Recent progress in non-opioid analgesic peptides.

Pain is a prevalent complex medical problem, characterized by physically debilitating and mentally destabilizing conditions. Current pain therapeutics mainly include non-steroidal anti-inflammatory drugs and narcotics (opioids), but they exhibit limitations in efficacy, unwanted side effects and the problem of drug abuse. To overcome these issues, the discovery of different molecular players within pain pathways could lead to new opportunities for therapeutic intervention. Among other strategies, peptides could be powerful pharmaceutical agents for effective opioid-free medications for pain treatment. This review is a compendium of representative non-opioid analgesic peptides acting directly or indirectly at different ion channels and receptors distributed in nociceptive pathways. They include peptides targeting Ca2+, Na+ and K+ voltage-gated ion channels, the neuronal nicotinic receptors (nAChR), transient receptor potential channels (TRP), and different non-opioid G-protein coupled receptors (GPCRs), like the calcitonin gen-related peptide (CGRP), cannabinoid, bradykinin and neurotensin receptors, among others. Peptides engineered from protein-protein interactions among pain-related receptors and regulatory proteins also led to new therapeutic approaches for pain management. Following some successful examples, already in the clinics or under clinical trials, the improved understanding of pain mechanisms, and the advances in peptide permeation and/or delivery, could afford new analgesic peptides in the near future.

Disulfide bonds versus TrpTrp pairs in irregular beta-hairpins: NMR structure of vammin loop 3-derived peptides as a case study.

Structural studies on model peptides have led to a good understanding of the rules behind the formation and stability of regular beta-hairpins. To test their applicability to the successful design of irregular beta-hairpins with long loops and/or beta-bulges at the strands, we mimicked loop 3 of vammin, a 4:6 beta-hairpin with a non-Gly beta-bulge. The most stabilising cross-strand pairs, disulfide bonds or/and TrpTrp pairs, were incorporated at non-hydrogen-bonded sites in peptides spanning the 69-80 region of vammin. According to NMR data, these modified peptides adopt beta-hairpin conformations as intended by design. The Trp-containing peptides reproduce even the unusual positive phi angle for the Gln residue, with the indole rings in the preferred edge-to-face orientation. For the first time the beta-hairpin-stabilising capacities of a disulfide bond and a TrpTrp pair are compared in the same model system. We found that the contribution to stability of the noncovalent indole-indole interaction is larger than that of the covalent disulfide bond, and that their combination gives rise to an even more stable beta-hairpin.

Further evidence for 2-alkyl-2-carboxyazetidines as gamma-turn inducers.

Reverse turns, a common motif in proteins and peptides, have attracted attention due to their relevance in a wide variety of biological processes. In an attempt to artificially imitate and stabilize these turns in short peptides, we have developed versatile synthetic methodologies for the preparation of 2-alkyl-2-carboxyazetidines and incorporated them into the i + 1 position of model tetrapeptides, where they have shown a tendency to induce gamma-turns. However, to ascertain the general utility of these restricted amino acids as gamma-type reverse turn inducers, it was then required to study the conformational preferences when located at other positions. To this end, model tetrapeptides R-CO-Ala-Xaa-NHMe, containing differently substituted azetidine moieties (Xaa = Aze, 2-MeAze, 2-BnAze) at the i + 2 position, were synthesized and subjected to a thorough conformational analysis. The theoretical and experimental results obtained, including the X-ray diffraction structure of a dipeptide derivative containing this skeleton, provide evidence that the 2-alkyl-2-carboxyazetidine scaffold is able to efficiently induce gamma-turns when incorporated into these short peptides, irrespective of their localization in the peptide chain.

Synthesis and biological properties of beta-turned Abeta(31-35) constrained analogues.

A series of constrained pentapeptide analogues of the fragment Abeta(31-35) has been prepared using solid phase synthesis protocols. The results of conformational studies and surface plasmon resonance (SPR) experiments seem to indicate that the affinity of these constrained analogues for immobilized Abeta(25-35) peptide could be related to their ability to adopt a Leu34N-Ile31O beta-turn-like folded conformation.

