Bioactivity of topologically confined gramicidin A dimers.

The d-/l-peptide gramicidin A (gA) is well known as a pivotal ion channel model and shows a broad spectrum of bioactivities such as antibiosis, antimalarial activity, as well as hemolysis. We applied inter-chain disulfide bonds to constrain the conformational freedom of gA into parallel and antiparallel dimeric topologies. Albeit the constructs were not found to be monoconformational, CD- and IR-spectroscopic studies suggested that this strategy indeed restricted the conformational space of the d-/l-peptide construct, and that ß-helical secondary structures prevail. Correlative testing of gA dimers in antimicrobial, antimalarial, and ion conduction assays suggested that the tail-to-tail antiparallel single stranded ß6.3 helix dominantly mediates the bioactivity of gA. Other conformers are unlikely to contribute to these activities. From these investigations, only weakly ion conducting gA dimers were identified that retained nM antimalarial activity.

Dynamic combinatorial enrichment of polyconformational D-/L-peptide dimers.

D-/L-peptides such as gramicidin A (gA) adopt unique dimeric ß-helical structures of different topologies. To overcome their conformational promiscuity and enrich individual components, a dynamic combinatorial approach assisted by thiol tags was developed. This method led to identification of the preferential formation of antiparallel dimers under a broad range of conditions, which was independent of peptide side-chain polarity. Exclusive formation of an antiparallel cyclic dimer was achieved in the presence of cesium ions.

Structure Elucidation and Activity of Kolossin†A, the D-/L-Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase.

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.

Iterative antimicrobial candidate selection from informed d-/l-Peptide dimer libraries.

Growing resistance to antibiotics, as well as newly emerging pathogens, stimulate the investigation of antimicrobial peptides (AMPs) as therapeutic agents. Here, we report a new library design concept based on a stochastic distribution of natural AMP amino acid sequences onto half-length synthetic peptides. For these compounds, a non-natural motif of alternating D- and L-backbone stereochemistry of the peptide chain predisposed for ß-helix formation was explored. Synthetic D-/L-peptides with permuted half-length sequences were delineated from a full-length starter sequence and covalently recombined to create two-dimensional compound arrays for antibacterial screening. Using the natural AMP magainin as a seed sequence, we identified and iteratively optimized hit compounds showing high antimicrobial activity against Gram-positive and Gram-negative bacteria with low hemolytic activity. Cryo-electron microscopy characterized the membrane-associated mechanism of action of the new D-/L-peptide antibiotics.

Activation of Mas and pGCA receptor pathways protects renal epithelial cell damage against oxidative-stress-induced injury.

Over-activation of the renin-angiotensin-aldosterone system (RAAS) is a leading cause of cardio-renal complications. Oxidative stress is one of the major contributing factors in the over-activation of RAAS. Angiotensin-converting enzyme2/Angiotensin1-7/MasR and natriuretic peptide/particulate guanylyl cyclase receptor-A pathways play a key role in cardiorenal disease protection. Even though individual activation of these pathways possesses cardiorenal protective effects. However, the dual activation of these pathways under stress conditions and the underlying mechanism has not been explored. The study aimed to investigate whether activation of these pathways by dual-acting peptide (DAP) shows a protective effect in-vitro in oxidative stress-induced renal epithelial cells. Oxidative stress was induced in renal epithelial NRK-52E cells with H2O2. Co-treatment with Ang 1-7, BNP, and DAP was given for 30 min. AT1, MasR, and pGCA expression were measured by RT-PCR. The markers of oxidative stress and apoptosis were measured by confocal microscopy and FACS analysis. A significant increase in AT1, renin, α-SMA, and NFk-ß expression and a significant decrease in MasR and pGCA expression was observed in H2O2-induced cells. DAP improved H2O2-induced pathological changes in NRK-52E cells. The effect of DAP was superior to that of Ang1-7 and BNP alone. Interestingly, MasR and pGCA inhibitors could block the effect of DAP in H2O2-induced cells. DAP shows superior anti-RAAS activity, and it is effective against H2O2-induced oxidative stress, apoptosis, fibrosis, and inflammation compared to Ang1-7 and BNP alone. The protective effect is mediated by the dual activation of MasR and pGCA.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

Anhydrous Hydrogen Fluoride Cleavage in Boc Solid Phase Peptide Synthesis.

