Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility.

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV ß-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

Chirality and Rigidity in Triazole-Modified Peptidomimetics Interacting with Neuropilin-1.

The interaction of Neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has been shown to promote angiogenesis under physiological and pathological conditions. Angiogenesis around tumors is a major factor allowing for their growth and spread. Disrupting NRP-1/VEGF complex formation is thus a promising pathway for the development of new anticancer pharmaceuticals. A large body of work has been produced in the last two decades detailing the development of inhibitors of NRP-1/VEGF complex formation. Among those were peptide A7R and its smaller derivatives KXXR and K(Har)XXR. It has been previously reported that replacement of the XX backbone with triazole residues has a positive effect on the proteolytic stability of inhibitors. It has also been reported that a higher dihedral angle range restriction of the XX backbone has a positive effect on the activity of inhibitors. In this work, we have designed new triazole derivatives of K(Har)XXR inhibitors with substitution allowing for higher range restriction of the XX backbone. The obtained peptidomimetics have greater activity than their less restricted counterparts. One of the newly obtained structures has greater affinity than the reference peptide A7R.

Moving out of CF3 -Land: Synthesis, Receptor Affinity, and in silico Studies of NK1 Receptor Ligands Containing a Pentafluorosulfanyl (SF5 ) Group.

The NK1 receptor (NK1R) is a molecular target for both approved and experimental drugs intended for a variety of conditions, including emesis, pain, and cancers. While contemplating modifications to the typical NK1R pharmacophore, we wondered whether the CF3 groups common for many NK1R ligands, could be replaced with some other moiety. Our attention was drawn by the SF5 group, and so we designed, synthesized, and tested ten novel SF5 -containing compounds for NK1R affinity. All analogues exhibit detectable NK1R binding, with the best of them, compound 5 a, (3-bromo-5-(pentafluoro-λ6 -sulfanyl)benzyl acetyl-L-tryptophanate) binding only slightly worse (IC50 =34.3 nM) than the approved NK1R-targeting drug, aprepitant (IC50 =27.7 nM). Molecular docking provided structural explanation of SAR. According to our analysis, the SF5 group in our compounds occupies a position similar to that of one of the CF3 groups of aprepitant as found in the crystal structure. Additionally, we checked whether the docking scoring function or energies derived from Fragment Molecular Orbital quantum chemical calculations may be helpful in explaining and predicting the experimental receptor affinities for our analogues. Both these methods produce moderately good results. Overall, this is the first demonstration of the utility of the SF5 group in the design of NK1R ligands.

Gαi-derived peptide binds the µ-opioid receptor.

BACKGROUND: G protein-coupled receptors (GPCRs) transduce external stimuli into the cell by G proteins via an allosteric mechanism. Agonist binding to the receptor stimulates GDP/GTP exchange within the heterotrimeric G protein complex, whereas recent structures of GPCR-G protein complexes revealed that the H5, S1 and S2 domains of Gα are involved in binding the active receptor, earlier studies showed that a short peptide analog derived from the C-terminus (H5) of the G protein transducin (Gt) is sufficient to stabilize rhodopsin in an active form. METHODS: We have used Molecular Dynamics simulations along with biological evaluation by means of radio-ligand binding assay to study the interactions between Gαi-derived peptide (G-peptide) and the µ-opioid receptor (µOR). RESULTS: Here, we show that a Gαi-derived peptide of 12 amino acids binds the µ-opioid receptor and acts as an allosteric modulator. The Gαi-derived peptide increases µOR affinity for its agonist morphine in a dose-dependent way. CONCLUSIONS: These results indicate that the GPCR-Gα peptide interaction observed so far for only rhodopsin can be extrapolated to µOR. In addition, we show that the C-terminal peptide of the Gαi subunit is sufficient to stabilize the active conformation of the receptor. Our approach opens the possibility to investigate the GPCR-G protein interface with peptide modification.

Docking is not enough: 17-trifluoromethylphenyl trinor PGF2α is only a very weak ligand of neurokinin-1 receptor.

