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1.
Chem Biol Drug Des ; 104(1): e14588, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-39048531

RESUMEN

Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.


Asunto(s)
Adrenomedulina , Dicroismo Circular , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Humanos , Adrenomedulina/química , Adrenomedulina/farmacología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Animales , Simulación por Computador , Precursores de Proteínas/química , Precursores de Proteínas/farmacología , Precursores de Proteínas/metabolismo , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Estructura Secundaria de Proteína
2.
Biochem Pharmacol ; 224: 116235, 2024 06.
Artículo en Inglés | MEDLINE | ID: mdl-38670438

RESUMEN

Calcitonin gene-related peptides alpha and beta (αCGRP, ßCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, -2, or -3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1-47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8-47) and AM2/IMD-53 exhibited even longer duration signaling than the 1-47 fragment. AM2/IMD(8-47) was a striking 8-fold longer acting than AM(13-52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1-52) and (13-52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. ßCGRP was âˆ¼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of ßCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.


Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Transducción de Señal , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/química , Humanos , Transducción de Señal/fisiología , Células HEK293 , AMP Cíclico/metabolismo , Adrenomedulina/metabolismo , Adrenomedulina/química , Adrenomedulina/genética , Secuencia de Aminoácidos
3.
J Biol Chem ; 299(6): 104785, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37146967

RESUMEN

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.


Asunto(s)
Adrenomedulina , Péptido Relacionado con Gen de Calcitonina , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Adrenomedulina , Receptores Acoplados a Proteínas G , Animales , Humanos , Adrenomedulina/química , Adrenomedulina/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/metabolismo , Chlorocebus aethiops , Células COS , AMP Cíclico/metabolismo , Células HEK293 , Modelos Moleculares , Simulación de Dinámica Molecular , Estabilidad Proteica , Proteínas Modificadoras de la Actividad de Receptores/química , Proteínas Modificadoras de la Actividad de Receptores/genética , Proteínas Modificadoras de la Actividad de Receptores/metabolismo , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal
4.
J Biochem ; 170(4): 445-451, 2021 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-33964134

RESUMEN

Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.


Asunto(s)
Adrenomedulina/análogos & derivados , Adrenomedulina/química , Albúmina Sérica Humana/química , Adrenomedulina/farmacocinética , Animales , Disponibilidad Biológica , Cromatografía en Gel/métodos , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Masculino , Maleimidas/metabolismo , Peso Molecular , Ratas , Ratas Wistar , Receptores de Adrenomedulina/metabolismo , Albúmina Sérica Humana/farmacocinética
5.
Biochem Biophys Res Commun ; 529(3): 778-783, 2020 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-32736707

RESUMEN

Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13-44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.


Asunto(s)
Adrenomedulina/química , Adrenomedulina/metabolismo , Trombina/metabolismo , Adrenomedulina/síntesis química , Adrenomedulina/farmacocinética , Animales , Células HEK293 , Humanos , Masculino , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacocinética , Proteolisis , Ratas Wistar
6.
Peptides ; 131: 170347, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32569606

RESUMEN

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids and plays a pivotal role in the regulation of many physiological processes, particularly those of the cardiovascular and lymphatic system. Like calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin (IMD) and amylin (AMY), it belongs to the CT/CGRP family of peptide hormones, which despite their low little sequence identity share certain characteristic structural features as well as a complex multicomponent receptor system. ADM, IMD and CGRP exert their biological effects by activation of the calcitonin receptor-like receptor (CLR) as a complex with one of three receptor activity-modifying proteins (RAMP), which alter the ligand affinity. Selectivity within the receptor system is largely mediated by the amidated C-terminus of the peptide hormones, which bind to the extracellular domains of the receptors. This enables their N-terminus consisting of a disulfide-bonded ring structure and a helical segment to bind within the transmembrane region and to induce an active receptor confirmation. ADM is expressed in a variety of tissues in the human body and is fundamentally involved in multitude biological processes. Thus, it is of interest as a diagnostic marker and a promising candidate for therapeutic interventions. In order to fully exploit the potential of ADM, it is necessary to improve its pharmacological profile by increasing the metabolic stability and, ideally, creating receptor subtype-selective analogs. While several successful attempts to prolong the half-life of ADM were recently reported, improving or even retaining receptor selectivity remains challenging.