Exceptional stereoselectivity in the synthesis of 1,3,4-trisubstituted 4-carboxy beta-lactam derivatives from amino acids.

[reaction: see text] The base-promoted cyclization of optically pure N-(p-methoxybenzyl)-N-(2-chloro)propionyl amino acid derivatives resulted in a diastereo- and enantioselective approach to valuable 1,3,4,4-tetrasubstituted beta-lactams. The stereochemical outcome of the reaction is exclusively governed by the configuration of the N-(2-chloro)propionyl moiety.

Nasopharyngeal carcinoma: 30-year experience of a single institution in a non-endemic area.

PURPOSE: Over the past years, radiotherapy techniques have changed significantly. The impact of these changes in the management of nasopharyngeal carcinoma (NPC) has not been fully evaluated. METHODS/PATIENTS: Between 1984 and 2014, 223 NPC were diagnosed in our hospital. Prior to 2000, patients were treated with 2D treatment plan (RT2D) that evolved to 3D schemes thereafter (RT3D). RESULTS: Tumors in the RT3D period showed significantly lower stages than those in the RT2D period. 5-year cause-specific survival improved from 55.7% (95% CI: 46.7-64.7%) in the RT2D period to 78.7% (95% CI: 68.7-88.7%) in the RT3D period (P = 0.006). This difference was greater for non-keratinizing NPC, where specific survival went from 63.2% (95% CI: 52.2-74.2%) to 84.4% (95% CI: 74.4-94.4%) (P = 0.014). CONCLUSION: Recent changes in treatment strategies including concurrent chemoradiation and 3D radiotherapy may have impacted in better survival for NPC. Improved imaging techniques may have contributed by earlier detection and better treatment planning.

Synthesis, high-throughput screening and pharmacological characterization of ß-lactam derivatives as TRPM8 antagonists.

The mammalian transient receptor potential melastatin channel 8 (TRPM8), highly expressed in trigeminal and dorsal root ganglia, mediates the cooling sensation and plays an important role in the cold hypersensitivity characteristic of some types of neuropathic pain, as well as in cancer. Consequently, the identification of selective and potent ligands for TRPM8 is of great interest. Here, a series of compounds, having a ß-lactam central scaffold, were prepared to explore the pharmacophore requirements for TRPM8 modulation. Structure-activity studies indicate that the minimal requirements for potent ß-lactam-based TRPM8 blockers are hydrophobic groups (benzyl preferentially or t Bu) on R1, R2, R3 and R5 and a short N-alkyl chain (≤3 carbons). The best compounds in the focused library (41 and 45) showed IC50 values of 46 nM and 83 nM, respectively, in electrophysiology assays. These compounds selectively blocked all modalities of TRPM8 activation, i.e. menthol, voltage, and temperature. Molecular modelling studies using a homology model of TRPM8 identified two putative binding sites, involving networks of hydrophobic interactions, and suggesting a negative allosteric modulation through the stabilization of the closed state. Thus, these ß-lactams provide a novel pharmacophore scaffold to evolve TRPM8 allosteric modulators to treat TRPM8 channel dysfunction.

Protein-Primed Replication of Bacteriophage Φ29 DNA.

The requirement of DNA polymerases for a 3'-hydroxyl (3'-OH) group to prime DNA synthesis raised the question about how the ends of linear chromosomes could be replicated. Among the strategies that have evolved to handle the end replication problem, a group of linear phages and eukaryotic and archaeal viruses, among others, make use of a protein (terminal protein, TP) that primes DNA synthesis from the end of their genomes. The replicative DNA polymerase recognizes the OH group of a specific residue in the TP to initiate replication that is guided by an internal 3' nucleotide of the template strand. By a sliding-back mechanism or variants of it the terminal nucleotide(s) is(are) recovered and the TP becomes covalently attached to the genome ends. Bacillus subtilis phage ϕ29 is the organism in which such a mechanism has been studied more extensively, having allowed to lay the foundations of the so-called protein-primed replication mechanism. Here we focus on the main biochemical and structural features of the two main proteins responsible for the protein-primed initiation step: the DNA polymerase and the TP. Thus, we will discuss the structural determinants of the DNA polymerase responsible for its ability to use sequentially a TP and a DNA as primers, as well as for its inherent capacity to couple high processive synthesis to strand displacement. On the other hand, we will review how TP primes initiation followed by a transition step for further DNA-primed replication by the same polymerase molecule. Finally, we will review how replication is compartmentalized in vivo.