17-trifluoromethylphenyl trinor prostaglandin F2α (17-CF3PTPGF2α) was reported recently to exhibit in vitro and in vivo anticancer activity. Based solely on the results of in silico molecular docking, it was claimed that this compound is NK1 receptor (NK1R) antagonist and that its activity is through this receptor. In this contribution we show that 17-CF3PTPGF2α is only a very weak NK1R ligand (IC50 > 200 µM). In connection with that we discuss the issue of this compound's molecular target. Finally, we briefly narrate on the proper use of molecular docking in biomedical research.

Correcting a widespread error: Neuroprotectant N-acetyl-L-tryptophan does not bind to the neurokinin-1 receptor.

N-acetyl-L-tryptophan (NAT) has been often shown to have neuroprotective action and other biological activities. In many of papers devoted to this compound, NAT is referenced to as an 'NK1 receptor (NK1R) antagonist' or a 'substance P (SP) antagonist'. Some of the studies treated NAT as a tool compound to interrogate NK1R function in a particular condition. Surprisingly however, no biochemical data on NAT/NK1R interaction have been available. On testing NAT in radioligand receptor binding assay, we found that this compound displays no significant binding to either human or rat NK1R up to millimolar concentrations. In light of this, the repeated claim of NAT being NK1R antagonist is an error. The use of NAT as a tool compound in NK1R-related studies should be discontinued. Some of the conclusions regarding the involvement of SP/NK1R axis in acute CNS injury or neurodegenerative diseases may then require careful rethinking. On the other hand, given interesting neuroprotective properties of NAT, the issue whether it acts by specific interactions with some molecular target or by unspecific physicochemical mechanisms needs be investigated.

Fentanyl Structure as a Scaffold for Opioid/Non-Opioid Multitarget Analgesics.

One of the strategies in the search for safe and effective analgesic drugs is the design of multitarget analgesics. Such compounds are intended to have high affinity and activity at more than one molecular target involved in pain modulation. In the present contribution we summarize the attempts in which fentanyl or its substructures were used as a µ-opioid receptor pharmacophoric fragment and a scaffold to which fragments related to non-opioid receptors were attached. The non-opioid 'second' targets included proteins as diverse as imidazoline I2 binding sites, CB1 cannabinoid receptor, NK1 tachykinin receptor, D2 dopamine receptor, cyclooxygenases, fatty acid amide hydrolase and monoacylglycerol lipase and σ1 receptor. Reviewing the individual attempts, we outline the chemistry, the obtained pharmacological properties and structure-activity relationships. Finally, we discuss the possible directions for future work.

In Vitro Biological Evaluation of Aprepitant Based 177Lu-Radioconjugates.

Currently, the search for promising NK1R-positive tumor-targeting radiopharmaceuticals based on the structure of small molecular antagonists of neurokinin-1 receptor can be observed. Following this trend, we continued our evaluation of aprepitant-based 177Lu-radioconjugates in terms of future oncological applications. For this purpose, three novel aprepitant homologues were synthesized to broaden the previously obtained derivative portfolio, functionalized with the DOTA chelator and labeled with 68Ga and 177Lu. The newly evaluated radioconjugates showed the intended significant increase in lipophilicity compared to the previous ones, while maintaining stability in the human serum. Then, in a receptor binding study to the human NK1 receptor, we compared the two series of 177Lu-radioconjugates of aprepitant with each other and with the reference Substance P derivative currently used in glioblastoma therapy, clearly indicating the high affinity and better binding capacity of the novel radioconjugates. The in vitro experimental results included in the presented study, supported by labeling optimization, radioconjugate characterization and docking modeling of new aprepitant-derived radioagents, confirm our assumptions about the usefulness of aprepitant as a NK1R targeting vector and point out the perspectives for the forthcoming first in vivo trials.

Novel NK1R-Targeted 68Ga-/177Lu-Radioconjugates with Potential Application against Glioblastoma Multiforme: Preliminary Exploration of Structure-Activity Relationships.

