YbiB: a novel interactor of the GTPase ObgE.

Obg is a widely conserved and essential GTPase in bacteria, which plays a central role in a large range of important cellular processes, such as ribosome biogenesis, DNA replication, cell division and bacterial persistence. Nevertheless, the exact function of Obg in these processes and the interactions it makes within the associated pathways remain largely unknown. Here, we identify the DNA-binding TrpD2 protein YbiB as an interactor of the Escherichia coli Obg (ObgE). We show that both proteins interact with high affinity in a peculiar biphasic fashion, and pinpoint the intrinsically disordered and highly negatively charged C-terminal domain of ObgE as a main driver for this interaction. Molecular docking and X-ray crystallography, together with site-directed mutagenesis, are used to map the binding site of this ObgE C-terminal domain within a highly positively charged groove on the surface of the YbiB homodimer. Correspondingly, ObgE efficiently inhibits the binding of DNA to YbiB, indicating that ObgE competes with DNA for binding in the positive clefts of YbiB. This study thus forms an important step for the further elucidation of the interactome and cellular role of the essential bacterial protein Obg.

Fast Self-Assembly Dynamics of a ß-Sheet Peptide Soft Material.

Peptide-based hydrogels are promising biocompatible materials for wound healing, drug delivery, and tissue engineering applications. The physical properties of these nanostructured materials depend strongly on the morphology of the gel network. However, the self-assembly mechanism of the peptides that leads to a distinct network morphology is still a subject of ongoing debate, since complete assembly pathways have not yet been resolved. To unravel the dynamics of the hierarchical self-assembly process of the model ß-sheet forming peptide KFE8 (Ac-FKFEFKFE-NH2 ), high-speed atomic force microscopy (HS-AFM) in liquid is used. It is demonstrated that a fast-growing network, based on small fibrillar aggregates, is formed at a solid-liquid interface, while in bulk solution, a distinct, more prolonged nanotube network emerges from intermediate helical ribbons. Moreover, the transformation between these morphologies has been visualized. It is expected that this new in situ and in real-time methodology will set the path for the in-depth unravelling of the dynamics of other peptide-based self-assembled soft materials, as well as gaining advanced insights into the formation of fibers involved in protein misfolding diseases.

Nature-Inspired Dual Purpose Strategy toward Cell-Adhesive PCL Networks: C(-linker-)RGD Incorporation via Thiol-ene Crosslinking.

In an attempt to mimic nature's ability to adhere cells, PCL is often coated with nature-derived polymers or its surface is functionalized with a cell-binding motif. However, said surface modifications are limited to the material's surface, include multiple steps, and are mediated by harsh conditions. Here, we introduce a single-step strategy toward cell-adhesive polymer networks where thiol-ene chemistry serves a dual purpose. First, alkene-functionalized PCL is crosslinked by means of a multifunctional thiol. Second, by means of a cysteine coupling site, the cell-binding motif C(-linker-)RGD is covalently bound throughout the PCL networks during crosslinking. Moreover, the influence of various linkers (type and length), between the cysteine coupling site and the cell-binding motif RGD, is investigated and the functionalization is assessed by means of static contact angle measurements and X-ray photoelectron spectroscopy. Finally, successful introduction of cell adhesiveness is illustrated for the networks by seeding fibroblasts onto the functionalized PCL networks.

Second generation Spautin-1 analogues targeting EGFR-mutant non-small cell lung cancer cells.

Treatment of advanced stage epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) is often complicated by the occurrence of acquired resistance, which emphasizes the need for improved treatment options. Based on a previously reported structure-activity relationship (SAR) study of Spautin-1, which resulted in the discovery of 10a, the search for more potent analogues was envisaged through optimization of the amine substituent. Our search led to the discovery of analogue 15b, harbouring the 2-[4-(4-fluoro-phenoxy)-phenyl]ethylamine substituent, among other potent and original analogues, with nanomolar activity towards EGFR-mutant NSCLC cells. Moreover, this compound 15b showed good selectivity for cancer cells over healthy lung epithelial cells and provides additive effects with food and drug administration (FDA) approved EGFR-tyrosine kinase inhibitors (TKIs), as proven by the co-administration of 15b with Afatinib. Altogether, we report promising lead compounds which show the potential to improve current treatment options.

Opening the amino acid toolbox for peptide-based NTS2-selective ligands as promising lead compounds for pain management.