Asunto(s)
Adrenomedulina/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcitonina/metabolismo , Enfermedades Cardiovasculares/metabolismo , Neoplasias/metabolismo , Hormonas Peptídicas/metabolismo , Adrenomedulina/química , Adrenomedulina/genética , Adrenomedulina/uso terapéutico , Animales , Sitios de Unión , Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/metabolismo , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Regulación de la Expresión Génica , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Sistema Linfático/efectos de los fármacos , Sistema Linfático/metabolismo , Sistema Linfático/patología , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Hormonas Peptídicas/genética , Unión Proteica , Transducción de Señal
7.
Biochem Biophys Res Commun ; 527(3): 744-750, 2020 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-32439180

RESUMEN

Proadrenomedullin N-terminal 20 peptide (PAMP) is a regulatory peptide that is found in various cell types. It is involved in many biological activities and is rich in basic and hydrophobic amino acids, a common feature of antimicrobial peptides (AMPs). In this study, the cell selectivity and antimicrobial mechanism of PAMP and its C-terminal peptide, PAMP(9-20), were investigated. PAMP and PAMP(9-20) displayed potent antimicrobial activity (minimum inhibitory concentration: 4-32 µM) against standard bacterial strains, but showed no hemolytic activity even at the highest tested concentration of 256 µM. PAMP(9-20) showed 2- to 4-fold increase in antimicrobial activity against gram-negative bacteria compared to PAMP. Cytoplasmic membrane depolarization, leakage of calcein dye from membrane mimic liposomes, SYTOX Green uptake, membrane permeabilization, and flow cytometry studies indicated that the major target of PAMP and PAMP(9-20) is not the microbial cell membrane. Interestingly, laser-scanning confocal microscopy demonstrated that FITC-labeled PAMP and PAMP(9-20) enter the cytoplasm of Escherichia coli similar to buforin-2, and gel retardation assay indicated that PAMP and PAMP(9-20) effectively bind to bacterial DNA. These results suggest that the intracellular target mechanism is responsible for the antimicrobial action of PAMP and PAMP(9-20). Collectively, PAMP and PAMP(9-20) could be considered promising candidates for the development of new antimicrobial agents.


Asunto(s)
Adrenomedulina/farmacología , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/farmacología , Adrenomedulina/química , Animales , Antibacterianos/química , Bacterias/metabolismo , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , ADN Bacteriano/metabolismo , Hemólisis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Ovinos
8.
Biochem Biophys Res Commun ; 523(4): 939-946, 2020 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-31964533

RESUMEN

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.


Asunto(s)
Adrenomedulina/uso terapéutico , Membrana Celular/metabolismo , Escherichia coli Enterohemorrágica/fisiología , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Inflamación/microbiología , Intestinos/microbiología , Sustancias Protectoras/farmacología , Adrenomedulina/química , Adrenomedulina/farmacología , Secuencia de Aminoácidos , Animales , Antiinfecciosos/farmacología , Antiinfecciosos/uso terapéutico , Membrana Celular/efectos de los fármacos , Citocinas/metabolismo , Escherichia coli Enterohemorrágica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Virulencia/genética
9.
Peptides ; 121: 170133, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31449828