Locoregionally administered, NK1 receptor (NK1R) targeted radionuclide therapy is a promising strategy for the treatment of glioblastoma multiforme. So far, the radiopharmaceuticals used in this approach have been based on the endogenous agonist of NK1R, Substance P or on its close analogues. Herein, we used a well-known, small molecular NK1R antagonist, L732,138, as the basis for the radiopharmaceutical vector. First, 14 analogues of this compound were evaluated to check whether extending the parent structure with linkers of different lengths would not deteriorate the NK1R binding. The tested analogues had affinity similar to or better than the parent compound, and none of the linkers had a negative impact on the binding. Next, five DOTA conjugates were synthesized and used for labelling with 68Ga and 177Lu. The obtained radioconjugates turned out to be fairly lipophilic but showed rather limited stability in human plasma. Evaluation of the receptor affinity of the (radio)conjugates showed that neither the chelator nor the metal negatively impacts the NK1R binding. The 177Lu-radioconjugates exhibited the binding characteristics towards NK1R similar or better than that of the 177Lu-labelled derivative of Substance P, which is in current clinical use. The experimental results presented herein, along with their structural rationalization provided by modelling, give insight for the further molecular design of small molecular NK1R-targeting vectors.

Bifunctional Opioid/Melanocortin Peptidomimetics for Use in Neuropathic Pain: Variation in the Type and Length of the Linker Connecting the Two Pharmacophores.

Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.

The solid state VCD of a novel N-acylhydrazone trifluoroacetate.

A novel N-acylhydrazone with pharmaceutical importance was subject of structural and IR/VCD investigations in the solid state. In the crystal structure, dimers of anion-cation pairs are stabilized by H-bonding and ionic interactions. Some less common interaction types, like C=N···C-NH3+ (σ-hole) interactions, hydrazone-aromatic interactions and dispersive contacts of the CF3 groups are also present in the crystal. Satisfactory reproduction of the solid state IR and VCD spectra required that quantum-chemical calculations be done on a tetramer (four cation-anion pairs) cut out from the crystal structure, exhibiting key intermolecular interactions. Ten DFT functionals were assessed as to the agreement between the calculated and experimental spectra. Various approaches to scaling of the calculated frequencies were applied. The best results were yielded with individual (optimized) frequency scaling factors (FSFs) and band half-widths at half maximum-(HWHM) for four separate spectral subregions. The best matching between the experimental and theoretical spectra (according to SimIR, SimVCD and SimVDF indices) was found for the B3PW91 functional, however, a few other functionals follow closely in the ranking. Based on the quantum chemical calculations, spectral assignments have been made.

In Vivo, In Vitro and In Silico Studies of the Hybrid Compound AA3266, an Opioid Agonist/NK1R Antagonist with Selective Cytotoxicity.

AA3266 is a hybrid compound consisting of opioid receptor agonist and neurokinin-1 receptor (NK1R) antagonist pharmacophores. It was designed with the desire to have an analgesic molecule with improved properties and auxiliary anticancer activity. Previously, the compound was found to exhibit high affinity for µ- and δ-opioid receptors, while moderate binding to NK1R. In the presented contribution, we report on a deeper investigation of this hybrid. In vivo, we have established that AA3266 has potent antinociceptive activity in acute pain model, comparable to that of morphine. Desirably, with prolonged administration, our hybrid induces less tolerance than morphine does. AA3266, contrary to morphine, does not cause development of constipation, which is one of the main undesirable effects of opioid use. In vitro, we have confirmed relatively strong cytotoxic activity on a few selected cancer cell lines, similar to or greater than that of a reference NK1R antagonist, aprepitant. Importantly, our compound affects normal cells to smaller extent what makes our compound more selective against cancer cells. In silico methods, including molecular docking, molecular dynamics simulations and fragment molecular orbital calculations, have been used to investigate the interactions of AA3266 with MOR and NK1R. Insights from these will guide structural optimization of opioid/antitachykinin hybrid compounds.

Antiproliferative effects of [D-Pro2, D-Trp7,9]-Substance P and aprepitant on several cancer cell lines and their selectivity in comparison to normal cells.