Transient Receptor Potential Melastatin 8 Channel (TRPM8) Modulation: Cool Entryway for Treating Pain and Cancer.

TRPM8 ion channels, the primary cold sensors in humans, are activated by innocuous cooling (<28 °C) and cooling compounds (menthol, icilin) and are implicated in sensing unpleasant cold stimuli as well as in mammalian thermoregulation. Overexpression of these thermoregulators in prostate cancer and in other life-threatening tumors, along with their contribution to an increasing number of pathological conditions, opens a plethora of medicinal chemistry opportunities to develop receptor modulators. This Perspective seeks to describe current known modulators for this ion channel because both agonists and antagonists may be useful for the treatment of most TRPM8-mediated pathologies. We primarily focus on SAR data for the different families of compounds and the pharmacological properties of the most promising ligands. Furthermore, we also address the knowledge about the channel structure, although still in its infancy, and the role of the TRPM8 protein signalplex to channel function and dysfunction. We finally outline the potential future prospects of the challenging TRPM8 drug discovery field.

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Mutational analysis of phi29 DNA polymerase residues acting as ssDNA ligands for 3'-5' exonucleolysis.

Here, three highly conserved amino acid residues have been characterized to function as ssDNA binding ligands at the 3'-5' exonuclease active site of phi29 DNA polymerase. One of these residues, Phe65, belongs to motif Exo II, previously described to contain an invariant aspartate and an invariant asparagine involved in catalysis and ssDNA binding, respectively. The other two residues, Ser122 and Leu123, form a newly identified motif "(S/T)Lx2h", and are the homologous counterparts of Pol I residues Asp457 and Met458, and of T4 DNA polymerase residues Ser286 and Leu287, the latter three residues shown to contact ssDNA at their corresponding cocrystal 3D structures. Site-directed mutagenesis and biochemical analysis of eight phi29 DNA polymerase mutant proteins at residues Phe65, Ser122 and Leu123 indicated their functional importance for: (1) a stable interaction with ssDNA; (2) 3'-5' exonucleolysis of ssDNA substrates; (3) proofreading of DNA polymerization errors. Extrapolation to the crystal structures of Klenow and T4 DNA polymerases indicates that the invariant aromatic ring contiguous to the catalytic aspartate of the Exo II motif, corresponding to Tyr423 in Klenow, Phe218 in T4, and Phe65 in phi29 DNA polymerase, appears to be critical to orient the ssDNA substrate in a stable conformation to allow 3'-5' exonucleolytic catalysis. This is the first time that the functional importance of this invariant residue, belonging to the Exo II motif, has been demonstrated.

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.

An invariant lysine residue is involved in catalysis at the 3'-5' exonuclease active site of eukaryotic-type DNA polymerases.

A lysine residue, contained in the motif "Kx2h", has been invariantly found in the eukaryotic-type (family B) class of DNA-dependent DNA polymerases with a proofreading function. The importance of this lysine has been assessed by site-directed mutagenesis in the corresponding residue (Lys143) of phi29 DNA polymerase. Substitution of this residue either by arginine or isoleucine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, giving a characteristic distributive pattern that contrasts with the processive pattern displayed by the wild-type phi29 DNA polymerase. Exonuclease assays carried out in the presence of a DNA trap, together with direct analysis of enzyme/ssDNA interaction, allowed us to conclude that this altered pattern was due to a reduction in the catalytic rate of these mutants, but not to a weakened association with ssDNA. These phenotypes indicate that the lysine residue of motif Kx2h plays an auxiliary role in catalysis of the exonuclease reaction, in very good agreement with recent crystallographic data showing that the lysine homologue of T4 DNA polymerase is indirectly involved in metal binding at the 3'-5' exonuclease active site. In agreement with a critical role in proofreading, substitution of Lys143 of phi29 DNA polymerase by arginine or isoleucine produced mutator enzymes that displayed a high frequency of misincorporation. Mutants at Lys143 also showed a reduced DNA polymerization capacity, but only when DNA synthesis was coupled to strand-displacement, an intrinsic property of phi29 DNA polymerase that is specifically affected by mutations at residues directly or indirectly involved in metal binding at the 3'-5' exonuclease active site.

