RESUMEN
BACKGROUND: Hemorphins are endogenous hemoglobin-derived peptides that belong to the family of "atypical" opioid peptides with both affinities to opioid receptors and ability to release other endogenous opioid peptides. OBJECTIVE: In the present study, peptide analogues of Valorphin (VV-hemorphin-5) containing amino phosphonate moiety synthesized by solid-phase peptide synthesis (Fmoc-strategy) were investigated for their potential antinociceptive activities and compared to the reference VV-H in formalin- induced model of acute and inflammatory pain in mice. METHODS: The hemorphin analogues were prepared by replacement of the one and/or two N-terminal Val in VV-hemorphin5 (VV-H) with ((dimethoxy phosphoryl) methyl)-L-valine and ((dimethoxy phosphoryl) methyl)-L-leucine to obtain the compounds pVV-H, pL-H, and pLV-H. Aiming to additionally prove the importance of amino acid valine, we introduced the ((dimethoxy phosphoryl) methyl)-L-leucine to the N-side of VV-hemorphin-5 (pLVV-H). The experiments were carried out on adult male ICR mice. All peptides were administered intracerebroventricularly at three doses (50, 25 and 12,5 µg/mouse). We have studied the effects of the peptides on acute (1st phase) and inflammatory (2nd phase) pain reaction using un experimental model with intraplantar formalin injection. RESULTS: VV-H showed a significant antinociceptive effect both in the acute and inflammatory phases of the test. Although Valorphin hexa-, hepta-, and octapeptide analogs demonstrated a significant antinociceptive effect, they showed substantial differences considering their effective dose and the phase of the test as compared to the Valorphin. DISCUSSION: Data showed that modified heptapeptides pVV-H and pLV-H exerted the same or better antinociception in acute and inflammatory pain, in comparison to the reference peptide, while pL-H and pLVV-H analogues were less effective. CONCLUSION: Our study contributes to the elucidation of the role of Valine and the number of amino acid residues in the structure of hemorphin peptide analogs in their effectiveness in suppressing both acute and inflammatory experimental pain.
Asunto(s)
Analgésicos , Formaldehído/toxicidad , Hemoglobinas , Dolor , Fragmentos de Péptidos , Analgésicos/química , Analgésicos/farmacología , Animales , Hemoglobinas/química , Hemoglobinas/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Dolor/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacologíaRESUMEN
In the present study, several new analogues of hemorphin-4, modified with unnatural conformationally restricted amino acids followed the structure Aaa-Tyr-Xxx-Trp-Thr-NH2, where Aaa is the low-molecular-weight lipophilic adamantyl building block, and Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) was synthesized, characterized and investigated for anticonvulsant activity in three seizure tests, the maximal electroshock test (MES), 6-Hz psychomotor seizure test and timed intravenous pentylenetetrazole infusion (ivPTZ) test. The acute neurological toxicity was determined using the rota-rod test. The new synthetic neuropeptide analogues were prepared by solid-phase peptide synthesis-Fmoc chemistry and were evaluated in three doses of 1, 3 and 5 µg, respectively, administered intracerebroventricularly in male ICR mice. The physicochemical properties of these peptide analogues were evaluated as pKa and pI values were calculated using potentiometry. The IR spectrum of the compounds was recorded and the characteristic lines of both adamantane moiety and the peptide backbone were registered in the wavelength range from 4000 to 400 cm-1. The hexapeptide Ang IV was used as a positive control. From the six synthesized peptide analogues, the P4-5 was the most active at doses of 1 and 3 µg in the three seizure tests. The order of potency of other peptides was as follows: P4 > P4-3 = P4-4 > P4-2 > Ang IV in MES, P4-4 ≥ P4-1 > P4-3 > P4-2 > P4 > Ang IV in 6-Hz test and P4-4 = P4-3 > P4-2 = P4 > Ang IV in ivPTZ test. None of the peptides displayed neurotoxicity in the rota-rod test. Docking study results suggest that direct H-bonding and ionic interactions between our synthetic ligands and residues, responsible for coordination of Zn2+ along with hydrophobic interactions between our ligands and IRAP active site are the most important for the ligand binding. The results propose that incorporation of adamantane and cycloalkane building blocks in the peptide chain of the hemorphin-4 scaffold is important for the potential high biological activity.
Asunto(s)
Anticonvulsivantes/química , Hemoglobinas/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/síntesis química , Sitios de Unión , Cistinil Aminopeptidasa/química , Hemoglobinas/administración & dosificación , Hemoglobinas/síntesis química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Infusiones Intraventriculares , Masculino , Ratones , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Convulsiones/prevención & control , Relación Estructura-ActividadRESUMEN
Herein, some new analogues of VV-hemorphin-7, modified at position 4 and 7 by the unnatural amino acids followed the structure Val-Val-Tyr-Xxx-Trp-Thr-Yyy-Arg-Phe-NH2, where Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) and Yyy is Dap (diaminopropanoic acid) or Dab (diaminobutanoic acid), were synthesized, characterized and investigated for anticonvulsant activity. The new synthetic peptide analogues were prepared by standard solid-phase peptide synthesis-Fmoc chemistry. A single intracerebroventricular (i.c.v.) injection at doses of 5, 10, and 20 µg/10 µl, respectively, was given before evaluation with timed intravenous pentylenetetrazole (ivPTZ) infusion test and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. To explain the structure-active properties of the modified peptides, some physicochemical characteristic was obtained. The FT-IR spectra and their second derivatives of the amide I, II, and III bands of the peptides show ß-sheet structure conformation. The calculation of isoelectric points, by potentiometric determination of dissociated constants, is in the range from 9.79 to 10.84. This study, for the first time, also reported on the reduction-oxidative potentials of the guanidine at Arg-moiety on such kind of peptides containing arginine and tyrosine residues in different medium and electrode surface. The VV-hemorphin-7 analogues 4 and 5 were the most active against the ivPTZ test, with the effect comparable to that of peptide 1 used as a positive control. Except compound 8, all other tested peptide analogues were ineffective to raise the threshold for the clonic seizures. The peptide analogue 5 showed 100% protection in the 6-Hz test, while the other seven VV-hemorphin-7 analogues have dose-dependent activity against psychomotor seizures comparable to 1. The novel peptides did not show neurotoxicity in the rotarod test.
