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G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.
BACKGROUND - The B receptor antagonist icatibant is approved for treatment of attacks of hereditary angioedema. Icatibant has been reported to decrease time-to-resolution of angiotensin-converting enzyme (ACE) inhibitor-associated angioedema in 1 study of European patients.
OBJECTIVE - We sought to test the hypothesis that a bradykinin B receptor antagonist would shorten time-to-resolution from ACE inhibitor-associated angioedema.
METHODS - Patients with ACE inhibitor-associated angioedema (defined as swelling of lips, tongue, pharynx, or face during ACE inhibitor use and no swelling in the absence of ACE inhibitor use) were enrolled at Vanderbilt University Medical Center from October 2007 through September 2015 and at Massachusetts General Hospital in 2012. C1 inhibitor deficiency and patients with bowel edema only were excluded. Patients were randomized within 6 hours of presentation to subcutaneous icatibant 30 mg or placebo at 0 and 6 hours later. Patients assessed severity of swelling using a visual analog scale serially following study drug administration or until discharge.
RESULTS - Thirty-three patients were randomized and 31 received treatment, with 13 receiving icatibant and 18 receiving placebo. One patient randomized to icatibant did not complete the visual analog scale and was excluded from analyses. Two-thirds of patients were black and two-thirds were women. Time-to-resolution of symptoms was similar in placebo and icatibant treatment groups (P = .19 for the primary symptom and P > .16 for individual symptoms of face, lip, tongue, or eyelid swelling). Frequency of administration of H1 and H2 blockers, corticosteroids, and epinephrine was similar in the 2 treatment groups. Time-to-resolution of symptoms was similar in black and white patients.
CONCLUSIONS - This study does not support clinical efficacy of a bradykinin B receptor antagonist in ACE inhibitor-associated angioedema.
Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Heart failure affects ≈5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, β-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the US Food and Drug Administration approved the first of a new class of drugs for the treatment of heart failure: Valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses 2 of the pathophysiological mechanisms of heart failure: activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared with enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacological properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension.
© 2016 American Heart Association, Inc.
BACKGROUND - Endothelial dysfunction occurs in patients with end-stage renal disease (ESRD) and is associated with increased cardiovascular morbidity and mortality. Asymmetric dimethylarginine (ADMA) contributes to endothelial dysfunction in ESRD. In the general population, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) decrease ADMA levels, but no study has compared the effect of these drugs in patients with ESRD on maintenance hemodialysis (MHD).
METHODS - We evaluated the effect of 1-week treatment with ramipril (5 mg/d), valsartan (160 mg/d), and placebo on ADMA levels in 15 patients on MHD in a double-blind, placebo-controlled, three x three cross-over study.
RESULTS - We found that ADMA levels were increased at baseline and throughout the dialysis session during ramipril treatment (p < 0.001 compared to both, placebo and valsartan). Ramipril did not increase ADMA levels in a study of patients without ESRD, suggesting that factors related to ESRD or hemodialysis contribute to the ACE inhibitor-induced increase in ADMA. We have previously shown that ACE inhibition increases bradykinin (BK) levels during hemodialysis. We therefore evaluated the effect of bradykinin on ADMA production in A549 cells; a cell line that expresses BK receptors. Incubation with BK increased intracellular ADMA concentration through BK B2-receptor stimulation.
CONCLUSION - These data indicate that short-term ACE inhibition increases ADMA in patients on MHD whereas ARBs do not. In vitro studies further suggest that this may occur through BK-mediated increase in ADMA production during ACE inhibition.
TRIAL REGISTRATION - Clinicaltrials.gov NCT00732069 August 6 2008 and NCT00607672 February 4 2008.
UNLABELLED - Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce postprandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme (ACE). During ACE inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, whereas bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when ACE is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg by mouth or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat; however, substance P increased heart rate and vascular release of norepinephrine during combined ACE and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined ACE and dipeptidyl peptidase-4 inhibition.
CLINICAL TRIAL REGISTRATION - - URL: http://www.clinicaltrials.gov. Unique identifier: NCT01413542.
