Intralesional Infiltrations of Arteriosclerotic Tissue Cells-Free Filtrate Reproduce Vascular Pathology in Healthy Recipient Rats.

Lower-extremity arterial disease is a major health problem with increasing prevalence, often leading to non-traumatic amputation, disability and mortality. The molecular mechanisms underpinning abnormal vascular wall remodeling are not fully understood. We hypothesized on the existence of a vascular tissue memory that may be transmitted through soluble signaling messengers, transferred from humans to healthy recipient animals, and consequently drive the recapitulation of arterial wall thickening and other vascular pathologies. We examined the effects of the intralesional infiltration for 6 days of arteriosclerotic popliteal artery-derived homogenates (100 µg of protein) into rats' full-thickness wounds granulation tissue. Animals infiltrated with normal saline solution or healthy brachial arterial tissue homogenate obtained from traumatic amputation served as controls. The significant thickening of arteriolar walls was the constant outcome in two independent experiments for animals receiving arteriosclerotic tissue homogenates. This material induced other vascular morphological changes including an endothelial cell phenotypic reprogramming that mirrored the donor's vascular histopathology. The immunohistochemical expression pattern of relevant vascular markers appeared to match between the human tissue and the corresponding recipient rats. These changes occurred within days of administration, and with no cross-species limitation. The identification of these "vascular disease drivers" may pave novel research avenues for atherosclerosis pathobiology.

Tetracycline Derivatives Inhibit Plasmodial Cysteine Protease Falcipain-2 through Binding to a Distal Allosteric Site.

Allosteric inhibitors regulate enzyme activity from remote and usually specific pockets. As they promise an avenue for less toxic and safer drugs, the identification and characterization of allosteric inhibitors has gained great academic and biomedical interest in recent years. Research on falcipain-2 (FP-2), the major papain-like cysteine hemoglobinase of Plasmodium falciparum, might benefit from this strategy to overcome the low selectivity against human cathepsins shown by active site-directed inhibitors. Encouraged by our previous finding that methacycline inhibits FP-2 noncompetitively, here we assessed other five tetracycline derivatives against this target and characterized their inhibition mechanism. As previously shown for methacycline, tetracycline derivatives inhibited FP-2 in a noncompetitive fashion, with Ki values ranging from 121 to 190 µM. A possible binding to the S' side of the FP-2 active site, similar to that described by X-ray crystallography (PDB: 6SSZ) for the noncompetitive inhibitor E-chalcone 48 (EC48), was experimentally discarded by kinetic analysis using a large peptidyl substrate spanning the whole active site. By combining lengthy molecular dynamics (MD) simulations that allowed methacycline to diffuse from solution to different FP-2 surface regions and free energy calculations, we predicted the most likely binding mode of the ligand. Of note, the proposed binding pose explains the low differences in Ki values observed for the tested tetracycline derivatives and the calculated binding free energies match the experimental values. Overall, this study has implications for the design of novel allosteric inhibitors against FP-2 and sets the basis for further optimization of the tetracycline scaffold to produce more potent and selective inhibitors.

Heberprot-P en el tratamiento de las úlceras del pie diabético / Heberprot-P in the treatment of diabetic foot ulcers

RESUMEN Introducción: El pie diabético es una de las complicaciones más temible de la diabetes mellitus; es el causante del 80 % de las amputaciones no traumáticas a nivel mundial. Durante décadas se han utilizados diferentes tratamientos para la cicatrización del pie diabético; los factores de crecimiento han revolucionado esta terapéutica. La participación de un equipo multidisciplinario con el cumplimiento de los protocolos de actuación y el empleo del Heberprot-P disminuyen el índice de amputaciones en pacientes con esta afección. Objetivo: Presentar la evolución de pacientes con úlceras del pie diabético, en diferentes estadios clínicos, tratados con Heberprot-P. Desarrollo: Se presentan 3 casos clínicos; el primero es un paciente masculino, de 56 años, diabético, con amputación transmetatarsiana abierta en la cual se realizaron 16 aplicaciones del Heberprot-P y se colocó un injerto de piel, la lesión cerró en 45 días. El segundo es un paciente de 58 años, diabético e hipertenso, con lesión en el pie derecho, con una cirugía vascular previa; se realizaron 19 aplicaciones del Heberprot-P y la lesión cicatrizó en 49 días. El tercer caso es un paciente de 55 años, que ingresa por la una lesión extensa en el pie derecho, se le realizaron 22 aplicaciones de Heberprot-P, se logró el cierre de la lesión en 85 días y se evitó la amputación. Conclusiones: Los casos presentados evolucionaron de forma favorable con el tratamiento de Heberprot-P.

