(L)-Monomethyl Tyrosine (Mmt): New Synthetic Strategy via Bulky 'Forced-Traceless' Regioselective Pd-Catalyzed C(sp2)-H Activation.

The enormous influence in terms of bioactivity, affinity, and selectivity represented by the replacement of (L)-2,6-dimethyl tyrosine (Dmt) instead of Phenylalanine (Phe) into Nociceptin/orphanin (N/OFQ) neuropeptide analogues has been well documented in the literature. More recently, the non-natural amino acid (L)-2-methyl tyrosine (Mmt), with steric hindrance included between Tyr and Dmt, has been studied because of the modulation of steric effects in opioid peptide chains. Here, we report a new synthetic strategy to obtain Mmt based on the well-known Pd-catalyzed ortho-C(sp2)-H activation approach, because there is a paucity of other synthetic routes in the literature to achieve it. The aim of this work was to force only the mono-ortho-methylation process over the double ortho-methylation one. In this regard, we are pleased to report that the introduction of the dibenzylamine moiety on a Tyr aromatic nucleus is a convenient and traceless solution to achieve such a goal. Interestingly, our method provided the aimed Mmt either as N-Boc or N-Fmoc derivatives ready to be inserted into peptide chains through solid-phase peptide synthesis (SPPS). Importantly, the introduction of Mmt in place of Phe1 in the sequence of N/OFQ(1-13)-NH2 was very well tolerated in terms of pharmacological profile and bioactivity.

Dimeric ferulic acid conjugate as a prodrug for brain targeting after nasal administration of loaded solid lipid microparticles.

OBJECTIVE: Ferulic acid (Fer) displays antioxidant/anti-inflammatory properties useful against neurodegenerative diseases. To increase Fer uptake and its central nervous system residence time, a dimeric prodrug, optimizing the Fer loading on nasally administrable solid lipid microparticles (SLMs), was developed. METHODS: The prodrug was synthesized as Fer dimeric conjugate methylated on the carboxylic moiety. Prodrug antioxidant/anti-inflammatory properties and ability to release Fer in physiologic environments were evaluated. Tristearin or stearic acid SLMs were obtained by hot emulsion technique. In vivo pharmacokinetics were quantified by HPLC. RESULTS: The prodrug was able to release Fer in physiologic environments (whole blood and brain homogenates) and induce in vitro antioxidant/anti-inflammatory effects. Its half-life in rats was 18.0 ± 1.9 min. Stearic acid SLMs, exhibiting the highest prodrug loading and dissolution rate, were selected for nasal administration to rats (1 mg/kg dose), allowing to obtain high prodrug bioavailability and prolonged residence in the cerebrospinal fluid, showing AUC (Area Under Concentration) values (108.5 ± 3.9 µg∙mL-1∙min) up to 30 times over those of Fer free drug, after its intravenous/nasal administration (3.3 ± 0.3/5.16 ± 0.20 µg∙mL-1∙min, respectively) at the same dose. Chitosan presence further improved the prodrug brain uptake. CONCLUSIONS: Nasal administration of prodrug-loaded SLMs can be proposed as a noninvasive approach for neurodegenerative disease therapy.

Impact of C- and N-terminal protection on the stability, metal chelation and antimicrobial properties of calcitermin.

The main limitation to the use of antimicrobial peptides (AMPs) as regular drugs, against antibiotic and antifungal resistance, mainly relates to their rapid degradation by proteolytic enzymes. The introduction of suitable structural changes in the peptide chain can make the peptide less susceptible to the action of proteases, thus overcoming this problem. To improve the plasma stability of calcitermin, a metal-chelating AMP present in the human respiratory tract and investigated in the present study, C- and/or N- terminal modifications have been introduced in the native sequence. Evaluation of peptide stability has been performed to determine the half-life times in human plasma of both native calcitermin and its derivatives. However, the protection of the peptide termini can also affect its metal coordination behaviour. Thus, the characterization of Zn2+ and Cu2+ complexes has been performed by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV-Vis, circular dichroism and EPR. On the basis of the obtained results, it was possible to compare the biological activity of the studied systems, taking into account both the metal-binding ability and the peptide stability to search for a link among them. A significant result of this study is that the N-terminal protection increases the calcitermin half-life over seven times and the formation of metal complexes confers resistance towards degradation almost doubling its half-life.

Schwann cell insulin-like growth factor receptor type-1 mediates metastatic bone cancer pain in mice.

Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to metastatic bone cancer pain (MBCP), but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve, and IGF-1-dependent stimulus/non-stimulus-evoked pain-like behaviors. Adeno-associated virus-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in Schwann cells, but not in dorsal root ganglion (DRG) neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. Schwann cell IGF-1R signaling promoted an endothelial nitric oxide synthase-mediated transient receptor potential ankyrin 1 (TRPA1) activation and release of reactive oxygen species that, via macrophage-colony stimulating factor-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment.

Novel Aryl Sulfonamide Derivatives as NLRP3 Inflammasome Inhibitors for the Potential Treatment of Cancer.

The NLRP3 inflammasome is a critical component of innate immunity that senses diverse pathogen- and host-derived molecules. However, its aberrant activation has been associated with the pathogenesis of multiple diseases, including cancer. In this study, we designed and synthesized a series of aryl sulfonamide derivatives (ASDs) to inhibit the NLRP3 inflammasome. Among these, compounds 6c, 7n, and 10 specifically inhibited NLRP3 activation at nanomolar concentrations without affecting the activation of the NLRC4 and AIM2 inflammasomes. Furthermore, we demonstrated that these compounds reduce interleukin-1ß (IL-1ß) production in vivo and attenuate melanoma tumor growth. Moreover, metabolic stability in liver microsomes of 6c, 7n, and 10 was studied along with plasma exposure in mice of the most interesting compound 6c. Therefore, we generated potent NLRP3 inflammasome inhibitors, which can be considered in future medicinal chemistry and pharmacological studies aimed at developing a new therapeutic approach for NLRP3 inflammasome-driven cancer.

Ageing Curtails the Diversity and Functionality of Nascent CD8+ T Cell Responses against SARS-CoV-2.

Age-related changes in the immune system are thought to underlie the vulnerability of elderly individuals to emerging viral diseases, such as coronavirus disease 2019 (COVID-19). In this study, we used a fully validated in vitro approach to determine how age impacts the generation of de novo CD8+ T cell responses against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19. Our data revealed a generalized deficit in the ability of elderly individuals to prime the differentiation of naïve precursors into effector CD8+ T cells defined by the expression of interferon (IFN)-γ and the transcription factor T-bet. As a consequence, there was an age-related decline in the diversity of newly generated CD8+ T cell responses targeting a range of typically immunodominant epitopes derived from SARS-CoV-2, accompanied by an overall reduction in the expression frequency of IFN-γ. These findings have potential implications for the development of new strategies to protect the elderly against COVID-19.

Non-neuronal TRPA1 encodes mechanical allodynia associated with neurogenic inflammation and partial nerve injury in rats.

BACKGROUND AND PURPOSE: The pro-algesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by a subpopulation of primary sensory neurons, has been implicated in various pain models in mice. However, evidence in rats indicates that TRPA1 conveys nociceptive signals elicited by channel activators, but not those associated with tissue inflammation or nerve injury. Here, in rats, we explored the TRPA1 role in mechanical allodynia associated with stimulation of peptidergic primary sensory neurons (neurogenic inflammation) and moderate (partial sciatic nerve ligation, pSNL) or severe (chronic constriction injury, CCI) sciatic nerve injury. EXPERIMENTAL APPROACH: Acute nociception and mechanical hypersensitivity associated with neurogenic inflammation and sciatic nerve injury (pSNL and CCI) were investigated in rats with TRPA1 pharmacological antagonism or genetic silencing. TRPA1 presence and function were analysed in cultured rat Schwann cells. KEY RESULTS: Hind paw mechanical allodynia (HPMA), but not acute nociception, evoked by local injection of capsaicin or allyl isothiocyanate, the TRP vanilloid 1 (TRPV1) or the TRPA1 activators was mediated by CGRP released from peripheral sensory nerve terminals. CGRP-evoked HPMA was sustained by a ROS-dependent TRPA1 activation, probably in Schwann cells. HPMA evoked by pSNL, but not that evoked by CCI, was mediated by ROS and TRPA1 without the involvement of CGRP. CONCLUSIONS AND IMPLICATIONS: As found in mice, TRPA1 mediates mechanical allodynia associated with neurogenic inflammation and moderate nerve injury in rats. The channel contribution to mechanical hypersensitivity is a common feature in rodents and might be explored in humans.

Effects of Microencapsulated Ferulic Acid or Its Prodrug Methyl Ferulate on Neuroinflammation Induced by Muramyl Dipeptide.

Ferulic acid (Fer) is known for its antioxidant and anti-inflammatory activities, which are possibly useful against neurodegenerative diseases. Despite the ability of Fer to permeate the brain, its fast elimination from the body does not allow its therapeutic use to be optimized. The present study proposes the preparation and characterization of tristearin- or stearic acid-based solid lipid microparticles (SLMs) as sustained delivery and targeting systems for Fer. The microparticles were produced by conventional hot emulsion techniques. The synthesis of the methyl ester of Fer (Fer-Me) allowed its encapsulation in the SLMs to increase. Fer-Me was hydrolyzed to Fer in rat whole blood and liver homogenate, evidencing its prodrug behavior. Furthermore, Fer-Me displayed antioxidant and anti-inflammatory properties. The amount of encapsulated Fer-Me was 0.719 ± 0.005% or 1.507 ± 0.014% in tristearin or stearic acid SLMs, respectively. The tristearin SLMs were able to control the prodrug release, while the stearic acid SLMs induced a significant increase of its dissolution rate in water. Jointly, the present results suggest that the tristearin SLMs loaded with Fer-Me could be a potential formulation against peripheral neuropathic pain; conversely, the stearic acid SLMs could be useful for Fer-Me uptake in the brain after nasal administration of the formulation.

Pharmacology of Kappa Opioid Receptors: Novel Assays and Ligands.

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and ß-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-ß-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3-10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.

Identification of small-molecule urea derivatives as PTPC modulators targeting the c subunit of F1/Fo-ATP synthase.

Maintaining a high percentage of living and functional cells in those pathologies in which excessive cell death occurs, such as neurodegenerative disorders and cardiovascular diseases, is one of the most intriguing challenges in the field of biochemical research for drug discovery. Here, mitochondrial permeability transition-driven regulated cell death is the main mechanism of mitochondrial impairment and cell fate; this pathway is still lacking of satisfying pharmacological treatments to counteract its becoming; for this reason, it needs continuous and intense research to find new compounds as modulator of the permeability transition pore complex (PTPC) activity. In this study, we report the identification of small-molecule urea derivatives able to inhibit PTPC opening following calcium overload and selected for future use in cytoprotection.

Synthesis of 2,6-Dimethyltyrosine-Like Amino Acids through Pinacolinamide-Enabled C-H Dimethylation of 4-Dibenzylamino Phenylalanine.

The synthesis of a small library of NH-Boc- or NH-Fmoc-protected l-phenylalanines carrying methyl groups at positions 2 and 6 and diverse functionalities at position 4 has been achieved. The approach, which took advantage of a Pd-catalyzed directed C-H dimethylation of picolinamide derivatives, allowed the electronic and steric properties of the resulting amino acid derivatives to be altered by appending a variety of electron-withdrawing, electron-donating, or bulky groups.

Thermodynamic Stability and Speciation of Ga(III) and Zr(IV) Complexes with High-Denticity Hydroxamate Chelators.