Bradykinin increases during cardiopulmonary bypass (CPB) and stimulates the release of nitric oxide, inflammatory cytokines, and tissue-type plasminogen activator (t-PA), acting through its B2 receptor. This study tested the hypothesis that endogenous bradykinin contributes to the fibrinolytic and inflammatory response to CPB and that bradykinin B2 receptor antagonism reduces fibrinolysis, inflammation, and subsequent transfusion requirements. Patients (N = 115) were prospectively randomized to placebo, ε-aminocaproic acid (EACA), or HOE 140, a bradykinin B2 receptor antagonist. Bradykinin B2 receptor antagonism decreased intraoperative fibrinolytic capacity as much as EACA, but only EACA decreased D-dimer formation and tended to decrease postoperative bleeding. Although EACA and HOE 140 decreased fibrinolysis and EACA attenuated blood loss, these treatments did not reduce the proportion of patients transfused. These data suggest that endogenous bradykinin contributes to t-PA generation in patients undergoing CPB, but that additional effects on plasmin generation contribute to decreased D-dimer concentrations during EACA treatment.
During inflammation, vascular permeability is increased by various proteolytic events, such as the generation of bradykinin, that augment local tissue responses by enabling tissue penetration of serum proteins, including complement and acute-phase proteins. Proteases also govern inflammatory responses by processing extracellular matrix proteins and soluble bioactive mediators. We quantified changes in the proteome and the nature of protein amino termini (the N-terminome) and the altered abundance of murine proteases and inhibitors during skin inflammation. Through analysis of the N-terminome by iTRAQ-TAILS, we identified cotranslational and posttranslational αN-acetylation motifs, quantitative increases in protein abundance, and qualitative changes in the proteolytic signature during inflammation. Of the proteins identified in normal skin, about half were cleaved, and phorbol ester-induced inflammation increased the proportion of cleaved proteins, including chemokines and complement proteins, that were processed at previously uncharacterized sites. In response to phorbol ester-induced inflammation, mice deficient in matrix metalloproteinase 2 (MMP2) showed reduced accumulation of serum proteins in the skin and exhibited different proteolytic networks from those of wild-type mice. We found that the complement 1 (C1) inhibitor attenuated the increase in serum protein accumulation in inflamed skin. Cleavage and inactivation of the C1 inhibitor by MMP2 increased complement activation and bradykinin generation in wild-type mice, leading to increased vessel permeability during inflammation, which was diminished in Mmp2(-/-) mice. Thus, our systems-level analysis of proteolysis dissected cleavage events associated with skin inflammation and demonstrated that loss of a single protease could perturb the proteolytic signaling network and enhance inflammation.
Preclinical pharmacological characterization of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) was investigated. We synthesized and evaluated a library of proline-based analogs as prospective recombinant PRCP (rPRCP) inhibitors and inhibitors of PRCP-dependent prekallikrein (PK) activation on human pulmonary artery endothelial cells (HPAEC). Among the newly synthesized compounds, UM8190 was further characterized in vivo using methods that encompassed a mouse carotid artery thrombosis model and animal model of food consumption. (S)-N-dodecyl-1-((S)-pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide [Compound 3 (UM8190)] was selected for further evaluation from the initial assessment of its PRCP inhibitory action (K(i)= 43 μM) coupled with its ability to block PRCP-dependent PK activation on HPAEC (K(i)= 34 μM). UM8190 demonstrated excellent selectivity against a panel of carboxypeptidases and serine proteases and blocked bradykinin (BK) generation and BK-induced permeability by 100%, suggesting that it may be useful in preventing the local production of large amounts of BK. Furthermore, UM8190 showed an anorexigenic effect when systemically administered to fasted mice, reducing food intake in a dose- and time-dependent manner. In a mouse carotid artery thrombosis model, it also demonstrated an antithrombotic effect. UM8190 is a selective PRCP inhibitor and it may represent a new anorexigenic, and antithrombotic drug, that works by inhibiting PRCP-mediated mechanisms.
The effects of angiotensin-converting enzyme (ACE) inhibition and angiotensin II type 1 receptor blockade (ARB) on fibrinolysis and inflammation after cardiopulmonary bypass (CPB) are uncertain. This study tested the hypothesis that ACE inhibition enhances fibrinolysis and inflammation to a greater extent than ARB in patients undergoing CPB. One week to 5 days before surgery, patients were randomized to ramipril 5 mg/day, candesartan 16 mg/day, or placebo. ACE inhibition increased intraoperative bradykinin and tissue-type plasminogen activator (t-PA ) concentrations as compared to AR B. Both ACE inhibition and AR B decreased the need for plasma transfusion relative to placebo, but only ACE inhibition decreased the duration of hospital stay. Neither ACE inhibition nor AR B significantly affected concentrations of plasminogen activator inhibitor-1 (PAI -1), interleukin (IL )-6, IL -8, or IL -10. ACE inhibition enhanced intraoperative fibrinolysis without increasing the likelihood of red-cell transfusion. By contrast, neither ACE inhibition nor ARB affected the inflammatory response. ACE inhibitors and ARBs may be safely continued until the day of surgery.