ABSTRACT Introduction: The diabetic foot is one of the most feared complications of diabetes mellitus; It is the cause of 80 % of non-traumatic amputations worldwide. For decades, different treatments have been used to heal diabetic foot; growth factors have revolutionized this therapy. The participation of a multidisciplinary team with compliance with the action protocols and the use of Heberprot-P decrease the rate of amputations in patients with this condition. Objective: To present the evolution of patients with diabetic foot ulcers, in different clinical stages, treated with Heberprot-P. Development: Three clinical cases are presented; The first is a male patient, 56 years old, diabetic, with open transmetatarsal amputation in which 16 applications of Heberprot-P were performed and a skin graft was placed, the lesion closed in 45 days. The second is a 58-year-old patient, diabetic and hypertensive, with a right foot injury, with previous vascular surgery; 19 applications of Heberprot-P were performed and the lesion healed in 49 days. The third case is a 55-year-old patient, who was admitted due to an extensive injury to his right foot, 22 applications of Heberprot-P were performed, closure of the injury was achieved in 85 days and amputation was avoided. Conclusions: The presented cases evolved favorably with Heberprot-P treatment.

d-Amino acid substitutions and dimerization increase the biological activity and stability of an IL-15 antagonist peptide.

Interleukin (IL)-15 plays an important role in several inflammatory diseases. We have previously identified an IL-15 antagonist called P8 peptide, which binds specifically to IL-15 receptor alpha subunit. However, the P8 peptide rapidly degraded by proteases, limiting its therapeutic application. Thus, we replaced each P8 peptide l-amino acid by its corresponding d-isomers. First, we determined the biological activity of the resulting peptides in a proliferation assay by using CTLL-2 cells. The substitution of l-Ala by d-Ala ([A4a]P8 peptide) increased the inhibitory effect of the P8 peptide in CTLL-2 cells in five-fold. In addition to that, the [A4a]P8 peptide dimer showed the most inhibitory effect. To protect the [A4a]P8 peptide and its dimer against exopeptidase activity, we acetylated the N-terminal of these peptides. At least a three-fold reduction in antagonist activity of acetylated peptides was exhibited. However, the substitution of the N-terminal l-Lys residue of [A4a]P8 peptide and its dimer by d-Lys ([K1k;A4a]P8 peptide) did not affect the antagonist effect of the aforementioned peptides. The [K1k;A4a]P8 peptide dimer was stable to the degradation of trypsin, chymotrypsin, and pepsin up until 48 min. Also, the safety and immunogenicity studies in healthy BALB/c mice demonstrated that the administration of this peptide did not affect the clinical parameters of the animals nor generated antipeptide antibodies. Our findings reveal that two distinct d-amino acid substitutions and dimerization increase the biological activity and stability of P8 peptide. The resulting peptide constitutes a novel IL-15 antagonist with potential applicability in inflammatory diseases.

In vitro and in vivo characterization of an interleukin-15 antagonist peptide by metabolic stability, 99m Tc-labeling, and biological activity assays.

Interleukin (IL)-15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL-15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with 99m Tc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL-2 cells, using 3 different additives to improve the solubility of these peptides. The half-life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with 99m Tc showed a yield of approximately 99.8%. The 99m Tc-labeled peptide was stable in a 30-fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL-15 overexpression.

Aminocatalysis-Mediated on-Resin Ugi Reactions: Application in the Solid-Phase Synthesis of N-Substituted and Tetrazolo Lipopeptides and Peptidosteroids.