From 1-acyl-beta-lactam human cytomegalovirus protease inhibitors to 1-benzyloxycarbonylazetidines with improved antiviral activity. A straightforward approach to convert covalent to noncovalent inhibitors.

Starting from the structure of known beta-lactam covalent human cytomegalovirus (HCMV) protease inhibitors and from the knowledge of the residues implicated in the active site of this enzyme, we designed a series of phenylalanine-derived 2-azetidinones bearing a 4-carboxylate moiety that could be apt for additional interactions with the guanidine group of the Arg165/Arg166 residues of the viral protease. Some compounds within this series showed anti-HCMV activity at 10-50 muM, but rather high toxicity. The presence of aromatic 1-acyl groups and a certain hydrophobic character in the region of the 4-carboxylate were stringent requirements for anti-HCMV activity. To go a step ahead into the search for effective HCMV medicines, we then envisaged a series of noncovalent inhibitors by simple deletion of the carbonyl group in the beta-lactam derivatives to provide the corresponding azetidines. This led to low micromolar inhibitors of HCMV replication, with 17 and 27 being particularly promising lead compounds for further investigation, although their toxicity still needs to be lowered.

Recurrence of thymic hyperplasia after thymectomy in myasthenia gravis. Its importance as a cause of failure of surgical treatment.

Persistence of symptoms in patients with myasthenia gravis who have undergone previous thymectomy has been attributed to thymus remnants. Patients with partial or no recovery were studied 40 +/- 31 months (mean +/- SD) after surgery, which had been carried out by the transcervical approach in 20 and trans-sternal approach in four. Lateral x-ray tomography of the mediastinum after injection of air showed images compatible with residual thymus gland in 18 patients (75 percent). Thirteen of these underwent reoperation by the trans-sternal approach, and thymic tissue was found in 11 (85 percent). After repeated thymectomy, 67 percent of the patients improved clinically. Therefore, it is quite reasonable to infer that incomplete removal of the thymus was responsible, at least partly, for failure of the first procedure.

Experimental study of a new porous tracheal prosthesis.

We studied the efficacy of a new tracheal prosthesis made of expanded polytetrafluorethylene reinforced with spiral silicone rings to repair circumferential tracheal defects in rabbits. Results showed an adequate consistency of prosthesis, adequate tolerance without producing tracheal stenoses, and impermeability to air, allowing a correct invasion by granulation tissue. This process was faster than any found in any other porous tracheal implant so far tested. We proved that epithelialization results from capillary invasion through the prosthetic pores and from growth from both tracheal ends. We conclude that this prosthetic material can be useful in repairing tracheal defects and may be the optimal tracheal graft for humans.

Induction of nitric oxide synthase in human mammary arteries in vitro.

This study investigated whether human mammary arteries express an inducible nitric oxide (NO) synthase and, if so, what its effects are on vascular tone. In human mammary artery pre-contracted with phenylephrine there was a gradual time-dependent loss of tone over an 8 h period. L-Arginine and lipopolysaccharide enhanced the rate but not the magnitude of this loss in tone, whereas NG-nitro-L-arginine, NG-monomethyl-L-arginine, dexamethasone, and polymyxin B inhibited these effects. These findings indicate that incubation of human mammary artery with lipopolysaccharide resulted in the expression of an inducible NO synthase. The induction of this enzyme in human vessels may be important in the pathogenesis of septic shock.