RESUMEN

Adrenomedullin (AM) is a multifunctional bioactive peptide. Recent studies have shown that AM has protective effects against ischemic brain damage. We recently prepared a long-acting human AM derivative that was conjugated with a 60 kDa polyethylene glycol (PEG-AM), which had an effect similar to that of native AM. In this study, we examined the effect of PEG-AM on four-vessel occlusion model rats, which exhibit vascular dementia. From day 10 to day 14 after surgery, the learning and memory abilities of the rats were examined using a Morris water maze. The rats were treated with a single subcutaneous injection of 1.0 or 10.0 nmol/kg of PEG-AM. PEG-AM treatment reduced the escape latency in the hidden platform test. Furthermore, the treatment increased the time spent in the platform quadrant in the probe test. The data showed that PEG-AM injection prevented memory loss and learning disorders in dose-dependent manner. On day 14, the immunoreactive AM concentration in plasma was 9.749 ±â€¯2.167 pM in the high-dose group (10.0 nmol/kg) and 0.334 ±â€¯0.073 pM in the low-dose group (1.0 nmol/kg). However, even in the low-dose group, a significant effect was observed in both tests. The present data indicate that PEG-AM is a possible therapeutic agent for the treatment of ischemic brain injury or vascular dementia.


Asunto(s)
Adrenomedulina/farmacología , Lesiones Encefálicas/tratamiento farmacológico , Demencia Vascular/tratamiento farmacológico , Glicoconjugados/farmacología , Nootrópicos/farmacología , Daño por Reperfusión/tratamiento farmacológico , Vasodilatadores/farmacología , Insuficiencia Vertebrobasilar/tratamiento farmacológico , Adrenomedulina/química , Adrenomedulina/farmacocinética , Animales , Lesiones Encefálicas/fisiopatología , Demencia Vascular/fisiopatología , Modelos Animales de Enfermedad , Esquema de Medicación , Glicoconjugados/química , Glicoconjugados/farmacocinética , Humanos , Inyecciones Subcutáneas , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria/efectos de los fármacos , Memoria/fisiología , Nootrópicos/química , Nootrópicos/farmacocinética , Polietilenglicoles/química , Ratas , Ratas Wistar , Daño por Reperfusión/fisiopatología , Resultado del Tratamiento , Vasodilatadores/química , Vasodilatadores/farmacocinética , Insuficiencia Vertebrobasilar/fisiopatología
10.
Biochemistry ; 58(32): 3468-3474, 2019 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-31328503

RESUMEN

Adrenomedullin 2 (AM2) is a peptide hormone with potent effects in the cardiovascular system. The N-terminal disulfide loop of AM2 is thought to be important for interacting with its receptors to initiate a signaling response. However, the relative contribution of each amino acid within this region is currently unknown. Thus, the region was investigated using an alanine scanning approach. Two AM2 peptides (AM2-47 and AM2-40) were directly compared at the CGRP, AM1, and AM2 receptors in transfected Cos7 cells and found to have equivalent activity. Analogues of AM2-40 were then synthesized, substituting each individual amino acid within the disulfide loop with alanine. The ability of these analogues to stimulate a cAMP response was evaluated at the CGRP, AM1, and AM2 receptors. AM2-40 L12A and T14A were less able to elicit cAMP responses through all tested receptors. In contrast, AM2-40 G13A was slightly more potent than the unmodified peptide at all tested receptors. Thus, it appears that residues within the disulfide loop region play differential roles in the ability of AM2 to stimulate cAMP production. The data provide the first structure-function investigation of AM2 agonism.


Asunto(s)
Adrenomedulina/química , Adrenomedulina/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , AMP Cíclico/biosíntesis , Humanos
11.
Sci Rep ; 9(1): 6609, 2019 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-31036871