Asunto(s)
Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Hemoglobinas/química , Hemoglobinas/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Aminoácidos Cíclicos/química , Animales , Anticonvulsivantes/síntesis química , Ácidos Ciclohexanocarboxílicos/química , Hemoglobinas/síntesis química , Ratones , Conformación Molecular , Oligopéptidos/química , Pentilenotetrazol , Fragmentos de Péptidos/síntesis química , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Técnicas de Síntesis en Fase Sólida , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-ActividadRESUMEN
A new series of N-modified analogues of the VV-hemorphin-5 with aminophosphonate moiety have been synthesized, characterized and investigated for anticonvulsant activity. The novel peptide analogues were prepared by solid-phase peptide synthesis-Fmoc-strategy and were evaluated in the timed intravenous pentylenetetrazole infusion test (ivPTZ) and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. The redox potentials at glass carbonic electrode (GC) and the acid/base properties as pKa values of these peptide analogues were compared with the electrochemical behaviour of tyrosine- and tryptophan-containing peptides using different voltamperometric modes. Among the five tested peptide analogues, V3p was the most active against the ivPTZ test with effect comparable to that of the VV-hemorphin-5 (V1) used as a positive control. Dose-dependent elevation of the seizure threshold for myoclonic twitch and generalized clonic seizures was observed after i.c.v. administration of V2p, V4p and V5p as well as for forelimbs tonus in V4p peptides. The peptide analogues V2p-V5p were able to suppress dose-dependent psychomotor seizures in the 6-Hz test. In contrast, the V6p peptide showed either a pro-convulsant effect in the iv PTZ test or was inactive in the 6-Hz test. No changes in motor coordination were noted with the novel peptides. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of aminophosphonate moiety at position 1 of the VV-hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.
Asunto(s)
Anticonvulsivantes/síntesis química , Anticonvulsivantes/uso terapéutico , Hemoglobinas/síntesis química , Hemoglobinas/uso terapéutico , Organofosfonatos/química , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/uso terapéutico , Animales , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hemoglobinas/química , Hemoglobinas/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Receptores Opioides kappa/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/metabolismo , Técnicas de Síntesis en Fase Sólida , Relación Estructura-ActividadRESUMEN
VV-Hemorphin-5 is an endogenous opioid peptide of the Hemorphin family with affinity at opioid receptors. A series of C-amide analogues have been synthesized, based on the structure of VV-Hemorphin-5, modified at position 1 and 7 by the un/natural amino acids (Aa8-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Aa1-NH2 ) using SPPS, Fmoc-chemistry. The peptide derivatives were evaluated for their anticonvulsant activity in three acute seizure tests in male ICR mice, the maximal electroshock (MES), the 6 Hz psychomotor seizure test, and the timed intravenous pentylenetetrazole (ivPTZ) infusion test. Their neurotoxicity was assessed in the rotarod test. Among the tested peptide analogues, V4 showed anticonvulsant activity in the three seizure tests that was comparable to the VV-Hemorphin-5 (V1) used as a positive control. While V5, V6, and V7 peptide derivatives exhibited anticonvulsant activity in the MES and 6 Hz test, they were inactive (V7) or showed pro-convulsant effect (V5 and V6) in the i.v. PTZ test. At a dose of 10 µg/mouse the peptide V2 was effective against clonic seizures induced by PTZ. Motor coordination was not affected by newly developed analogues of VV-Hemorphin-5. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of nonproteinogenic and/or natural amino acids at position 1 and 7 of the VV-Hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.
Asunto(s)
Anticonvulsivantes/farmacología , Hemoglobinas/farmacología , Péptidos Opioides/farmacología , Fragmentos de Péptidos/farmacología , Convulsiones/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Electrochoque , Hemoglobinas/síntesis química , Hemoglobinas/química , Masculino , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Estructura Molecular , Péptidos Opioides/síntesis química , Péptidos Opioides/química , Pentilenotetrazol , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Unión Proteica , Desempeño Psicomotor/efectos de los fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Relación Estructura-ActividadRESUMEN
In the present study, some new analogues of VV-hemorphin-5, modified at position 1 and 7 by the non-proteinogenic and/or natural amino acids followed the structures Xxx-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Yyy-NH2, where Xxx is Ile or Aib and Yyy is Lys/Orn/Dap/Dab were synthesized to investigate their potential antinociceptive activities. We report also the redox potentials and the acid/base properties as pKa values of these peptide analogues which were compared toward electrochemical behaviour of tryptophan containing peptides. All analogues showed a short lasting initial antinociceptive effect, however H2 hemorphin analogue is characterized with prolong and strong antinociceptive effect, while the other peptide analogues exerted more variable effects on the visceral nociception depending on the dose or time after the intracerebral injection.