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1.
Mar Drugs ; 20(12)2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36547897

RESUMO

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABABR)-coupled N-type calcium channel (CaV2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure-activity relationships of 4/7 type α-CTxs targeting GABABR-coupled CaV2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2-3-fold increase in the inhibition of GABABR-coupled CaV2.2 (IC50 is 0.74 nM); substitutions of position 9-12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABABR-coupled CaV2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of "1-4, 2-3" (ribbon), which differs from the "1-3, 2-4" (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity "1-4, 2-3," are also potent inhibitors for GABABR-coupled CaV2.2, exhibiting potent analgesic activity.


Assuntos
Conotoxinas , Receptores Nicotínicos , Aminoácidos , Conotoxinas/química , Analgésicos/farmacologia , Analgésicos/química , Canais de Cálcio Tipo N/metabolismo , Dissulfetos/química , Ácido gama-Aminobutírico , Receptores Nicotínicos/metabolismo
2.
ACS Chem Biol ; 17(9): 2483-2494, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36048451

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels widely distributed in the central peripheral nervous system and muscles which participate in rapid synaptic transmission. The α9α10 nAChR is an acetylcholine receptor subtype and is involved in chronic pain. In the present study, a new A-superfamily conotoxin Bt14.12 cloned from Conus betulinus was found to selectively inhibit α9α10 nAChRs with an IC50 of 62.3 nM. Unlike α-conotoxins and other A-superfamily conotoxins, Bt14.12 contains a four Cys (C-C-C-C) framework with a unique disulfide bond connection "C1-C4, C2-C3". The structure-activity studies of Bt14.12 demonstrate that all amino acid residues contribute to its potency. Interestingly, mutation experiments show that the deletion of Asp2 or the addition of three Arg residues at the N-terminus of Bt14.12 significantly enhances its inhibitory activity (IC50 is 21.9 nM or 12.7 nM, respectively) by 2- or 4-fold compared to the wild-type Bt14.12. The NMR structure of Bt14.12 shows that it contains α-helix- and ß-turn-like elements, and further computational modelings of the interaction between Bt14.12 and the α9α10 nAChR demonstrate that Bt14.12 possesses a distinctive mode of action and displays a different structure-activity relationship from known α9α10 nAChR targeting α-conotoxins. Our findings provide a novel conotoxin that potently targets α9α10 nAChRs and a new motif for designing potent inhibitors against α9α10 nAChRs.


Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Sequência de Aminoácidos , Aminoácidos , Animais , Conotoxinas/química , Conotoxinas/farmacologia , Caramujo Conus/química , Caramujo Conus/metabolismo , Dissulfetos/metabolismo , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo
3.
Bioorg Med Chem Lett ; 73: 128913, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35914651

RESUMO

A series of novel conjugates of benzoselenazole or selenazole and CPI-1 were designed, synthesized, and evaluated for inhibitory activities against the botulinum neurotoxin A (BoNT/A) light chain (LC) and BoNT/A in vivo. The results show that these compounds exhibit potent inhibitory activities to the LC with IC50 of 0.5-4.1 µM. The reaction kinetics and the mass spectra of the reaction products of LC with benzoselenazole- or selenazole- coupled CPI-1 demonstrate that the benzoselenazole group of most inhibitors is coupled to the LC of BoNT/A. These data indicate that the CPI-1 conjugates can inhibit both the active center of BoNT/A LC as well as Cys165, therefore functioning as irreversible bifunctional inhibitors. The detoxification activities in vivo show that one of the benzoselenazole-CPI-1 compounds prolongs the survival time of mice challenged by 2 × LD50 of BoNT/A. This work provides a new strategy to design potent antidotes of BoNT/A.


Assuntos
Toxinas Botulínicas Tipo A , Animais , Camundongos , Cinética , Ligação Proteica
4.
Front Pharmacol ; 13: 881732, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35754473

RESUMO

A novel 4/8 subtype α-conotoxin, Vt1.27 (NCCMFHTCPIDYSRFNC-NH2), was identified from Conus vitulinus in the South China Sea by RACE methods. The peptide was synthesized and structurally characterized. Similar to other α-conotoxins that target neuronal nicotinic acetylcholine receptor (nAChR) subtypes, Vt1.27 inhibited the rat α3ß2 nAChR subtype (IC50 = 1160 nM) and was inactive at voltage-gated sodium and potassium channels in rat sensory neurons. However, Vt1.27 inhibited high voltage-activated N-type (CaV2.2) calcium channels expressed in HEK293T cells with an IC50 of 398 nM. An alanine scan of the peptide showed that residues Phe5, Pro9, Ile10, and Ser13 contribute significantly to the inhibitory activity of Vt1.27. The molecular dockings indicate that Vt1.27 inhibits the transmembrane region of CaV2.2, which is different from that of ω-conotoxins. Furthermore, Vt1.27 exhibited potent anti-allodynic effect in rat partial sciatic nerve injury (PNL) and chronic constriction injury (CCI) pain models at 10 nmol/kg level with the intramuscular injection. The pain threshold elevation of Vt1.27 groups was higher than that of α-conotoxin Vc1.1 in CCI rat models. These findings expand our knowledge of targets of α-conotoxins and potentially provide a potent, anti-allodynic peptide for the treatment of neuropathic pain.

5.
Toxicon ; 208: 53-61, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35104533

RESUMO

α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab')2 to MI by synthesizing a new MI antigen through the coupling of alkynyl-modified MI and azide-modified bovine serum albumin (BSA), followed by immunization into mouse and horse. The new MI-BSA antigen generated high titers of mouse and horse antiserum (1:204,800 and 1:51,200, respectively), and both the antiserum as well as the horse F(ab')2 displayed highly potent neutralization and detoxification efficacy. 12.5 µL of mouse or horse antiserum preincubated with MI could completely neutralize a lethal dose of the MI (0.4 µg, 1.7 × LD50), while 6.25 µL (mouse) or 10.41 µL (horse) of the antiserum could exert complete detoxification of mice injected with 1.7 × LD50 of MI. Moreover, the mouse and horse antiserum exhibited medium cross-reactivity for highly toxic α-CTX GI. These results demonstrate that the integrity of MI's antigen epitope and carrier effect of BSA can improve MI's immunogenicity, and provides an effective detoxification treatment for highly toxic α-conotoxins as well as an effective method for the preparation of antiserum of small peptide toxins.


Assuntos
Conotoxinas , Receptores Nicotínicos , Animais , Conotoxinas/toxicidade , Cavalos , Soros Imunes , Antagonistas Nicotínicos , Peptídeos
6.
Mar Drugs ; 19(12)2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34940704

RESUMO

α-Conotoxins GI and MI belong to the 3/5 subfamily of α-conotoxins and potently inhibit muscular nicotinic acetylcholine receptors (nAChRs). To date, no 3/4- or 3/6-subfamily α-conotoxins have been reported to inhibit muscular nAChRs. In the present study, a series of new 3/4-, 3/6-, and 3/7-subfamily GI and MI variants were synthesized and functionally characterized by modifications of loop2. The results show that the 3/4-subfamily GI variant GI[∆8G]-II and the 3/6-subfamily variants GI[+13A], GI[+13R], and GI[+13K] displayed potent inhibition of muscular nAChRs expressed in Xenopus oocytes, with an IC50 of 45.4-73.4 nM, similar to or slightly lower than that of wild-type GI (42.0 nM). The toxicity of these GI variants in mice appeared to be about a half to a quarter of that of wild-type GI. At the same time, the 3/7-subfamily GI variants showed significantly lower in vitro potency and toxicity. On the other hand, similar to the 3/6-subfamily GI variants, the 3/6-subfamily MI variants MI[+14R] and MI[+14K] were also active after the addition of a basic amino acid, Arg or Lys, in loop2, but the activity was not maintained for the 3/4-subfamily MI variant MI[∆9G]. Interestingly, the disulfide bond connectivity "C1-C4, C2-C3" in the 3/4-subfamily variant GI[∆8G]-II was significantly more potent than the "C1-C3, C2-C4" connectivity found in wild-type GI and MI, suggesting that disulfide bond connectivity is easily affected in the rigid 3/4-subfamily α-conotoxins and that the disulfide bonds significantly impact the variants' function. This work is the first to demonstrate that 3/4- and 3/6-subfamily α-conotoxins potently inhibit muscular nAChRs, expanding our knowledge of α-conotoxins and providing new motifs for their further modifications.


Assuntos
Conotoxinas/farmacologia , Caramujo Conus , Antagonistas Nicotínicos/farmacologia , Animais , Animais não Endogâmicos , Organismos Aquáticos , Conotoxinas/química , Feminino , Concentração Inibidora 50 , Masculino , Camundongos , Antagonistas Nicotínicos/química , Oócitos/efeitos dos fármacos , Ratos , Receptores Nicotínicos/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
7.
Acta Pharm Sin B ; 11(9): 2685-2693, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34589389

RESUMO

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple ß-strand. The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.

8.
J Neurochem ; 159(1): 90-100, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34008858

RESUMO

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3ß2ß3 and rα3ß2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3ß2ß3 and hα3ß2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3ß2ß3 subtype compared to rα3ß2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3ß2ß3 and α3ß2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3ß2ß3 nAChRs.


