Agonists of galanin subtype 2 receptor may prevent pancreatic cancer and agonists of angiotensin II type 2 receptor may prevent colorectal cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a dreadful disease with poor prognosis. While the prognosis of colorectal carcinoma (CRC) is better than that of PDAC, it still is the second-leading cause of cancer deaths worldwide. Recently, a (methyl)lanthionine-stabilized, highly receptor-specific agonist of galanin subtype 2 (GAL2) receptor inhibited the growth of GAL2 receptor-expressing patient-derived xenografts (PDX) of pancreatic cancer.

Evolving views on the first two ligands of the angiotensin II type 2 receptor. From putative antagonists to potential agonists?

The renin-angiotensin system is one of the most complex regulatory systems that controls multiple organ functions. One of its key components, angiotensin II (Ang II), stimulates two G-protein coupled class A receptors: the Ang II type 1 (AT1) receptor and the Ang II type 2 (AT2) receptor. While stimulation of the AT1 receptor causes G-protein-dependent signaling and arrestin recruitment, the AT2 receptor seems to have a constitutively active-like conformation and appears to act via G-protein-dependent and -independent pathways.

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

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 10 μg/kg/d, whereas animals from a therapeutic arm received this LP2 treatment from day 7.

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

Galanin is a 30 amino acid peptide that stimulates three subtype receptors (GALR). M89b is a lanthionine-stabilized, C-terminally truncated galanin analog that specifically stimulates GALR. We investigated the potential of M89b as a therapeutic for pancreatic ductal adenocarcinoma (PDAC) and assessed its safety.

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

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 (GALR). 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.

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

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 ATR 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.

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

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.

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

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.

High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB.

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.

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

Lipid II is an essential precursor for 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.

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

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.

Genetically Encoded Libraries of Constrained Peptides.

Many therapeutic peptides can still be improved with respect to target specificity, target affinity, resistance to peptidases/proteases, physical stability, and capacity to pass through membranes required for oral delivery. Several modifications can improve the peptides' properties, in particular those that impose (a) conformational constraint(s). Screening of constrained peptides and the identification of hits is greatly facilitated by the generation of genetically encoded libraries.

Efficacy of lanthionine-stabilized angiotensin-(1-7) in type I and type II diabetes mouse models.

Native angiotensin-(1-7) exerts many therapeutic effects. However, it is rapidly degraded by ACE and other peptidases. This drawback is largely eliminated for lanthionine-stabilized angiotensin-(1-7), termed cAng-(1-7), which is fully resistant to ACE and has strongly increased resistance to other peptidases.

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display.

Semi-microbiological synthesis of an active lysinoalanine-bridged analog of glucagon-like-peptide-1.

Some modified glucagon-like-peptide-1 (GLP-1) analogs are highly important for treating type 2 diabetes. Here we investigated whether GLP-1 analogs expressed in Lactococcus lactis could be substrates for modification and export by the nisin dehydratase and transporter enzyme. Subsequently we introduced a lysinoalanine by coupling a formed dehydroalanine with a lysine and investigated the structure and activity of the formed lysinoalanine-bridged GLP-1 analog.

Mutagenesis of nisin's leader peptide proline strongly modulates export of precursor nisin.

The lantibiotic nisin is produced by Lactococcus lactis as a precursor peptide comprising a 23 amino acid leader peptide and a 34 amino acid post-translationally modifiable core peptide. We previously demonstrated that the conserved FNLD part of the leader is essential for intracellular enzyme-catalyzed introduction of lanthionines in the core peptide and also for transporter-mediated export, whereas other positions are subject to large mutational freedom. We here demonstrate that, in the absence of the extracellular leader peptidase, NisP, export of precursor nisin via the modification and transporter enzymes, NisBTC, is strongly affected by multiple substitutions of the leader residue at position -2, but not by substitution of positions in the vicinity of this site.

Discovery, Production and Modification of Five Novel Lantibiotics Using the Promiscuous Nisin Modification Machinery.

To find the right conditions to isolate natively expressed antimicrobial peptides from a wide range of different microorganisms can be a challenge. Here, we exploited a heterologous expression system to produce and characterize several novel lantibiotics. We identified 54 novel putative class I and class II lantibiotics after inspecting all publicly available prokaryotic genomes using the in-house developed mining tool BAGEL3.

Identification of essential amino acid residues in the nisin dehydratase NisB.

Nisin is a posttranslationally-modified antimicrobial peptide that has the ability to induce its own biosynthesis. Serines and threonines in the modifiable core peptide part of precursor nisin are dehydrated to dehydroalanines and dehydrobutyrines by the dehydratase NisB, and subsequently cysteines are coupled to the dehydroamino acids by the cyclase NisC. In this study, we applied extensive site-directed mutagenesis, together with direct binding studies, to investigate the molecular mechanism of the dehydratase NisB.

Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC.

Nisin is the most prominent and applied bacteriocin that serves as a model for class I lantibiotics. The nisin leader peptide importantly determines interactions between precursor nisin and its modification enzymes NisB and NisC that mature nisin posttranslationally. NisB dehydrates serines and threonines, while NisC catalyzes the subsequent coupling of the formed dehydroamino acids to form lanthionines.

Mechanistic aspects of lanthipeptide leaders.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides produced by microorganisms. The name lanthipeptide is derived from lanthionine, a thioether-bridged amino acid installed by dedicated modification enzymes. Serines and threonines are dehydrated and subsequently coupled to cysteines, thus forming intramolecular lanthionine rings.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Determining sites of interaction between prenisin and its modification enzymes NisB and NisC.

Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L.

Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7) or an angiotensin II receptor antagonist.

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome. Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin (Ang) II have been implicated in the pathogenesis of ARDS. A counter-regulatory enzyme of ACE, ie ACE2 that degrades Ang II to Ang-(1-7), offers a promising novel treatment modality for this syndrome.

Bacterial display and screening of posttranslationally thioether-stabilized peptides.

A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable.

Substrate recognition and specificity of the NisB protein, the lantibiotic dehydratase involved in nisin biosynthesis.

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification.