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.

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.

Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds.

Non-cationic and amphipathic indoloazepinone-constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l-Aia-Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc-[l-Aia-Gly]2,4 -OBn oligomers 12 and 13 and Boc-[l-Aia-ß3 -h-l-Ala]2,4 -OBn oligomers 16 and 17 were totally or partially disordered, Boc-[l-Aia-l-Ala]2 -OBn (14) induced a typical turn stabilized by C5 - and C7 -membered H-bond pseudo-cycles and aromatic interactions. Boc-[l-Aia-l-Ala]4 -OBn (15) exhibited a unique structure with remarkable T-shaped π-stacking interactions involving the indole rings of the four l-Aia residues forming a dense hydrophobic cluster. All of the proposed FITC-6-Ahx-[l-Aia-Xxx]4 -NH2 oligomers 19-23, with the exception of FITC-6-Ahx-[l-Aia-Gly]4 -NH2 (18), were internalized by MDA-MB-231 cells with higher efficiency than the positive references penetratin and Arg8 . In parallel, the compounds of this series were successfully explored in an in vitro blood-brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac-[l-Aia-Xxx]4 -NH2 oligomers in the PAMPA model, Ac-[l-Aia-l-Arg]4 -NH2 (26) showed significant permeation in the in vitro cell-based human model of the BBB, suggesting an active mechanism of cell penetration.

Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

Stabilisation of a short α-helical VIP fragment by side chain to side chain cyclisation: a comparison of common cyclisation motifs by circular dichroism.

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α-helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross-link side chain residues in a 'helical' fragment.

Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes.

Both, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Novel DOTA-neurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors.

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.

Conformational constraints in angiotensin IV to probe the role of Tyr², Pro5 and Phe6.

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of ß-Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr¹, while only e-ß-MePhe6 substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP-N or the AT1 receptor. This indicates an important role of the orientation of the Phe6 for inducing selectivity. Pro5 replacement with 2-aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT1 affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides.

Comprehensive overview of biased pharmacology at the opioid receptors: biased ligands and bias factors.

One of the main challenges in contemporary medicinal chemistry is the development of safer analgesics, used in the treatment of pain. Currently, moderate to severe pain is still treated with the "gold standard" opioids whose long-term often leads to severe side effects. With the discovery of biased agonism, the importance of this area of pharmacology has grown exponentially over the past decade. Of these side effects, tolerance, opioid misuse, physical dependence and substance use disorder (SUD) stand out, since these have led to many deaths over the past decades in both USA and Europe. New therapeutic molecules that induce a biased response at the opioid receptors (MOR, DOR, KOR and NOP receptor) are able to circumvent these side effects and, consequently, serve as more advantageous therapies with great promise. The concept of biased signaling extends far beyond the already sizeable field of GPCR pharmacology and covering everything would be vastly outside the scope of this review which consequently covers the biased ligands acting at the opioid family of receptors. The limitation of quantifying bias, however, makes this a controversial subject, where it is dependent on the reference ligand, the equation or the assay used for the quantification. Hence, the major issue in the field of biased ligands remains the translation of the in vitro profiles of biased signaling, with corresponding bias factors to in vivo profiles showing the presence or the lack of specific side effects. This review comprises a comprehensive overview of biased ligands in addition to their bias factors at individual members of the opioid family of receptors, as well as bifunctional ligands.

Structure-Based Design of Melanocortin 4 Receptor Ligands Based on the SHU-9119-hMC4R Cocrystal Structure†.

The melanocortin receptors (MC1R-MC5R) belong to class A G-protein-coupled receptors (GPCRs) and are known to have receptor-specific roles in normal and diseased states. Selectivity for MC4R is of particular interest due to its involvement in various metabolic disorders, including obesity, feeding regulation, and sexual dysfunctions. To further improve the potency and selectivity of MC4R (ant)agonist peptide ligands, we designed and synthesized a series of cyclic peptides based on the recent crystal structure of MC4R in complex with the well-characterized antagonist SHU-9119 (Ac-Nle4-c[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2). These analogues were pharmacologically characterized in vitro, giving key insights into exploiting binding site subpockets to deliver more selective ligands. More specifically, the side chains of the Nle4, DNal(2')7, and Trp9 residues in SHU-9119, as well as the amide linkage between the Asp5 and Lys10 side chains, were found to represent structural features engaging a hMC4R/hMC3R selectivity switch.

PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects.

BACKGROUND: The clinical treatment of various types of pain relies upon the use of opioid analgesics. However most of them produce, in addition to the analgesic effect, several side effects such as the development of dependence and addiction as well as sedation, dysphoria, and constipation. One solution to these problems are chimeric compounds in which the opioid pharmacophore is hybridized with another type of compound to incease antinociceptive effects. Neurotensin-induced antinociception is not mediated through the opioid system. Therefore, hybridizing neurotensin with opioid elements may result in a potent synergistic antinociceptor. RESULTS: Using the known structure-activity relationships of neurotensin we have synthesized a new chimeric opioid-neurotensin compound PK20 which is characterized by a very strong antinociceptive potency. The observation that the opioid antagonist naltrexone did not completely reverse the antinociceptive effect, indicates the partial involvement of the nonopioid component in PK20 in the produced analgesia. CONCLUSIONS: The opioid-neurotensin hybrid analogue PK20, in which opioid and neurotensin pharmacophores overlap partially, expresses high antinociceptive tail-flick effects after central as well as peripheral applications.

The use of time-averaged 3JHH restrained molecular dynamics (tar-MD) simulations for the conformational analysis of five-membered ring systems: methodology and applications.

Because of its presence in many molecules of biological relevance, the conformational analysis of five-membered rings using (3)J(HH) scalar coupling data from NMR is a topic of considerable interest. Typically, conformational analysis involves the use of a well-established mathematical procedure, originally developed by de Leeuw et al., that fits two rigid conformations to the available experimental data. This so-called pseudorotation analysis approach is not without problems, however, as chemically unrealistic conformations are sometimes generated from the data. Here, we present our investigations in the use of time-averaged restrained molecular dynamics simulations as a generic tool to determine the conformations that agree with experimental (3)J(HH) scalar coupling data. For this purpose, a set of six ribose-based molecules has been used as model compounds. The influence of several modeling parameters is assessed and optimized values are proposed. The results obtained with the tar-MD approach are compared to those obtained from the two conformer fitting procedure. Interpretation of the latter is facilitated by the introduction of a fitting error analysis that allows mapping the solution space of the fitting procedure. The relative merits of both methods and the advantages that result from the use of a force field and a time-averaged restraint potential for the experimental data are discussed. When combined, both techniques allow an enhanced understanding of the molecules' conformational behavior and prevent possible overinterpretation. In view of the very reasonable computational burden of a tar-MD simulation for the systems investigated here, the approach should be generally applicable.

Radical stability directs electron capture and transfer dissociation of ß-amino acids in peptides.

We report on the characteristics of the radical-ion-driven dissociation of a diverse array of ß-amino acids incorporated into α-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for ß-amino acids than for their α-form counterparts. In particular, only aromatic (e.g., ß-Phe), and to a substantially lower extent, carbonyl-containing (e.g., ß-Glu and ß-Gln) amino acid side chains, lead to N-Cß bond cleavage in the corresponding ß-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α-amino acids, ECD of peptides composed mainly of ß-amino acids reveals a shift in cleavage priority from the N-Cß to the Cα-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven ß-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds.

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced delta/mu antagonists, as determined by the functional [S(35)]GTPgammaS binding assay, the dimerization of potent Aia-based 'parent' ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds.

Development of a new microwave-assisted cleavable backbone amide linker (BAL): a comparative study.

A thorough comparative study to demonstrate the properties of a new microwave labile backbone amide linker is presented. A cyclic pentapeptide, cyclo(Trp-Gln-Gly-beta-Ala-Phe), was used as a model and synthesized following 16 different conditions. The new backbone amide linker is stable towards acid and base at r.t., and can be cleaved at elevated temperature in trifluoroacetic acid under microwave irradiation, avoiding the use of aggressive reagents like HF. The new linker is compatible with Boc- as well as Fmoc-strategy and allows the cleavage of acid labile side chain protective groups at r.t., prior to cleavage of the cyclic pentapeptide from the resin.

New tetracyclic tetrahydro-beta-carbolines as tryptophan-derived peptidomimetics.

The Pictet-Spengler reaction is known as a useful tool for the synthesis of constrained analogs of tryptophan. Herein, we present the further cyclization of 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl esters with 1-(1'-aminoalkyl) side chain. These transformations lead to heterocyclic structures which can find useful applications in medicinal chemistry as peptide mimetics.

Neuromedin U and Structural Analogs: An Overview of their Structure, Function and Selectivity.

The neuromedin U peptide sequence is highly conserved between various species. Neuromedin U is involved in a variety of physiological processes. It exerts its effects via two neuromedin U receptors, NMUR1 and NMUR2. These receptors are characterized by a distinct, yet complementary, tissue distribution with NMUR1 mostly found in the periphery, while NMUR2 is most abundant in the central nervous system. The capability of the neuropeptide to reduce food intake in rodents triggered the design and synthesis of a broad range of modified peptide ligands. The purpose of these ligands is to develop novel therapeutics which could be beneficial in the treatment of obesity and diabetes. Most compounds are derived either from the full-length neuromedin U sequence or are based on the truncated orthologs of this neuropeptide. Only a few non-peptidic ligands were developed. This review provides an overview on various neuromedin U analogs and mimetics that have been reported to date.

Optimized Opioid-Neurotensin Multitarget Peptides: From Design to Structure-Activity Relationship Studies.

Fusion of nonopioid pharmacophores, such as neurotensin, with opioid ligands represents an attractive approach for pain treatment. Herein, the µ-/δ-opioid agonist tetrapeptide H-Dmt-d-Arg-Aba-ß-Ala-NH2 (KGOP01) was fused to NT(8-13) analogues. Since the NTS1 receptor has been linked to adverse effects, selective MOR-NTS2 ligands are preferred. Modifications were introduced within the native NT sequence, particularly a ß3-homo amino acid in position 8 and Tyr11 substitutions. Combination of ß3hArg and Dmt led to peptide 7, a MOR agonist, showing the highest NTS2 affinity described to date (Ki = 3 pM) and good NTS1 affinity (Ki = 4 nM), providing a >1300-fold NTS2 selectivity. The (6-OH)Tic-containing analogue 9 also exhibited high NTS2 affinity (Ki = 1.7 nM), with low NTS1 affinity (Ki = 4.7 µM), resulting in an excellent NTS2 selectivity (>2700). In mice, hybrid 7 produced significant and prolonged antinociception (up to 8 h), as compared to the KGOP01 opioid parent compound.

Novel neurotensin analogues for radioisotope targeting to neurotensin receptor-positive tumors.

The increased expression of the neurotensin (NT) receptor NTS1 by different cancer cells, such as pancreatic adenocarcinoma and ductal breast cancer cells, as compared to normal epithelium, offers the opportunity to target these tumors with radiolabeled neurotensin analogues for diagnostic or therapeutic purposes. The aim of the present study was to design and synthesize new neurotensin radioligands and to select a lead molecule with high in vivo tumor selectivity for further development. Two series of neurotensin analogues bearing DTPA were tested: a series of NT(8-13) analogues, with DTPA coupled to the α-NH(2), sharing the same peptide sequence with analogues previously developed for radiolabeling with technetium or rhenium, as well as an NT(6-13) series in which DTPA was coupled to the ε-NH(2) of Lys(6). Changes were introduced to stabilize the bonds between Arg(8)-Arg(9), Pro(10)-Tyr(11), and Tyr(11)-Ile(12) to provide metabolic stability. Structure-activity studies of NT analogues have shown that the attachment of DTPA induces an important loss of affinity unless the distance between the chelator and the NT(8-13) sequence, which binds to the NTS1 receptor, is increased. The doubly stabilized DTPA-NT-20.3 exhibits a high affinity and an elevated stability to enzymatic degradation. It shows specific tumor uptake and high tumor to blood, to liver, and to intestine activity uptake ratios and affords high-contrast planar and SPECT images in an animal model. The DTPA-NT-20.3 peptide is a promising candidate for imaging neurotensin receptor-positive tumors, such as pancreatic adenocarcinoma and invasive ductal breast cancer. Analogues carrying DOTA are being developed for yttrium-90 or lutetium-177 labeling.