Increasing attention has been recently devoted to 89Zr(IV) and 68Ga(III) radionuclides, due to their favorable decay characteristics for positron emission tomography (PET). In the present paper, a deep investigation is presented on Ga(III) and Zr(IV) complexes with a series of tri-(H3L1, H3L3, H3L4 and desferrioxamine E, DFOE) and tetrahydroxamate (H4L2) ligands. Herein, we describe the rational design and synthesis of two cyclic complexing agents (H3L1 and H4L2) bearing three and four hydroxamate chelating groups, respectively. The ligand structures allow us to take advantage of the macrocyclic effect; the H4L2 chelator contains an additional side amino group available for a possible further conjugation with a biomolecule. The thermodynamic stability of Ga(III) and Zr(IV) complexes in solution has been measured using a combination of potentiometric and pH-dependent UV-vis titrations, on the basis of metal-metal competition. The Zr(IV)-H4L2 complex is characterized by one of the highest formation constants reported to date for a tetrahydroxamate zirconium chelate (log ß = 45.9, pZr = 37.0), although the complex-stability increase derived from the introduction of the fourth hydroxamate binding unit is lower than that predicted by theoretical calculations. Solution studies on Ga(III) complexes revealed that H3L1 and H4L2 are stronger chelators in comparison to DFOB. The complex stability obtained with the new ligands is also compared with that previously reported for other hydroxamate ligands. In addition to increasing the library of the thermodynamic stability data of Ga(III) and Zr(IV) complexes, the present work allows new insights into Ga(III) and Zr(IV) coordination chemistry and thermodynamics and broadens the selection of available chelators for 68Ga(III) and 89Zr(IV).

Impaired Priming of SARS-CoV-2-Specific Naive CD8+ T Cells in Older Subjects.

Advanced age is associated with severe symptoms and death upon SARS-CoV-2 infection. Virus-specific CD8+ T-cell responses have shown to be protective toward critical COVID-19 manifestations, suggesting that suboptimal cellular immunity may contribute to the age-pattern of the disease. The induction of a CD8+ T-cell response against an emerging pathogen like SARS-CoV-2 relies on the activation of naive T cells. To investigate whether the primary CD8+ T-cell response against this virus is defective in advanced age, we used an in vitro approach to prime SARS-CoV-2-specific naive CD8+ T cells from healthy, unexposed donors of different age groups. Compared to younger adults, older individuals display a poor SARS-CoV-2-specific T-cell priming capacity in terms of both magnitude and quality of the response. In addition, older subjects recognize a lower number of epitopes. Our results implicate that immune aging is associated with altered primary SARS-CoV-2-specific CD8+ T-cell responses.

Use of a Novel Peptide Welding Technology Platform for the Development of B- and T-Cell Epitope-Based Vaccines.

Peptide vaccines incorporating B- and T-cell epitopes have shown promise in the context of various cancers and infections. These vaccines are relatively simple to manufacture, but more immunogenic formulations are considered a priority. We developed tetrabranched derivatives for this purpose based on a novel peptide welding technology (PWT). PWTs provide molecular scaffolds for the efficient synthesis of ultrapure peptide dendrimers, which allow the delivery of multiple ligands within a single macromolecular structure. Peptide vaccines incorporating T-cell epitopes derived from melanoma and B-cell epitopes derived from human immunodeficiency virus, synthesized using this approach, elicited primary immune responses in vitro and in vivo. Subcutaneous administration of the B-cell epitope-based vaccines also elicited more potent humoral responses than subcutaneous administration of the corresponding peptides alone. Highly immunogenic peptide epitope-based vaccines can therefore be generated quickly and easily using a novel PWT.

Novel Mixed NOP/Opioid Receptor Peptide Agonists.

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with the noncanonical amino acid Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

Structure-Activity Relationship Studies on Oxazolo[3,4-a]pyrazine Derivatives Leading to the Discovery of a Novel Neuropeptide S Receptor Antagonist with Potent In Vivo Activity.

Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure-activity relationships and provide an updated model of ligand/NPSR interactions.

Biased Agonism at Nociceptin/Orphanin FQ Receptors: A Structure Activity Study on N/OFQ(1-13)-NH2.

