C5a receptor antagonism coming of age for vascular pathology.

The Food and Drug Administration recently approved the new drug avacopan for a relatively rare disease, anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis. Avacopan is an antagonist of receptor-1 for anaphylatoxin C5a (C5aR) that is the first one to meet all expectations of an orally bioavailable drug. Pharmacological effects of C5a on vascular tissue are reviewed; they are essentially indirect, via resident or infiltrating leukocytes, and largely mediated by vasoconstrictor prostanoids that are potentially thrombogenic.

Are noscapine and raloxifene ligands of the bradykinin B receptor? An assessment based on the human umbilical vein contractility assay.

The centrally acting antitussive opiate derivative, noscapine, has been claimed to be a non-competitive bradykinin B receptor antagonist. Raloxifene, a selective estrogen receptor modulator, was predicted to bind the bradykinin B receptor and to exert a partial agonist activity. These intriguing claims suggest that new molecular scaffolds ("chemotypes") may be identified for small molecule ligands of kinin receptors and that some off-target effects of noscapine or raloxifene may be mediated by bradykinin B receptors.

Picomolar Sensitivity Analysis of Multiple Bradykinin-Related Peptides in the Blood Plasma of Patients With Hereditary Angioedema in Remission: A Pilot Study.

Background: Hereditary angioedema (HAE) is a rare autosomal dominant disease; the most well understood forms concern the haplodeficiency of C1 esterase inhibitor (C1INH) and a gain of function mutation of factor XII (FXII). The acute forms of these conditions are mediated by an excessive bradykinin (BK) formation by plasma kallikrein.

Methods: A validated LC-MS/MS platform of picomolar sensitivity developed for the analysis of eleven bradykinin-related peptides was applied to the plasma of HAE-C1INH and HAE-FXII sampled during remission.

In vitro pharmacological profile of PHA-022121, a small molecule bradykinin B receptor antagonist in clinical development.

PHA-022121 is a novel small molecule bradykinin B receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B receptor with K values of 0.

A Robust Bioassay of the Human Bradykinin B Receptor that Extends Molecular and Cellular Studies: The Isolated Umbilical Vein.

Bradykinin (BK) has various physiological and pathological roles. Medicinal chemistry efforts targeted toward the widely expressed BK B receptor (BR), a G-protein-coupled receptor, were primarily aimed at developing antagonists. The only BR antagonist in clinical use is the peptide icatibant, approved to abort attacks of hereditary angioedema.

In Vitro Modeling of Bradykinin-Mediated Angioedema States.

Kinins (peptides related to bradykinin, BK) are formed from circulating substrates, the kininogens, by the action of two proteases, the kallikreins. The only clinical application of a BK receptor ligand, the B receptor antagonist icatibant, is the treatment of the rare hereditary angioedema (HAE) caused by the deficiency of C1-esterase inhibitor (C1-INH). Less common forms of HAE (genetic variants of factor XII, plasminogen, kininogen) are presumably mediated by increased BK formation.

Measurement of Bradykinin Formation and Degradation in Blood Plasma: Relevance for Acquired Angioedema Associated With Angiotensin Converting Enzyme Inhibition and for Hereditary Angioedema Due to Factor XII or Plasminogen Gene Variants.

Bradykinin (BK)-mediated angioedema (AE) states are rare acquired or hereditary conditions involving localized edema of the subcutaneous and submucosal tissues. Citrated plasma from healthy volunteers or patients with hereditary angioedema (HAE) with normal level of C1-inhibitor (C1-INH) was used to investigate pathways of BK formation and breakdown relevant to AE physiopathology. The half-life of BK (100 nM) added to normal plasma was 34 s, a value that was increased ~12-fold when the angiotensin converting enzyme (ACE) inhibitor enalaprilat (130 nM) was added (enzyme immunoassay measurements).

Pharmacological Profile of a New Small Molecule Bradykinin B Receptor Antagonist.

We here report the discovery and early characterization of Compound 3, a representative of a novel class of small molecule bradykinin (BK) B receptor antagonists, and its superior profile to the prior art B receptor antagonists Compound 1 and Compound 2. Compound 3, Compound 2, and Compound 1 are highly potent antagonists of the human recombinant B receptor (K values 0.24, 0.

The development of a targeted and more potent, anti-Inflammatory derivative of colchicine: Implications for gout.

Background: Colchicine is routinely used for its anti-inflammatory properties to treat gout and Familial Mediterranean fever. More recently, it was also shown to be of therapeutic benefit for another group of diseases in which inflammation is a key component, namely, cardiovascular disease. Whilst there is considerable interest in repurposing this alkaloid, it has a narrow therapeutic index and is associated with undesirable side effects and drug interactions.

Bradykinin receptors: Agonists, antagonists, expression, signaling, and adaptation to sustained stimulation.

Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein-coupled receptors, the B and B receptors (BR, BR). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed herein, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and non-peptide antagonists and fluorescent ligands have been produced for each receptor.

Increased fibrinolysis-induced bradykinin formation in hereditary angioedema confirmed using stored plasma and biotechnological inhibitors.

Objective: We recently investigated the pathways of immunoreactive bradykinin (iBK) formation in fresh blood of normal volunteers and of patients with hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-1/-2). Herein, we adapted the techniques to small volumes (200 μl) of previously frozen citrated plasma and further analyzed the mechanisms of iBK formation with additional biotechnological inhibitors.

