72 research outputs found

    Galanin 2 Receptor: A Novel Target for a Subset of Pancreatic Ductal Adenocarcinoma

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    Galanin is a 30 amino acid peptide that stimulates three subtype receptors (GAL1–3R). M89b is a lanthionine-stabilized, C-terminally truncated galanin analog that specifically stimulates GAL2R. We investigated the potential of M89b as a therapeutic for pancreatic ductal adenocarcinoma (PDAC) and assessed its safety. The anti-tumor activity of subcutaneously injected M89b on the growth of patient-derived xenografts of PDAC (PDAC–PDX) in mice was investigated. In addition, the safety of M89b was assessed in vitro using a multi-target panel to measure the off-target binding and modulation of enzyme activities. In a PDAC–PDX with a high GAL2R expression, M89b completely inhibited the growth of the tumor (p GAL2R expression, low or negligeable inhibition of tumor growth was measured, and in the PDX without GAL2R expression no influence on the tumor growth was observed. The M89b treatment of the GAL2R high-PDAC–PDX-bearing mice led to a reduction in the expression of RacGap1 (p PCNA (p MMP13 (p 2R is a safe and valuable target for treating PDACs with high GAL2R expression

    AT2 receptor agonist LP2 restores respiratory function in a rat model of bleomycin-induced lung remodelling

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    This study aimed to evaluate the prophylactic and therapeutic potential of angiotensin II type 2 receptor peptide agonist LP2 in bleomycin-induced airway and cardiac remodeling in rats. Male Wistar rats were intratracheally instillated with bleomycin. Animals of a prophylactic arm received LP2 from day 0 at intraperitoneal doses of 1, 3 or 10μg/kg/d, whereas animals from a therapeutic arm received this LP2 treatment from day 7. On day 28 direct lung mechanics were determined and cardiac and lung tissues were collected and (histo)morphologically assessed. Prophylactic LP2 at 1µg/kg/d with bleomycin, versus bleomycin alone, significantly improved the airway pressure responses at fixed inflation of 4ml (p&lt;0.05) and 7ml volume (p&lt;0.05), static compliance (p&lt;0.01), inspiratory capacity (p&lt;0.05), lung tolerance of increased volume (p&lt;0.0001), right to left ventricular hypertrophy (p&lt;0.05). Therapeutic regime showed a similar trend as the prophylactic arm but was less effective, mostly lacking significance. However, and importantly, therapeutic LP2 at 1µg/kg/d significantly decreased mRNA expression of collagen 1A1 (p&lt;0.01), of Connective Tissue Growth Factor 1 (p&lt;0.05) and of Tissue MetalloPeptidase inhibitor 1 (p&lt;0.05). In conclusion, a very low dose of 1µg/kg/d LP2 has capacity to counter bleomycin-induced impairment of lung functioning and consequent cardiac remodeling.</p

    LP2, the first lanthipeptide GPCR agonist in a human pharmacokinetics and safety study

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    Introduction of a lanthionine into a peptide may enhance target affinity, target specificity and proteolytic resistance. This manuscript reports preclinical safety studies and the first-in-human study with the lanthipeptide AT 2R agonist LP2, a structural analog of cAng-(1–7), whose N-terminus was protected against aminopeptidases by the presence of a D-lysine. None of the preclinical studies, including an in vitro multitarget panel, behavioral, respiratory and cardiovascular measurements, genotoxicity and toxicity studies in rat and dog, posed any safety concern. Due to lack of toxicity the maximum tolerated dose was not reached neither in rat nor in dog. In the human dose escalation study, healthy male volunteers received a single 1 mL subcutaneous injection (0.001 mg, 0.01 mg or 0.1 mg) of LP2 or matching placebo. In contrast to angiotensin II which has a T 1/2 in plasma of < 1 min, LP2 has a T 1/2 of approximately 2.1–2.6 hours. The fraction of the dose excreted unchanged in urine ranged from 84.73 ± 10.4 % at a dose of 0.001 mg to 66.4 ± 3.9 % at 0.1 mg. There were no deaths, serious adverse events or subject withdrawals as a result of an adverse event. The incidence of adverse events was 16.7 %; each was mild in severity. One adverse event, peripheral coldness, was considered to be possibly related to LP2 at 0.001 mg LP2. None of the results was considered to pose a clinically relevant safety concern. This study supports the potential for the therapeutic use of lanthipeptides

    Second asymptomatic carotid surgery trial (ACST-2) : a randomised comparison of carotid artery stenting versus carotid endarterectomy

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    Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86-1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91-1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable

    Intranasal Delivery of a Methyllanthionine-Stabilized Galanin Receptor-2-Selective Agonist Reduces Acute Food Intake

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    The regulatory (neuro)peptide galanin is widely distributed in the central and peripheral nervous systems, where it mediates its effects via three G protein-coupled receptors (GAL(1-3)R). Galanin has a vast diversity of biological functions, including modulation of feeding behavior. However, the clinical application of natural galanin is not practicable due to its rapid in vivo breakdown by peptidases and lack of receptor subtype specificity. Much effort has been put into the development of receptor-selective agonists and antagonists, and while receptor selectivity has been attained to some degree, most ligands show overlapping affinity. Therefore, we aimed to develop a novel ligand with specificity to a single galanin receptor subtype and increased stability. To achieve this, a lanthionine amino acid was enzymatically introduced into a galanin-related peptide. The residue’s subsequent cyclization created a conformational constraint which increased the peptide’s receptor specificity and proteolytic resistance. Further exchange of certain other amino acids resulted in a novel methyllanthionine-stabilized galanin receptor agonist, a G1pE-T3N-S6A-G12A-methyllanthionine[13–16]-galanin-(1–17) variant, termed M89b. M89b has exclusive specificity for GAL(2)R and a prolonged half-life in serum. Intranasal application of M89b to unfasted rats significantly reduced acute 24 h food intake inducing a drop in body weight. Combined administration of M89b and M871, a selective GAL(2)R antagonist, abolished the anorexigenic effect of M89b, indicating that the effect of M89b on food intake is indeed mediated by GAL(2)R. This is the first demonstration of in vivo activity of an intranasally administered lanthipeptide. Consequently, M89b is a promising candidate for clinical application as a galanin-related peptide-based therapeutic. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01155-x

    Does activation of the protective Renin-Angiotensin System have therapeutic potential in COVID-19?