Chronic pain is one of the most critical health issues worldwide. Despite considerable efforts to find therapeutic alternatives, opioid drugs remain the gold standard for pain management. The administration of µ-opioid receptor (MOR) agonists is associated with detrimental and limiting adverse effects. Overall, these adverse effects strongly overshadow the effectiveness of opioid therapy. In this context, the development of neurotensin (NT) ligands has shown to be a promising approach for the management of chronic and acute pain. NT exerts its opioid-independent analgesic effects through the binding of two G protein-coupled receptors (GPCRs), NTS1 and NTS2. In the last decades, modified NT analogues have been proven to provide potent analgesia in vivo. However, selective NTS1 and nonselective NTS1/NTS2 ligands cause antinociception associated with hypothermia and hypotension, whereas selective NTS2 ligands induce analgesia without altering the body temperature and blood pressure. In light of this, various structure-activity relationship (SAR) studies provided findings addressing the binding affinity of ligands towards NTS2. Herein, we comprehensively review peptide-based NTS2-selective ligands as a robust alternative for future pain management. Particular emphasis is placed on SAR studies governing the desired selectivity and associated in vivo results.

Introduction of constrained Trp analogs in RW9 modulates structure and partition in membrane models.

Over the past decades, many cell-penetrating peptides (CPP) have been studied for their capacity to cross cellular membranes, mostly in order to improve cellular uptake of therapeutic agents. Even though hydrophobic and anionic CPPs have been described, many of them are polycationic, due to the presence of several arginine (Arg) residues. Noteworthy, however, the presence of aromatic amino acids such as tryptophan (Trp) within CPPs seems to play an important role to reach high membranotropic activity. RW9 (RRWWRRWRR) is a designed CPP derived from the polyarginine R9 presenting both features. In general, when interacting with membranes, CPPs adopt an optimal conformation for membrane interactions - an amphipathic helical secondary structure in the case of RW9. Herein, we assumed that the incorporation of a locally constrained amino acid in the peptide sequence could improve the membranotropic activity of RW9, by facilitating its structuration upon contact with a membrane, while leaving a certain plasticity. Therefore, two cyclized Trp derivatives (Tcc and Aia) were synthesized to be incorporated in RW9 as surrogates of Trp residues. Thus, a series of peptides containing these building blocks has been synthesized by varying the type, position, and number of modifications. The membranotropic activity of the RW9 analogs was studied by spectrofluorescence titration of the peptides in presence of liposomes (DMPG), allowing to calculate partition coefficients (Kp). Our results indicate that the partitioning of the modified peptides depends on the type, the number and the position of the modification, with the best sequence being [Aia4]RW9. Interestingly, both NMR analysis and molecular dynamic (MD) simulations indicate that this analog presents an extended conformation similar to the native RW9, but with a much-reduced structural flexibility. Finally, cell internalization properties were also confirmed by confocal microscopy.

Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide 10Panx1.

Pannexin1 channels facilitate paracrine communication and are involved in a broad spectrum of diseases. Attempts to find appropriate pannexin1 channel inhibitors that showcase target-selective properties and in vivo applicability remain nonetheless scarce. However, a promising lead candidate, the ten amino acid long peptide mimetic 10Panx1 (H-Trp1-Arg2-Gln3-Ala4-Ala5-Phe6-Val7-Asp8-Ser9-Tyr10-OH), has shown potential as a pannexin1 channel inhibitor in both in vitro and in vivo studies. Nonetheless, structural optimization is critical for clinical use. One of the main hurdles to overcome along the optimization process consists of subduing the low biological stability (10Panx1 t1/2 = 2.27 ± 0.11 min). To tackle this issue, identification of important structural features within the decapeptide structure is warranted. For this reason, a structure-activity relationship study was performed to proteolytically stabilize the sequence. Through an Alanine scan, this study demonstrated that the side chains of Gln3 and Asp8 are crucial for 10Panx1's channel inhibitory capacity. Guided by plasma stability experiments, scissile amide bonds were identified and stabilized, while extracellular adenosine triphosphate release experiments, indicative of pannexin1 channel functionality, allowed to enhance the in vitro inhibitory capacity of 10Panx1.

Nanobody Loop Mimetics Enhance Son of Sevenless 1-Catalyzed Nucleotide Exchange on RAS.

RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active guanosine triphosphate (GTP)-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14 (Nb14), which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of Nb14 with an EC50 value of 29 µM. Altogether, this study demonstrated that peptides able to modulate a protein-protein interaction can be obtained by structural mimicry of a Nb paratope.

Genome-Based Characterization of a Plasmid-Associated Micrococcin P1 Biosynthetic Gene Cluster and Virulence Factors in Mammaliicoccus sciuri IMDO-S72.

Analysis of the de novo assembled genome of Mammaliicoccus sciuri IMDO-S72 revealed the genetically encoded machinery behind its earlier reported antibacterial phenotype and gave further insight into the repertoire of putative virulence factors of this recently reclassified species. A plasmid-encoded biosynthetic gene cluster was held responsible for the antimicrobial activity of M. sciuri IMDO-S72, comprising genes involved in thiopeptide production. The compound encoded by this gene cluster was structurally identified as micrococcin P1. Further examination of its genome highlighted the ubiquitous presence of innate virulence factors mainly involved in surface colonization. Determinants contributing to aggressive virulence were generally absent, with the exception of a plasmid-associated ica cluster. The native antibiotic resistance genes sal(A) and mecA were detected within the genome, among others, but were not consistently linked with a resistance phenotype. While mobile genetic elements were identified within the genome, such as an untypeable staphylococcal cassette chromosome (SCC) element, they proved to be generally free of virulence- and antibiotic-related genes. These results further suggest a commensal lifestyle of M. sciuri and indicate the association of antibiotic resistance determinants with mobile genetic elements as an important factor in conferring antibiotic resistance, in addition to their unilateral annotation. IMPORTANCEMammaliicoccus sciuri has been put forward as an important carrier of virulence and antibiotic resistance genes, which can be transmitted to clinically important staphylococcal species such as Staphylococcus aureus. As a common inhabitant of mammal skin, this species is believed to have a predominant commensal lifestyle, although it has been reported as an opportunistic pathogen in some cases. This study provides an extensive genome-wide description of its putative virulence potential taking into consideration the genomic context in which these genes appear, an aspect that is often overlooked during virulence analysis. Additional genome and biochemical analysis linked M. sciuri with the production of micrococcin P1, gaining further insight into the extent to which these biosynthetic gene clusters are distributed among different related species. The frequent plasmid-associated character hints that these traits can be horizontally transferred and might confer a competitive advantage to its recipient within its ecological niche.

Downsizing antibodies: Towards complementarity-determining region (CDR)-based peptide mimetics.

Monoclonal antibodies emerged as an important therapeutic drug class with remarkable specificity and binding affinity. Nonetheless, these heterotetrameric immunoglobulin proteins come with high manufacturing and therapeutic costs which can take extraordinary proportions, besides other limitations such as their limited in cellulo access imposed by their molecular size (ca. 150 kDa). These drawbacks stimulated the development of downsized functional antibody fragments (ca. 15-50 kDa), together with smaller synthetic peptides (ca. 1-3 kDa) derived from the antibodies' crucial complementarity-determining regions (CDR). Despite the general lack of success in the literal translation of CDR loops in peptide mimetics, rational structure-based and computational approaches have shown their potential for obtaining functional CDR-based peptide mimetics. In this review, we describe the efforts made in the development of antibody and nanobody paratope-derived peptide mimetics with particular focus on the used design strategies, in addition to highlighting the challenges associated with their development.

Effects of Drugs Formerly Suggested for COVID-19 Repurposing on Pannexin1 Channels.

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.

Bifunctional Peptidomimetic G Protein-Biased Mu-Opioid Receptor Agonist and Neuropeptide FF Receptor Antagonist KGFF09 Shows Efficacy in Visceral Pain without Rewarding Effects after Subcutaneous Administration in Mice.

There is still an unmet clinical need to develop new pharmaceuticals for effective and safe pain management. Current pharmacotherapy offers unsatisfactory solutions due to serious side effects related to the chronic use of opioid drugs. Prescription opioids produce analgesia through activation of the mu-opioid receptor (MOR) and are major contributors to the current opioid crisis. Multifunctional ligands possessing activity at more than one receptor represent a prominent therapeutic approach for the treatment of pain with fewer adverse effects. We recently reported on the design of a bifunctional MOR agonist/neuropeptide FF receptor (NPFFR) antagonist peptididomimetic, KGFF09 (H-Dmt-DArg-Aba-ßAla-Bpa-Phe-NH2), and its antinociceptive effects after subcutaneous (s.c.) administration in acute and persistent pain in mice with reduced propensity for unwanted side effects. In this study, we further investigated the antinociceptive properties of KGFF09 in a mouse model of visceral pain after s.c. administration and the potential for opioid-related liabilities of rewarding and sedation/locomotor dysfunction following chronic treatment. KGFF09 produced a significant dose-dependent inhibition of the writhing behavior in the acetic acid-induced writhing assay with increased potency when compared to morphine. We also demonstrated the absence of harmful effects caused by typical MOR agonists, i.e., rewarding effects (conditioned-place preference test) and sedation/locomotor impairment (open-field test), at a dose shown to be highly effective in inhibiting pain behavior. Consequently, KGFF09 displayed a favorable benefit/side effect ratio regarding these opioid-related side effects compared to conventional opioid analgesics, such as morphine, underlining the development of dual MOR agonists/NPFFR antagonists as improved treatments for various pain conditions.

Using conformational constraints at position 6 of Angiotensin II to generate compounds with enhanced AT2R selectivity and proteolytic stability.

The Renin-Angiotensin System (RAS) plays a crucial role in numerous pathological conditions. Two of the critical RAS players, the angiotensin receptors AT1R and AT2R, possess differential functional profiles, although they share high sequence similarity. Although the main focus has been placed on AT1R, several epidemiological studies have evidenced that activation of AT2R could operate as a multimodal therapeutic target for different diseases. Thus, the development of selective AT2R ligands could have a high clinical potential for different therapeutic directions. Furthermore, they could serve as a powerful tool to interrogate the molecular mechanisms that are mediated by AT2R. Based on our recently established high affinity and AT2R selective compound [Y]6-AII we developed several analogues through modifying aminoacids located at positions 6 and 7 with various conformationally constrained analogues to enhance both the selectivity and stability. We report the development of high-affinity AT2R binders, which displayed high selectivity for AT2R versus AT1R. Furthermore, all analogues presented enhanced stability in human plasma with respect to the parent hormone Angiotensin II as also [Y]6-AII.

Structure-Activity Relationship (SAR) Study of Spautin-1 to Entail the Discovery of Novel NEK4 Inhibitors.

Lung cancer is one of the most frequently diagnosed cancers accounting for the highest number of cancer-related deaths in the world. Despite significant progress including targeted therapies and immunotherapy, the treatment of advanced lung cancer remains challenging. Targeted therapies are highly efficacious at prolonging life, but not curative. In prior work we have identified Ubiquitin Specific Protease 13 (USP13) as a potential target to significantly enhance the efficacy of mutant EGFR inhibition. The current study aimed to develop lead molecules for the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) by developing potent USP13 inhibitors initially starting from Spautin-1, the only available USP13 inhibitor. A SAR study was performed which revealed that increasing the chain length between the secondary amine and phenyl group and introducing a halogen capable of inducing a halogen bond at position 4' of the phenyl group, dramatically increased the activity. However, we could not confirm the binding between Spautin-1 (or its analogues) and USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but do not exclude binding under physiological conditions. Nevertheless, we found that the anti-proliferative activity displayed by Spautin-1 towards EGFR-mutant NSCLC cells in vitro was at least partially associated with kinase inhibition. In this work, we present N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. These analogues are significantly more effective towards EGFR-mutant NSCLC cells than Spautin-1 and act as potent never in mitosis A related kinase 4 (NEK4) inhibitors (IC50~1 µM) with moderate selectivity over other kinases.

Antinociceptive Efficacy of the µ-Opioid/Nociceptin Peptide-Based Hybrid KGNOP1 in Inflammatory Pain without Rewarding Effects in Mice: An Experimental Assessment and Molecular Docking.

Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund's adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional µ/δ-Opioid Agonist-Neurokinin Antagonist Peptidomimetics.

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic µ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual µ/δ opioid agonist H-Dmt-d-Arg-Aba-ßAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range µ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

Development of Generic G Protein Peptidomimetics Able to Stabilize Active State Gs Protein-Coupled Receptors for Application in Drug Discovery.