Results: Measurable iBK formation was observed under stimulation with tissue kallikrein (KLK-1, 10 nM), the particulate material Kontact-APTT (concentration reduced to 2% v/v) or recombinant tissue plasminogen activator (tPA, 169 nM), with little background in unstimulated plasma incubated for up to 2 h.

Comparing Pathways of Bradykinin Formation in Whole Blood From Healthy Volunteers and Patients With Hereditary Angioedema Due to C1 Inhibitor Deficiency.

Multiple pathways have been proposed to generate bradykinin (BK)-related peptides from blood. We applied various forms of activation to fresh blood obtained from 10 healthy subjects or 10 patients with hereditary angioedema (HAE-1 or -2 only) to investigate kinin formation. An enzyme immunoassay for BK was applied to extracts of citrated blood incubated at 37°C under gentle agitation for 0-2 h in the presence of activators and/or inhibitory agents.

Bifunctional ligands of the bradykinin B and B receptors: An exercise in peptide hormone plasticity.

Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B receptor (BR) and to the inducible B receptor (BR). BRs and BRs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for BRs, Lys-des-Arg-BK for BR).

D-Arg-Bradykinin-Arg-Arg, a Latent Vasoactive Bradykinin B Receptor Agonist Metabolically Activated by Carboxypeptidases.

We previously reported hypotensive and vasodilator effects from C-terminally extended bradykinin (BK) sequences that behave as B receptor (BR) agonists activated by vascular or plasma peptidases. D-Arg-BK-Arg-Arg (r-BK-RR) is a novel prodrug peptide hypothetically activated by two catalytic cycles of Arg-carboxypeptidases (CPs) to release the direct agonist D-Arg-BK. N-terminally extending the BK sequence with D-Arg in the latter peptide was meant to block the second kinin inactivation pathway in importance, aminopeptidase P.

Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B receptor.

To support bradykinin (BK) B receptor (BR) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat BRs (immunoblots, epifluorescence microscopy). APEX2-(NG)-MK is a bona fide agonist of the rat, but not of the human BR (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric BR detection, cell well plate format).

Production and evaluation of parathyroid hormone receptor ligands with intrinsic or assembled peroxidase domains.

Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH receptor (PTHR). We developed fusion protein ligands with enzymatic activity to probe PTHRs at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2).

S100A9 potentiates the activation of neutrophils by the etiological agent of gout, monosodium urate crystals.

Gout is one of the most painful types of arthritis that arises when the body mounts an acute inflammatory reaction against a crystallized form of uric acid known as monosodium urate crystals (MSUs). Although MSUs are known to activate neutrophils, the most abundant leukocyte in the synovial fluid of patients with gout, few studies have investigated the effect on neutrophils of the simultaneous stimulation with MSU and proinflammatory mediators in the inflamed joint. Herein, we focused on a protein that is highly expressed in the synovium in gout, S100A9.

Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B receptor and peptidase resistance in rats.

Maximakinin (MK), an amphibian peptide possessing the C-terminal sequence of bradykinin (BK), is a BK B receptor (BR) agonist eliciting prolonged signaling. We reinvestigated this 19-mer for species-specific pharmacologic profile, confirmation of resistance to inactivation by angiotensin converting enzyme (ACE), value as a module for the design of fusion proteins that bind to the BR in mammalian species and potential activity as a histamine releaser. Competition of the binding of [H]BK to recombinant human myc-BRs in cells that express these receptors revealed that MK possessed a tenuous fraction (<0.

Infrared-emitting, peptidase-resistant fluorescent ligands of the bradykinin B receptor: application to cytofluorometry and imaging.

Background: We have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B receptor (BR) ligands conjugated with fluorophores such as fluorescein derivatives at their N-terminus. To take advantage of the high penetration of infrared light into living tissues and their low autofluorescence in this region of the spectrum, additional probes conjugated with cyanine dye 7 (Cy7) were synthesized and characterized.

Results: The antagonist B-9430 (D-Arg-[Hyp,Igl,D-Igl,Oic]-BK) and the agonist B-9972 (D-Arg-[Hyp,Igl,Oic,Igl]-BK) were N-terminally extended with the infrared fluorophore Cy7, producing the peptides B-10665 and B-10666, respectively.

Lysosomotropic cationic drugs induce cytostatic and cytotoxic effects: Role of liposolubility and autophagic flux and antagonism by cholesterol ablation.

Cation trapping in acidic cell compartments determines an antiproliferative effect that has a potential interest in oncology, as shown by clinical data and trials involving chloroquine and hydroxychloroquine. To further characterize the mechanism of this effect, we studied a series of 6 substituted triethylamine (s-Et3N) drugs that encompasses a wide range of liposolubility (amiodarone, quinacrine, chloroquine, hydroxychloroquine, lidocaine, and procainamide). Three tumor cell lines and primary human endothelial cells were exploited in proliferation assays (48h, cell counts).

The isolated human umbilical vein as a bioassay for kinin-generating proteases: An in vitro model for therapeutic angioedema agents.

Aims: The isolated human umbilical vein is a robust contractile bioassay for ligands of the bradykinin (BK) B2 receptor (B2R), also extendable to B1 receptor (B1R) pharmacology. We hypothesized that, as a freshly isolated vessel, it also contains traces of plasma proteins that may confer responses to exogenous proteases via the formation of kinins.

Main Methods: Rings of human umbilical veins were mounted in organ baths containing Krebs buffer maintained at 37°C and purified proteases were introduced in the bathing fluid along with additional drugs/proteins that permit mechanistic analysis of effects.