RESUMEN

Endothelial dysfunction is a core pathophysiologic process in pulmonary arterial hypertension (PAH). We developed PulmoBind (PB), a novel imaging biomarker of the pulmonary vascular endothelium. 99mTechnetium (99mTc)-labelled PB binds to adrenomedullin receptors (AM1) densely expressed in the endothelium of alveolar capillaries. We evaluated the effect of sildenafil on AM1 receptors activity using 99mTc-PB. PAH was induced in rats using the Sugen/hypoxia model and after 3 weeks, animals were allocated to sildenafil (25 or 100 mg/kg/day) for 4 weeks. 99mTc-PB uptake kinetics was assessed by single-photon emission computed tomography. PAH caused right ventricular (RV) hypertrophy that was decreased by low and high sildenafil doses. Sildenafil low and high dose also improved RV function measured from the tricuspid annulus plane systolic excursion. Mean integrated pulmonary uptake of 99mTc-PB was reduced in PAH (508% · min ± 37, p < 0.05) compared to controls (630% · min ± 30), but unchanged by sildenafil at low and high doses. Lung tissue expressions of the AM1 receptor components were reduced in PAH and also unaffected by sildenafil. In experimental angio-proliferative PAH, sildenafil improves RV dysfunction and remodeling, but does not modify pulmonary vascular endothelium dysfunction assessed by the adrenomedullin receptor ligand 99mTc-PB.


Asunto(s)
Adrenomedulina/análogos & derivados , Biomarcadores/metabolismo , Endotelio Vascular/metabolismo , Hipertensión Pulmonar/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Citrato de Sildenafil/farmacología , Adrenomedulina/química , Adrenomedulina/aislamiento & purificación , Animales , Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/patología , Hipertensión Pulmonar/diagnóstico por imagen , Hipertensión Pulmonar/patología , Pulmón/diagnóstico por imagen , Pulmón/metabolismo , Pulmón/patología , Masculino , Fragmentos de Péptidos/química , Arteria Pulmonar/diagnóstico por imagen , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Ratas , Receptores de Adrenomedulina/química , Receptores de Adrenomedulina/genética , Tecnecio/farmacología
12.
J Biochem ; 166(2): 157-162, 2019 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-30895298

RESUMEN

Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful anti-inflammatory effects. However, treatment required continuous administration of hAM, as the half-life of native hAM is quite short in blood. To resolve this problem, we designed two kinds of human IgG1 Fc fusion proteins containing either full-length hAM (IgG1-AM) or hAM residues 6-52 [IgG1-AM (6-52)]. A DNA construct was constructed by connecting DNA sequences encoding hAM and the IgG1 Fc region with a DNA sequence encoding a (GGGGS)3 linker. The molecular weights of IgG1-AM and IgG1-AM (6-52) were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. By protein sequencing, the N-terminal sequence of both recombinant AM-Fc fusions showed the expected human IgG1 sequence. Sufficient concentrations of both AM-Fc fusions were observed in blood 2 days after a single subcutaneous administration. IgG1-AM and IgG1-AM (6-52) stimulated cAMP production in human embryonic kidney-293 cells stably expressing the AM1 receptor. The activity of IgG1-AM (6-52) was higher than that of IgG1-AM. Treatment with IgG1-AM (6-52) inhibited blood pressure increase in spontaneously hypertensive rats. In addition, IgG1-AM (6-52) reduced total inflammation scores in the dextran sulfate sodium colitis model. Therefore, AM-IgG1 Fc fusions represent potential novel therapeutic agents.


Asunto(s)
Adrenomedulina/inmunología , Colitis/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Inflamación/inmunología , Proteínas Recombinantes/inmunología , Adrenomedulina/química , Adrenomedulina/aislamiento & purificación , Animales , Células Cultivadas , Colitis/terapia , Sulfato de Dextran , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Inflamación/inducido químicamente , Masculino , Pliegue de Proteína , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
13.
J Pept Sci ; 25(3): e3147, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30680847

RESUMEN

Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM-1 receptor (AM1 R). A disulfide-bonded ring structure consisting of six amino acids between Cys16 and Cys21 has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t-Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam-based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether-based mimetics to be well accepted with full AM1 R activity. While a reduced selectivity over the calcitonin gene-related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild-type-like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM1 R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.