G protein-coupled receptors (GPCRs) represent an important group of membrane proteins that play a central role in modern medicine. Unfortunately, conformational promiscuity hampers full therapeutic exploitation of GPCRs, since the largest population of the receptor will adopt a basal conformation, which subsequently challenges screens for agonist drug discovery programs. Herein, we describe a set of peptidomimetics able to mimic the ability of G proteins in stabilizing the active state of the ß2 adrenergic receptor (ß2 AR) and the dopamine 1 receptor (D1R). During fragment-based screening efforts, these (un)constrained peptide analogues of the α5 helix in Gs proteins, were able to identify agonism pre-imprinted fragments for the examined GPCRs, and as such, they behave as a generic tool, enabling an engagement in agonist earmarked discovery programs.

Connexin and Pannexin (Hemi)Channels: Emerging Targets in the Treatment of Liver Disease.

Connexin proteins are the building blocks of hemichannels, which dock further between adjacent cells to form gap junctions. Gap junctions control the intercellular exchange of critical homeostasis regulators. By doing so, gap junctions control virtually all aspects of the hepatic life cycle. In the last decade, it has become clear that connexin hemichannels also provide a pathway for cellular communication on their own independent of their role as structural precursors of gap junctions, namely between the cytosol of an individual cell and its extracellular environment. In contrast to gap junctions, connexin hemichannels become particularly active in liver disease by facilitating inflammation and cell death. This equally holds true for cellular channels composed of pannexins, being connexin-like proteins recently identified in the liver that gather in structures reminiscent of hemichannels. This paper gives an overview of the involvement of connexin-based and pannexin-based channels in noncancerous liver disease.

Effects of neuromedin U-8 on stress responsiveness and hypothalamus-pituitary-adrenal axis activity in male C57BL/6J mice.

Neuromedin U (NMU) is a highly conserved neuropeptide that has been implicated in the stress response. To better understand how it influences various aspects of the stress response, we studied the effects of intracerebroventricular NMU-8 administration on stress-related behavior and activity of the hypothalamus-pituitary-adrenal (HPA) axis in male C57BL/6J mice. We investigated these NMU-8 effects when mice remained in their home cage and when they were challenged by exposure to forced swim stress. NMU-8 administration resulted in increased grooming behavior in mice that remained in their home cage and in a significant increase in c-Fos immunoreactivity in the paraventricular hypothalamus (PVH) and arcuate nucleus (ARC). Surprisingly, NMU-8 administration significantly decreased plasma corticosterone concentrations. Furthermore, NMU-8 administration increased immobility in the forced swim test in both naïve mice and mice that were previously exposed to swim stress. The effect of NMU-8 on c-Fos immunoreactivity in the PVH was dependent on previous exposure to swim stress given that we observed no significant changes in mice exposed for the first time to swim stress. In contrast, in the ARC we observed a significant increase in c-Fos immunoreactivity regardless of previous stress exposure. Interestingly, NMU-8 administration also significantly decreased plasma corticosterone concentrations in mice that were exposed to single forced swim stress, while this effect was no longer observed when mice were exposed to forced swim stress for a second time. Taken together, our data indicate that NMU-8 regulates stress responsiveness and suggests that its effects depend on previous stress exposure.

Mechanistic Understanding of Peptide Analogues, DALDA, [Dmt1]DALDA, and KGOP01, Binding to the mu Opioid Receptor.

The mu opioid receptor (MOR) is the primary target for analgesia of endogenous opioid peptides, alkaloids, synthetic small molecules with diverse scaffolds, and peptidomimetics. Peptide-based opioids are viewed as potential analgesics with reduced side effects and have received constant scientific interest over the years. This study focuses on three potent peptide and peptidomimetic MOR agonists, DALDA, [Dmt1]DALDA, and KGOP01, and the prototypical peptide MOR agonist DAMGO. We present the first molecular modeling study and structure-activity relationships aided by in vitro assays and molecular docking of the opioid peptide analogues, in order to gain insight into their mode of binding to the MOR. In vitro binding and functional assays revealed the same rank order with KGOP01 > [Dmt1]DALDA > DAMGO > DALDA for both binding and MOR activation. Using molecular docking at the MOR and three-dimensional interaction pattern analysis, we have rationalized the experimental outcomes and highlighted key amino acid residues responsible for agonist binding to the MOR. The Dmt (2',6'-dimethyl-L-Tyr) moiety of [Dmt1]DALDA and KGOP01 was found to represent the driving force for their high potency and agonist activity at the MOR. These findings contribute to a deeper understanding of MOR function and flexible peptide ligand-MOR interactions, that are of significant relevance for the future design of opioid peptide-based analgesics.