility and sensitivity. Further, the utility of this assay to determine target engagement by chemokine receptor antagonists was demonstrated using a CCR2 antagonist in rhesus monkeys. Blockade of CCR2 on whole blood monocytes was demonstrated ex vivo on blood samples collected from rhesus monkeys administered a small molecule CCR2 antagonist (MK-0812). Using a delayed-type hypersensitivity reaction to elicit monocyte recruitment to the skin of rhesus monkeys, we also evaluated the ability of MK-0812 to block monocyte migration in vivo. Blockade of CCR2 stimulation of whole blood monocytes was correlated with the inhibition of monocyte recruitment to the skin, validating the potential to use this approach in the evaluation of dose selection for chemokine receptor antagonists clinically.

An initial appraisal of the clinical significance of Roseomonas species associated with human infections.

We reviewed laboratory, clinical, and epidemiologic data on 35 patients from whom organisms belonging to the genus Roseomonas, a pink-pigmented gram-negative coccobacillus, were isolated over a 22-year period (1972-1994). Roseomonas strains were most commonly isolated from middle-aged women with one of several underlying conditions, including cancer and diabetes. Roseomonas was most commonly isolated from the blood, in association with clinical signs of sepsis. Approximately 60% of all isolates were judged to be of possible clinical significance, either as primary or secondary pathogens; 75% of all strains were recovered in pure culture. Roseomonas gilardii was the most frequently isolated species and was significantly associated with septicemia and underlying immunocompromised conditions; the species of 29% of all Roseomonas isolates could not be unequivocally identified with presently available differential tests. Genomospecies 5, currently an unnamed taxon within the genus Roseomonas, was primarily recovered as a commensal from young adults attending a sexually transmitted diseases clinic. The findings suggest that although this genus appears to have an overall low pathogenic potential for humans, Roseomonas species-in particular, R. gilardii-may be significant pathogens in persons with underlying medical complications.

Tertiary interactions between the fifth and sixth transmembrane segments of rhodopsin.

We have used cysteine scanning mutagenesis and disulfide cross-linking in a split rhodopsin construct to investigate the secondary structure and tertiary contacts of the fifth (TM5) and sixth (TM6) transmembrane segments of rhodopsin. Using a simple increase in pH to promote disulfide bond formation, three cross-links between residues on the extracellular side of TM5 (at positions 198, 200, and 204) and TM6 (at position 276) have been identified and characterized. The helical pattern of cross-linking observed indicates that the fifth transmembrane helix extends through residue 200 but does not include residue 198. Rhodopsin mutants containing these disulfides demonstrate nativelike absorption spectra and light-dependent activation of transducin, suggesting that large movements on the extracellular side of TM5 with respect to TM6 are not required for receptor activation.

G protein-coupled receptor activation: analysis of a highly constrained, "straitjacketed" rhodopsin.

G protein-coupled receptor (GPCR) activation is generally assumed to result in a significant structural rearrangement of the receptor, presumably involving the rigid body movement of transmembrane helices. We have investigated the activation of the GPCR rhodopsin by the construction and analysis of a mutant which contains a total of four disulfide bonds connecting the cytoplasmic ends of helices 1 and 7, and 3 and 5, and the extracellular ends of helices 3 and 4, and 5 and 6. Despite the constraints imposed by four disulfides, this "straitjacketed" receptor retains the ability to activate the G protein transducin and, therefore, provides insight into the molecular mechanism of the initial step in signal transduction of this important class of receptors.

Design and NMR analyses of compact, independently folded BBA motifs.

BACKGROUND: Small folded polypeptide motifs represented highly simplified systems for theoretical and experimental studies on protein structure and folding. We have recently reported the design and characterization of a metal-ion-independent 23-residue peptide with a beta beta alpha structure (BBA1), based on the zinc finger domains. To understand better the determinants of structure for this small peptide, we investigated the conformational role of the synthetic residue 3-(1, 10-phenanthrol-2-yl)-L-alanine (Fen) in BBA1. RESULTS: NMR analysis revealed that replacing the Fen residue of peptide BBA1 by either of the natural amino acids tyrosine (BBA2) or tryptophan (BBA3) resulted in conformational flexibility in the sheet and loop regions of the structure. This conformational ambiguity was eliminated in peptides BBA4 and BBA5 by including charged residues on the exterior of the beta hairpin designed to both select against the undesired fold and stabilize the desired structure. The evaluation of two additional peptides (BBA6 and BBA7) provided further insight into the specific involvement of the surface polar residues in the creation of well-defined structure in BBA4 and BBA5. The sequences of BBA5, BBA6 and BBA7 include only one non-standard amino acid (D-proline), which constrains a critical engineered type II' turn. CONCLUSIONS: Manipulation of residues on the exterior of small beta beta alpha motifs has led to the design of 23-residue polypeptides that adopt a defined tertiary structure in the absence of synthetic amino acids, increasing the availability and expanding the potential uses of the BBA motif. The importance of negative design concepts to the creation of structured polypeptides is also highlighted.

Oral disopyramide dosage regimes in ischaemic heart disease.

The serum concentration/time profiles resulting from two oral disopyramide dosage regimes were studied in ten patients with ischaemic heart disease. Conventional dosing on a 6 r 8 hourly basis consistently achieved disopyramide concentrations within the accepted therapeutic range of 2-7 micrograms/ml. In contrast, a twice daily regime was associated in some patients with trough levels below the minimum effective concentration. The mean elimination half-life was 5.8 h; this does not substantiate previous reports of significantly prolonged disopyramide half-lives in patients with ischaemic heart disease. Unless significant renal impairment or cardiac failure is present, or a sustained release preparation is used, the dosage interval for oral disopyramide should not exceed 8 h.