Asunto(s)
Adrenomedulina/química , Adrenomedulina/farmacología , Disulfuros/química , Receptores de Adrenomedulina/agonistas , Receptores de Adrenomedulina/metabolismo , Adrenomedulina/síntesis química , Disulfuros/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad
14.
Nucl Med Biol ; 67: 36-42, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30388434

RESUMEN

INTRODUCTION: Adrenomedullin receptors are highly expressed in human alveolar capillaries and provide a molecular target for imaging the integrity of pulmonary microcirculation. In this work, we aimed to develop a NOTA-derivatized adrenomedullin analog (DFH17), radiolabeled with [18F]AlF, for PET imaging of pulmonary microcirculation. METHODS: Highly concentrated [18F](AlF)2+ (15 µL) was produced from purified fluorine-18 in NaCl 0.9%. Various complexation experiments were carried out at Al-to-NOTA molar ratios ranging from 1:1 to 1:40 to assess optimal radiolabeling conditions before using the peptide. DFH17 peptide (2 mM, pH 4) was radiolabeled with [18F](AlF)2+ for 15 min at 100 °C in a total volume of 60 µL. As part of the radiolabeling process, parameters such as fluorine-18 activity (~37 and 1480 MBq), concentration of AlCl3 (0.75, 2, 3, 6 or 10 mM) and the effects of hydrophilic organic solvent (aqueous vs ethanol 50%) were studied. The final formulation was tested for purity, identity and stability in saline. Initial in vivo evaluation of [18F]AlF-DFH17 was performed in normal rats by PET/CT. RESULTS: The scaled-up production of [18F]AlF-DFH17 was performed in high radiochemical and chemical purities in an overall radiochemical yield of 22-38% (at end-of-synthesis) within 60 min. The final formulation was stable in saline at different radioactive concentrations for 8 h. PET evaluation in rats revealed high lung-to-background ratios and no defluorination in vivo up to 1 h post-injection. CONCLUSION: The novel radioconjugate [18F]AlF-DFH17 appears to be a promising PET ligand for pulmonary microcirculation imaging.


Asunto(s)
Adrenomedulina/química , Radioisótopos de Flúor/química , Compuestos Heterocíclicos/química , Tomografía de Emisión de Positrones/métodos , Circulación Pulmonar , Adrenomedulina/farmacocinética , Estabilidad de Medicamentos , Radioisótopos de Flúor/farmacocinética , Compuestos Heterocíclicos con 1 Anillo , Marcaje Isotópico , Distribución Tisular
15.
ChemMedChem ; 13(17): 1797-1805, 2018 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-29979487

RESUMEN

Adrenomedullin (ADM) is a peptide hormone of the calcitonin gene-related peptide (CGRP) family. It is involved in the regulation of cardiovascular processes such as angiogenesis, vasodilation, and the reduction of oxidative stress. ADM mediates its effects by activation of the ADM-1 and -2 receptors (AM1 R/AM2 R), but also activates the CGRP receptor (CGRPR) with reduced potency. It binds to the extracellular domains of the receptors with its C-terminal binding motif (residues 41-52). The activation motif, consisting of a disulfide-bonded ring structure (residues 16-21) and an adjacent helix (residues 22-30), binds to the transmembrane region and stabilizes the receptor conformation in the active state. While it was shown that the binding motif of ADM guides AM1 R selectivity, there is little information on the activation motif itself. Here, we demonstrate that Thr22 of ADM contributes to the selectivity. By using solid-phase peptide synthesis and cAMP-based signal transduction, we studied the effects of analogues in the activation motif of ADM on AM1 R and CGRPR activity. Our results indicate that Thr22 terminates the α-helix and orients the ring segment by hydrogen bonding. Using olefin stapling, we showed that the α-helical arrangement of the ring segment leads to decreased AM1 R activity, but does not affect CGRPR activation. These results demonstrate that the conformation of the ring segment of ADM has a strong impact on the selectivity within the receptor system.