A neuropeptide, Substance P (SP), has mitogenic action in many types of cancer cells mediated via the neurokinin-1 receptor (NK1R). Small molecular NK1R antagonists have been frequently shown to possess anticancer activity both in vivo and in vitro, but there are only a few papers on such activity regarding peptide antagonists. In order to extend the data on this class of compounds, we have compared the effects of a peptide antagonist, [D-Pro2, D-Trp7,9]-Substance P, and a small molecular antagonist, aprepitant on the proliferation of five cancer and three normal cell lines. The comparison was based on three assays: cell proliferation test, MTT test and assay for colony formation. Consistently with earlier reports, aprepitant potently reduced cell proliferation in cancer cell lines in all assays, but in contrast to previous works, the compound was not selective and it affected normal cell lines to a similar degree. The studied peptide antagonist, [D-Pro2, D-Trp7,9]-Substance P, was able to decrease proliferation only in a few cell lines, and only in the highest concentration (100 µM). In a lower concentration, a slight pro-proliferative effect was observed in a few cell lines. No statistically significant effects on colony formation were found for this compound.

Antinociceptive and Cytotoxic Activity of Opioid Peptides with Hydrazone and Hydrazide Moieties at the C-Terminus.

In the present contribution, we analyze the influence that C-terminal extension of short opioid peptide sequences by organic fragments has on receptor affinity, in vivo analgesic activity, and antimelanoma properties. The considered fragments were based on either N-acylhydrazone (NAH) or N'-acylhydrazide motifs combined with the 3,5-bis(trifluoromethyl)phenyl moiety. Eleven novel compounds were synthesized and subject to biological evaluation. The analyzed compounds exhibit a diversified range of affinities for the µ opioid receptor (MOR), rather low δ opioid receptor (DOR) affinities, and no appreciable neurokinin-1 receptor binding. In three out of four pairs, N-acylhydrazone-based derivatives bind MOR better than their N'-acylhydrazide counterparts. The best of the novel derivatives have similar low nanomolar MOR binding affinity as the reference opioids, such as morphine and biphalin. The obtained order of MOR affinities was compared to the results of molecular docking. In vivo, four tested compounds turned out to be relatively strong analgesics. Finally, the NAH-based analogues reduce the number of melanoma cells in cell culture, while their N'-acylhydrazide counterparts do not. The antimelanoma properties are roughly correlated to the lipophilicity of the compounds.

Radiochemical Synthesis and Evaluation of Novel Radioconjugates of Neurokinin 1 Receptor Antagonist Aprepitant Dedicated for NK1R-Positive Tumors.

Aprepitant, a lipophilic and small molecular representative of neurokinin 1 receptor antagonists, is known for its anti-proliferative activity on numerous cancer cell lines that are sensitive to Substance P mitogen action. In the presented research, we developed two novel structural modifications of aprepitant to create aprepitant conjugates with different radionuclide chelators. All of them were radiolabeled with 68Ga and 177Lu radionuclides and evaluated in terms of their lipophilicity and stability in human serum. Furthermore, fully stable conjugates were examined in molecular modelling with a human neurokinin 1 receptor structure and in a competitive radioligand binding assay using rat brain homogenates in comparison to the aprepitant molecule. This initial research is in the conceptual stage to give potential theranostic-like radiopharmaceutical pairs for the imaging and therapy of neurokinin 1 receptor-overexpressing cancers.

Can Keratin Scaffolds be used for Creating Three-dimensional Cell Cultures?

Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

The analgesic hybrid of dermorphin/substance P and analog of enkephalin improve wound healing in streptozotocin-induced diabetic rats.

The purpose of this study was to investigate the effect of the peptide analgesic hybrid compounds: AWL3106 analog of dermorphin and substance P (7-11), and biphalin enkephalin analog on wound healing in streptozotocin-induced diabetic rats. The diabetes was induced in 6-7 week-old male Wistar rats by intraperitoneal injection of streptozotocin. After 70 days, the wounds were created on the back of the rats and then, once a day for 21 days, the dressing containing lanolin ointment, 10% of keratin scaffolds, and 1 mM of AWL3106 or biphalin was applied. The wounds histology were analyzed by hematoxylin and eosin staining. The orientation and organization of collagen was analyzed by Masson's trichome staining. The number of macrophages, blood vessels, and fibroblasts were visualized by CD68, CD34, and vimentin immunoreactivity, respectively. Our results demonstrated that the wound area of AWL3106- and biphalin-treated groups was greatly reduced (up to 47% on the 7 day) in comparison with untreated diabetic groups. The immunohistochemical staining of macrophages demonstrated that AWL3106 and biphalin accelerated inflammatory progression and subsequently decreased persistent inflammation. The histological analysis showed that the structure of tissue in the groups under the study was very similar to the one of wound tissue in N-DM group. The H&E and Masson's trichome staining demonstrated that the orientation and organization of collagen as well as the number and shape of blood vessels were better in 3106- and BIF-treated group than in DM group. In conclusion, the obtained data suggested that our hybrid peptides enhanced wound healing, particularly by accelerating the inflammatory phase and promoted the wound closure.