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    Infection of lung cells by the corona virus results in a loss of the balance between, on the one hand, angiotensin II-mediated stimulation of the angiotensin II type 1 receptor and, on the other hand, stimulation of the angiotensin II type 2 receptor and/or the Mas receptor. The unbalanced enhanced stimulation of the angiotensin II type 1 receptor causes inflammation, edema and contributes to the pathogenesis of severe acute respiratory distress syndrome. Here we hypothesize that stable, receptor-specific agonists of the angiotensin II type 2 receptor and of the Mas receptor are molecular medicines to treat COVID-19 patients. These agonists have therapeutic potential in the acute disease but in addition may reduce COVID-19-associated long-term pulmonary dysfunction and overall end-organ damage of this disease

    An Engineered Double Lipid II Binding Motifs-Containing Lantibiotic Displays Potent and Selective Antimicrobial Activity against Enterococcus faecium

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    Lipid II is an essential precursor of the bacterial cell wall biosynthesis and thereby an important target for various antibiotics. Several lanthionine-containing peptide antibiotics target lipid II with lanthionine-stabilized lipid II-binding motifs. Here, we used the biosynthesis system of the lantibiotic nisin to synthesize a two lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal lipid II binding motif of nisin and the distinct C-terminal lipid II binding motif of one peptide of the two-component haloduracin (i.e. HalA1). Further characterization demonstrated that (i) TL19 exerts 64-fold stronger antimicrobial activity against E. faecium than nisin (1-22), which has only one lipid II binding site, and (ii) both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19, as evidenced by mutagenesis of each single and double domains. These results show the feasibility of a new approach to synthesize potent lantibiotics with two different lipid II binding motifs to treat specific antibiotic-resistant pathogens

    Biosynthesis of lanthionine-constrained agonists of G protein-coupled receptors

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    The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as hydrogen bridges or is induced by ligand-receptor interactions. The conformational freedom of a peptide can be constrained by intramolecular cross-links. Conformational constraints enhance the receptor specificity, may lead to biased activity and confer proteolytic resistance to peptidic GPCR agonists. Chemical synthesis allows to introduce a variety of cross-links into a peptide and is suitable for bulk production of relatively simple lead peptides. Lanthionines are thioether bridged alanines of which the two alanines can be introduced at different distances in chosen positions in a peptide. Thioether bridges are much more stable than disulfide bridges. Biosynthesis of lanthionine-constrained peptides exploiting engineered Gram-positive or Gram-negative bacteria that contain lanthionine-introducing enzymes constitutes a convenient method for discovery of lanthionine-stabilized GPCR agonists. The presence of an N-terminal leader peptide enables dehydratases to dehydrate serines and threonines in the peptide of interest after which a cyclase can couple the formed dehydroamino acids to cysteines forming (methyl)lanthionines. The leader peptide also guides the export of the formed lanthionine-containing precursor peptide out of Gram-positive bacteria via a lanthipeptide transporter. An engineered cleavage site in the C-terminus of the leader peptide allows to cleave off the leader peptide yielding the modified peptide of interest. Lanthipeptide GPCR agonists are an emerging class of therapeutics of which a few examples have demonstrated high efficacy in animal models of a variety of diseases. One lanthipeptide GPCR agonist has successfully passed clinical Phase Ia

    Cyclic angiotensin-(1-7) contributes to rehabilitation of animal performance in a rat model of cerebral stroke

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    Peptidase-resistant, lanthionine-stabilized angiotensin-(1-7), termed cAng-(1-7), has shown therapeutic efficacy in animal models of cardiovascular, metabolic, kidney and pulmonary disease. Goal of the present study was testing the capacity of subcutaneously administered cAng-(1-7) to induce rehabilitation of animal performance in the transient middle cerebral artery occlusion rat model of cerebral stroke. 24 h after ischemic stroke induction, cAng-(1-7) was administered for 28 days at a dose of 500 μg/kg/day, either daily via subcutaneous injection or continuously via an alzet pump. Both ways of administration of cAng-(1-7) were equally effective. Measurements were continued until day 50. Compared to vehicle, cAng-(1-7) clearly demonstrated significantly increased capillary density (p < 0.01) in the affected hemisphere and improved motor and somatosensory functioning. The modified neurological severity score (p < 0.001 at days 15 and 50), stepping test (p < 0.001 at days 36–50), forelimb placement test (p < 0.001 at day 50), body swing test (p < 0.001 at days 43 and 50) all demonstrated that cAng-(1-7) caused significantly improved animal performance. Taken together the data convincingly indicate rehabilitating capacity of subcutaneously injected cAng-(1-7) in cerebral ischemic stroke

    High-throughput screening for substrate specificity-adapted mutants of the nisin dehydratase NisB

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    Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially displayed lanthionine-constrained streptavidin ligands, which have a much higher affinity for streptavidin than linear ligands. The designed NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration but less effectively via wild-type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisB-containing bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity
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