Asunto(s)
Adrenomedulina/farmacología , Proteína Similar al Receptor de Calcitonina/antagonistas & inhibidores , Cardiotónicos/farmacología , Treonina/química , Adrenomedulina/química , Proteína Similar al Receptor de Calcitonina/metabolismo , Cardiotónicos/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad
16.
Mol Neurobiol ; 55(12): 8799-8814, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29600350

RESUMEN

Neurodegenerative diseases represent a heterogeneous group of disorders whose common characteristic is the progressive degeneration of neuronal structure and function. Although much knowledge has been accumulated on the pathophysiology of neurodegenerative diseases over the years, more efforts are needed to understand the processes that underlie these diseases and hence to propose new treatments. Adrenomedullin (AM) is a multifunctional peptide involved in vasodilation, hormone secretion, antimicrobial defense, cellular growth, and angiogenesis. In neurons, AM and related peptides are associated with some structural and functional cytoskeletal proteins that interfere with microtubule dynamics. Furthermore, AM may intervene in neuronal dysfunction through other mechanisms such as immune and inflammatory response, apoptosis, or calcium dyshomeostasis. Alterations in AM expression have been described in neurodegenerative processes such as Alzheimer's disease or vascular dementia. This review addresses the current state of knowledge on AM and its possible implication in neurodegenerative diseases.


Asunto(s)
Adrenomedulina/antagonistas & inhibidores , Terapia Molecular Dirigida , Enfermedades Neurodegenerativas/terapia , Adrenomedulina/química , Animales , Citoesqueleto/metabolismo , Humanos , Modelos Neurológicos , Neuronas/metabolismo
17.
Breastfeed Med ; 12(9): 561-565, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28956619

RESUMEN

INTRODUCTION: Under some circumstances human milk (HM) extraction and refrigerated storage may be necessary. Depending on the length and on the type of cold storage, milk may lose some important properties, but current advices on safe HM storage are discordant. Moreover until now no data in literature were present on the effect of prolonged cold storage on biologically active components of the HM such as adrenomedullin (AM). This important peptide is involved in response to hypoxia and inflammation, associated with neovascularization, in several tissues. The aim is to evaluate: (a) the presence of AM in preterm and term HM and (b) the concentration of AM in refrigerated milk at 4°C at 24-hour intervals, up to 96 hours of storage. MATERIALS AND METHODS: The experiment was repeated four times. Immediately after collection, each HM sample deriving from each mother was divided into two parts as follows: "Pool" line and "Single Mother" line. One part (Pool line) was pooled and then divided into five aliquots. The other part (Single Mother line) was divided into five aliquots. From each line, one aliquot was analyzed within 3 hours, while the others were stored in the refrigerator for 24, 48, 72, and 96 hours, respectively, and then analyzed. AM levels were determined using a specific ELISA test. RESULTS: AM was detectable in all samples. Its concentration was significantly higher in preterm milk with respect to term milk (p < 0.05). Significant differences were observed during the cold storage: the AM levels decreased steadily during the storage and the remaining concentration at 96 hours is ∼2%. DISCUSSION: This study provides evidences regarding the presence of AM in HM, regardless of the gestational age. In particular, the refrigeration of fresh HM in controlled conditions significantly affected its bioactivity and nutritional quality related with AM, already at 24 hours.


Asunto(s)
Adrenomedulina/química , Frío/efectos adversos , Almacenamiento de Alimentos/métodos , Leche Humana/química , Refrigeración , Adrenomedulina/análisis , Femenino , Humanos , Valor Nutritivo , Estabilidad Proteica , Factores de Tiempo
18.
Drug Dev Res ; 78(3-4): 129-134, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28449192