Solid phase peptide synthesis using tert-butyloxycarbonyl/benzyl chemistry (Boc-SPPS) is important for producing peptides for fundamental research as well as for clinical use. During Boc-SPPS, liquid anhydrous hydrogen fluoride (HF) is used to remove the side chain protecting groups of the assembled peptide and to release it from the resin. Here, we provide a detailed protocol for "HF cleavage," aiming to improve accessibility and the use of this valuable and well-validated technique.

Up-regulation of PKR pathway contributes to L-NAME induced hypertension and renal damage.

OBJECTIVE: Hypertension induced kidney damage is often associated with fibrosis and tubular apoptosis. Double-stranded protein kinase (PKR) is a well recognized inducer of inflammation and apoptosis. However, role of PKR in hypertension coupled renal damage is still not explored. Therefore here we sought to investigate the role of PKR in the pathogenesis of L-NAME induced hypertension and renal damage in Wistar rats and the underneath molecular mechanism. METHODS: L-NAME (40 mg/kg, p.o) and imoxin (0.5 mg/kg, i.p) was given to Wistar rats for 4 weeks. Increased eNOS expression, serum creatinine, BUN and changes in mean arterial pressure confirmed for hypertensive renal damage. Western blot and immunohistochemistry was carried out for PKR and markers for fibrosis and apoptosis. Morphological alterations were assessed by H&E staining. Sirius red and Masson's Trichrome staining was performed for collagen and fibrosis. TUNEL assay was done for tubular cell death and apoptosis. RESULTS: Increased expression of PKR and its downstream markers were reported in L-NAME rats, attenuation was observed with imoxin treatment. L-NAME treated rats showed a significant increase in MAP, serum calcium, creatinine and BUN along with the significant morphological changes, attenuation was reported with the imoxin treatment. CONCLUSION: PKR is a core contributor in the pathogenesis of L-NAME induced renal damage and tubular apoptosis. Therapeutically targeting of PKR could be an attractive approach to treat the renal complications associated with hypertension.

PKR inhibitor imoxin prevents hypertension, endothelial dysfunction and cardiac and vascular remodelling in L-NAME-treated rats.

AIMS: Hypertension is one of the leading causes of cardiovascular mortality and morbidity. It is associated with severe cardiac and vascular dysfunction. Double-stranded RNA-dependent protein kinase (PKR), is a known inducer of inflammation and apoptosis. However, no research has been done to elucidate the role of the PKR in an experimental model of hypertension, and related cardiovascular complications. MAIN METHODS: L-NAME (NG-Nitro-L-arginine-methyl ester) was used to induce the hypertension. Imoxin treatment was given to Wistar rats for the four weeks along with the L-NAME, to investigate the influence on the hypertension. Changes in physiological parameter were assessed by recording non-invasive blood pressure. Expression of PKR and downstream markers for inflammation, fibrosis, and vascular damage in rat heart and aorta was determined by western blot and immunohistochemistry. Histological examination and fibrosis assessment were done by using assay kits. Vascular reactivity was determined by ex-vivo isometric tension studies on rat aortic rings. KEY FINDINGS: L-NAME-treated rats showed a significant increase in PKR expression followed by cardiac damage and vascular alterations compared to that of control animals. Results of western blot and immunohistochemistry indicate a significant increase in the inflammatory markers downstream to PKR. Endothelium-dependent vascular relaxation was significantly impaired in L-NAME administered rats. All effects of the L-NAME were attenuated by selective inhibition of PKR by imoxin. SIGNIFICANCE: Alterations in the heart and vasculature could be mediated in part by activation of the PKR pathway. Hence selective inhibition of PKR has therapeutic potential for combating hypertension and associated cardiovascular complications.