Fentanyl Family at the Mu-Opioid Receptor: Uniform Assessment of Binding and Computational Analysis.

Interactions of 21 fentanyl derivatives with µ-opioid receptor (µOR) were studied using experimental and theoretical methods. Their binding to µOR was assessed with radioligand competitive binding assay. A uniform set of binding affinity data contains values for two novel and one previously uncharacterized derivative. The data confirms trends known so far and thanks to their uniformity, they facilitate further comparisons. In order to provide structural hypotheses explaining the experimental affinities, the complexes of the studied derivatives with µOR were modeled and subject to molecular dynamics simulations. Five common General Features (GFs) of fentanyls' binding modes stemmed from these simulations. They include: GF1) the ionic interaction between D147 and the ligands' piperidine NH⁺ moiety; GF2) the N-chain orientation towards the µOR interior; GF3) the other pole of ligands is directed towards the receptor outlet; GF4) the aromatic anilide ring penetrates the subpocket formed by TM3, TM4, ECL1 and ECL2; GF5) the 4-axial substituent (if present) is directed towards W318. Except for the ionic interaction with D147, the majority of fentanyl-µOR contacts is hydrophobic. Interestingly, it was possible to find nonlinear relationships between the binding affinity and the volume of the N-chain and/or anilide's aromatic ring. This kind of relationships is consistent with the apolar character of interactions involved in ligand⁻receptor binding. The affinity reaches the optimum for medium size while it decreases for both large and small substituents. Additionally, a linear correlation between the volumes and the average dihedral angles of W293 and W133 was revealed by the molecular dynamics study. This seems particularly important, as the W293 residue is involved in the activation processes. Further, the Y326 (OH) and D147 (Cγ) distance found in the simulations also depends on the ligands' size. In contrast, neither RMSF measures nor D114/Y336 hydrations show significant structure-based correlations. They also do not differentiate studied fentanyl derivatives. Eventually, none of 14 popular scoring functions yielded a significant correlation between the predicted and observed affinity data (R < 0.30, n = 28).

Opioid Tripeptides Hybridized with trans-1-Cinnamylpiperazine as Proliferation Inhibitors of Pancreatic Cancer Cells in Two- and Three-Dimensional in†vitro Models.

According to the World Health Organization, the mortality rate among patients with pancreatic cancer will increase in the upcoming years. Gemcitabine is the first choice for treatment of pancreatic malignancy, but increasing resistance to this drug is decreasing its overall efficacy. Studies on new therapies that target metabolic pathways, growth factor inhibitors, and tumor stroma or tumor stem cells are currently underway in many research groups. Herein we report the bioactive properties (cytotoxicity and hemolytic activity) of synthetic peptidomimetics containing an opioid tripeptide fragment (Tyr-R1 -R2 -; where R1 is d-Ala or d-Thr, and R2 is Phe or Trp) hybridized with trans-1-cinnamylpiperazine. These compounds are stable in plasma up to 96 h and exhibit low hemotoxicity and good inhibitory effects on cancer cell growth in two- and three-dimensional in vitro models of pancreatic cancer.

SYNTHESIS AND ANTIMICROBIAL PROPERTIES OF NEW MANDELATE IONIC LIQUIDS.

Novel mandelate ionic liquids with quartenary ammonium cations were synthesized and characterized. The compounds exhibit antimicrobial activity and the most potent one is of similar efficacy against Gram+ bacteria as its counterpart chloride. On the other hand, the mandelates are much less active against Gram-bacteria and fungi. QSAR models suggest that, with respect to cation, their potency depends on lipophilicity. The synthesized ionic liquids are also quite cytotoxic against mammalian cells.