A new solid-phase protocol for the synthesis of N-substituted and tetrazolo peptides is described. The strategy relies on the combination of aminocatalysis-mediated on-resin Ugi reactions and peptide couplings for the N-alkylation of peptides at selected sites, including the N-terminal double lipidation, the simultaneous lipidation/biotinylation, and the steroid/lipid conjugation via tetrazole ring formation. The solid-phase Ugi four-component reactions were enabled by on-resin transimination steps prior to addition of the acid and isocyanide components. The strategy proved to be suitable for the feasible incorporation of complex N-substituents at both termini and at internal positions, which is not easily achievable by other solid-phase methods.

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae).

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.

LPS inmobilization on porous and non-porous supports as an approach for the isolation of anti-LPS host-defense peptides.

Lipopolysaccharides (LPSs) are the major molecular component of the outer membrane of Gram-negative bacteria. This molecule is recognized as a sign of bacterial infection, responsible for the development of local inflammatory response and, in extreme cases, septic shock. Unfortunately, despite substantial advances in the pathophysiology of sepsis, there is no efficacious therapy against this syndrome yet. As a consequence, septic shock syndrome continues to increase, reaching mortality rates over 50% in some cases. Even though many preclinical studies and clinical trials have been conducted, there is no Food and Drug Administration-approved drug yet that interacts directly against LPS. Cationic host-defense peptides (HDPs) could be an alternative solution since they possess both antimicrobial and antiseptic properties. HDPs are small, positively charged peptides which are evolutionarily conserved components of the innate immune response. In fact, binding to diverse chemotypes of LPS and inhibition of LPS-induced pro-inflammatory cytokines from macrophages have been demonstrated for different HDPs. Curiously, none of them have been isolated by their affinity to LPS. A diversity of supports could be useful for such biological interaction and suitable for isolating HDPs that recognize LPS. This approach could expand the rational search for anti-LPS HDPs.

Functional characterization of a synthetic hydrophilic antifungal peptide derived from the marine snail Cenchritis muricatus.

Antimicrobial peptides have been found in mollusks and other sea animals. In this report, a crude extract of the marine snail Cenchritis muricatus was evaluated against human pathogens responsible for multiple deleterious effects and diseases. A peptide of 1485.26 Da was purified by reversed-phase HPLC and functionally characterized. This trypsinized peptide was sequenced by MS/MS technology, and a sequence (SRSELIVHQR), named Cm-p1 was recovered, chemically synthesized and functionally characterized. This peptide demonstrated the capacity to prevent the development of yeasts and filamentous fungi. Otherwise, Cm-p1 displayed no toxic effects against mammalian cells. Molecular modeling analyses showed that this peptide possible forms a single hydrophilic α-helix and the probable cationic residue involved in antifungal activity action is proposed. The data reported here demonstrate the importance of sea animals peptide discovery for biotechnological tools development that could be useful in solving human health and agribusiness problems.

Bibliotecas combinatorias en fase sólida / Combinatorial libraries on solid phase

La búsqueda de nuevos fármacos requiere de la síntesis y la evaluación de muchos compuestos diferentes. Un factor que limita estos estudios ha sido el tiempo y el esfuerzo empleado en la obtención de estas moléculas. Por tanto es importante disponer de bibliotecas combinatorias de compuestos que se caractericen| por una gran variedad en cuanto a un factor determinado: composición química, estructura etc. Se conocen cuatro grupos de biblotecas combinatorias: las biológicas, restringidas a la obtención de compuestos naturales (proteínas, péptidos, anticuerpos, etc.), las que se alcanzan por síntesis paralela de estructuras definidas y que se aplican a la obtención de péptidos oligonucleótidos y moléculas orgánicas pequeñas(tiadiazinas, didantoinas, etc.); las bibliotecas con deconvolución de las mezclas, con similares usos y las bibliotecas inmovilizadas sobre un soporte sólido que se emplea fundamentalmente para la obtención de péptidos y péptidos modificados(AU)