RESUMEN

Preclinical Research Human adrenomedullin (hAM), a hypotensive peptide, also has anti-inflammatory effects. hAM can reduce the severity of the dextran sulphate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in animal models. Furthermore, in a clinical study, hAM treatment reduced the Disease Activity Index in ulcerative colitis. However, these therapeutic effects required continuous administration of hAM as the half-life of native hAM is quite short in blood. To resolve this problem, hAM N-terminal was conjugated with two kinds of polyethylene glycol (PEG); 5 kDa PEG or 60 kDa PEG (5 kDa PEG-hAM and 60 kDa PEG-hAM respectively). In a previous study, 5 kDa PEG-hAM stimulated cAMP production and prolonged the plasma half-life compared with native hAM. Herein we examine the effect of PEG-hAM in the DSS colitis model. Treatment with both PEG-hAM preparations reduced the total inflammation score. In addition, the plasma half-life of 60 kDa PEG-hAM was much longer than 5 kDa PEG-hAM. In summary, a single subcutaneous administration of 60 kDa PEG-hAM reduced the total inflammation score in mice with DSS-induced colitis. Therefore, these results suggest that 60 kDa PEG-hAM is a possible therapeutic agent for the treatment of inflammatory bowel disease. Drug Dev Res 78 : 129-134, 2017. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Adrenomedulina/administración & dosificación , Antiinflamatorios/administración & dosificación , Colitis Ulcerosa/tratamiento farmacológico , Sulfato de Dextran/efectos adversos , Polietilenglicoles/química , Adrenomedulina/química , Adrenomedulina/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Semivida , Humanos , Inyecciones Subcutáneas , Masculino , Ratones
19.
Biosci Trends ; 10(6): 500-506, 2017 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-27840371

RESUMEN

Retinal pericytes play an important role in the maintenance of retinal microvascular homeostasis. We performed a secretory peptidome of primary human retinal pericytes. Using liquid chromatography-tandem mass spectrometry analysis in the culture medium of retinal pericytes, we identified 256 peptides derived from 114 proteins, and identified a novel partial fragment Leu163-His183 (termed ΔADT) of adrenotensin (ADT). To elucidate the role of ΔADT as a soluble mediator of pericyte-endothelial cell interactions, we investigated the bioactivity of ΔADT in human retinal microvascular endothelial cells (HRMVECs). The cell proliferation assay indicated that the proliferation of HRMVECs was promoted by ADT or ΔADT. Moreover, ΔADT had a greater growth promoting effect than ADT in HRMVECs and induced migration and tube formation of HRMVECs. We also observed actin reorganization and that the levels of phosphorylated focal adhesion kinase in ΔADT stimulated HRMVECs. These results showed that ΔADT induces profound actin reorganization and increases the levels of phosphorylated focal adhesion kinase. Collectively, our study showed that ΔADT has an angiogenic activity, and suggested that ΔADT is a novel angiogenic peptide.


Asunto(s)
Adrenomedulina/química , Fragmentos de Péptidos/farmacología , Pericitos/metabolismo , Actinas/metabolismo , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Fragmentos de Péptidos/química , Fosforilación/efectos de los fármacos , Retina/citología
20.
J Med Chem ; 59(12): 5695-705, 2016 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-27166982

RESUMEN

The 52 amino acid peptide hormone adrenomedullin (ADM) plays a major role in the development and regulation of the cardiovascular and lymphatic system and has therefore gained significant interest for clinical applications. Because adrenomedullin exhibits low metabolic stability, enhancement of the plasma half-life is essential for peptide-based drug design. Fluorescently labeled ADM analogues synthesized by Fmoc/t-Bu solid phase peptide synthesis were used to analyze their enzymatic degradation and specific fragmentation pattern in human blood plasma. The determination of important cleavage sites allowed the development of selectively modified peptides in a rational approach. By combination of palmitoylation, lactam-bridging, and Nα-methylation, ADM analogues protected from enzymatic cleavage in human blood were developed and revealed an explicitly elongated half-life of 5 days in comparison to the wild-type in vitro. This triple-modification did not alter the selectivity of the analogues at the AM1 receptor, highlighting their potential for therapeutic applications.


Asunto(s)
Adrenomedulina/metabolismo , Adrenomedulina/sangre , Adrenomedulina/química , Adrenomedulina/farmacología , Células Cultivadas , Estabilidad de Medicamentos , Células HEK293 , Semivida , Humanos , Estructura Molecular , Receptores de Adrenomedulina/agonistas , Receptores de Adrenomedulina/metabolismo , Relación Estructura-Actividad
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