Nociceptin/orphanin FQ (N/OFQ) controls different biological functions via selective stimulation of the N/OFQ peptide (NOP) receptor. The pleiotropic actions of N/OFQ may limit the development of NOP ligands as innovative drugs in different therapeutic areas. The pharmacological concept of functional selectivity (aka biased agonism) might be useful for amplifying beneficial actions and/or counteracting side effects. Thus, molecules with large bias factors toward G protein or ß arrestin are required for investigating the translational value of NOP biased modulation. Herein, the biased behavior of a heterogeneous library of NOP-targeting peptide derivatives was evaluated in vitro with the aim to provide possible insights into the structural determinants that govern the selective activation of G protein versus ß-arrestin. Our results demonstrate that lipidation of N/OFQ(1-13)-NH2 is a useful strategy for obtaining G protein biased agonists for the NOP receptor.

Cu(II) coordination to His-containing linear peptides and related branched ones: Equalities and diversities.

The two branched peptides (AAHAWG)4-PWT2 and (HAWG)4-PWT2 where synthesized by mounting linear peptides on a cyclam-based scaffold (PWT2), provided with four maleimide chains, through a thio-Michael reaction. The purpose of this study was primarily to verify if the two branched ligands had a Cu(II) coordination behavior reproducing that of the single-chain peptides, namely AAHAWG-NH2, which bears an Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) Motif, and HAWG-NH2, which presents a His residue as the N-terminal amino acid, in a wide pH range. The study of Cu(II) binding was performed by potentiometric, spectroscopic (UV-vis absorption, CD, fluorescence) and ESI-MS techniques. ATCUN-type ligands ((AAHAWG)4-PWT2 and AAHAWG-NH2) were confirmed to bind one Cu(II) per peptide fragment at both pH 7.4 and pH 9.0, with a [NH2, 2N-, NIm] coordination mode. On the other hand, the ligand HAWG-NH2 forms a [CuL2]2+ species at neutral pH, while, at pH 9, the formation of 1:2 Cu(II):ligand adducts is prevented by amidic nitrogen deprotonation and coordination, to give rise solely to 1:1 species. Conversely, Cu(II) binding to (HAWG)4-PWT2 resulted in the formation of 1:2 copper:peptide chain also at pH 9: hence, through the latter branched peptide we obtained, at alkaline pH, the stabilization of a specific Cu(II) coordination mode which results unachievable using the corresponding single-chain peptide. This behavior could be explained in terms of high local peptide concentration on the basis of the speciation of the Cu(II)/single-chain peptide systems.

Tetrabranched Hetero-Conjugated Peptides as Bifunctional Agonists of the NOP and Mu Opioid Receptors.

The general aim of the work was the validation of a new synthetic methodology designed for obtaining bifunctional heterotetrabranched peptide ligands. Applying an easily accessible synthetic route, we provided a small series of heteromultimeric peptide conjugates targeting the nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) and mu opioid receptors. Among these, H-PWT1-N/OFQ-[Dmt1]dermorphin demonstrated a similar and high agonist potency at the NOP and mu receptors. The achieved results confirmed the robustness of the approach that is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity can be combined for generating dual acting multimeric compounds. These innovative pharmacological tools will be extremely helpful for investigating the consequences of the simultaneous activation and/or blockage of different peptidergic receptors.

Synthesis and Biological Activity of Peptide α-Ketoamide Derivatives as Proteasome Inhibitors.

Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudodi/tripeptides bearing at the C-terminal position different α-ketoamide moieties as pharmacophoric units for the interaction with the catalytic threonine residue that sustains the proteolytic action of the proteasome. Among these, we identified the 1-naphthyl derivative 13c as a potent and selective inhibitor of the ß5 subunit of the 20S proteasome, exhibiting nanomolar potency in vitro (ß5 IC50 = 7 nM, ß1 IC50 = 60 µM, ß2 IC50 > 100 µM). Furthermore, it significantly inhibited proliferation and induced apoptosis of the human colorectal carcinoma cell line HCT116.