Assuntos
Conotoxinas/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Conotoxinas/química , Conotoxinas/isolamento & purificação , Caramujo Conus , Relação Dose-Resposta a Droga , Feminino , Humanos , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/isolamento & purificação , Oócitos , Estrutura Terciária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/genética , Xenopus laevis
9.
Mar Drugs ; 19(1)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478061

RESUMO

N-methyl-D-aspartate receptor (NMDAR) antagonists have been found to be effective to inhibit morphine dependence. However, the discovery of the selective antagonist for NMDAR GluN2B with low side-effects still remains challenging. In the present study, we report a selective NMDAR GluN2B antagonist con-T[M8Q](a conantokin-T variant) that potently inhibits the naloxone-induced jumping and conditioned place preference of morphine-dependent mice at nmol/kg level, 100-fold higher than ifenprodil, a classical NMDAR NR2B antagonist. Con-T[M8Q] displays no significant impacts on coordinated locomotion function, spontaneous locomotor activity, and spatial memory mice motor function at the dose used. Further molecular mechanism experiments demonstrate that con-T[M8Q] effectively inhibited the transcription and expression levels of signaling molecules related to NMDAR NR2B subunit in hippocampus, including NR2B, p-NR2B, CaMKII-α, CaMKII-ß, CaMKIV, pERK, and c-fos. The high efficacy and low side effects of con-T[M8Q] make it a good lead compound for the treatment of opiate dependence and for the reduction of morphine usage.


Assuntos
Conotoxinas/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Dependência de Morfina/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Conotoxinas/administração & dosagem , Conotoxinas/toxicidade , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/toxicidade , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Dependência de Morfina/fisiopatologia , Naloxona/farmacologia , Piperidinas/farmacologia , Memória Espacial/efeitos dos fármacos
10.
Mar Drugs ; 17(5)2019 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-31083641

RESUMO

As the first in a new class of non-opioid drugs, ω-Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA's ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.


Assuntos
Analgésicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas Recombinantes de Fusão/farmacocinética , ômega-Conotoxinas/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Analgésicos/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Feminino , Masculino , Camundongos , Dor/tratamento farmacológico , Dor/metabolismo , Medição da Dor/efeitos dos fármacos , Peptídeos/administração & dosagem , Peptídeos/química , Proteína FUS de Ligação a RNA , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Tremor/tratamento farmacológico , Tremor/metabolismo , ômega-Conotoxinas/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem
11.
Biochemistry ; 57(44): 6349-6355, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30281282

RESUMO

As an ω-conopeptide originally discovered from Conus striatus, SO-3 contains 25 amino acid residues and three disulfide bridges. Our previous study has shown that this peptide possesses potent analgesic activity in rodent pain models (mouse and rat), and it specifically inhibits an N-type calcium ion channel (Cav2.2). In the study presented here, we investigated the key amino acid residues for their inhibitory activity against Cav2.2 expressed in HEK 293 cells and analgesic activity in mice. To improve the inhibitory activity of SO-3, we also evaluated the effects of some amino acid residues derived from the corresponding residues of ω-peptide MVIIA, CVID, or GVIA. Our data reveal that Lys6, Ile11, and Asn14 are the important functional amino acid residues for SO-3. The replacement of some amino acid residues of SO-3 in loop 1 with the corresponding residues of CVID and GVIA improved the inhibitory activity of SO-3. The binding mode of Cav2.2 with SO-3 amino acids in loop 1 and loop 2 may be somewhat different from that of MVIIA. This study expanded our knowledge of the structure-activity relationship of ω-peptides and provided a new strategy for improving the potency of Cav2.2 inhibitors.


Assuntos
Analgésicos/farmacologia , Comportamento Animal/efeitos dos fármacos , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/metabolismo , Dor/tratamento farmacológico , Peptídeos/farmacologia , Analgésicos/química , Animais , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Dor/metabolismo , Peptídeos/química , Conformação Proteica , Ratos , Relação Estrutura-Atividade
12.
J Med Chem ; 61(22): 10198-10205, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30358401

RESUMO

α-Conotoxins exhibiting analgesic activity, such as Vc1.1, have been shown to inhibit α9α10 nicotinic acetylcholine receptors (nAChRs) and GABAB-receptor (GABABR) coupled N-type (CaV2.2) calcium channels. Here, we report two Vc1.1 variants, Vc1.1[N9R] and benzoyl-Vc1.1[N9R], that selectively inhibit CaV2.2 channels via GABABR activation but exhibit reduced inhibitory activity at α9α10 and other neuronal nAChR subtypes compared with Vc1.1. Surprisingly, the analgesic activity of Vc1.1[N9R] and benzoyl-Vc1.1[N9R] was more potent than that of Vc1.1 when tested in partial sciatic nerve ligation injury and chronic constriction injury models. Vc1.1[N9R] and benzoyl-Vc1.1[N9R] exhibited either similar or tenfold higher activity of GABABR-mediated CaV2.2 inhibition but no activity at CaV2.2 alone; however, the mechanism of increased analgesic activity is unknown. The effects on analgesic activity and α9α10 nAChR of other Vc1.1 variations at position 9 and the N-terminus were also determined. Our findings provide new insights for designing potent inhibitors for GABABR-coupled N-type (CaV2.2) calcium channels.


Assuntos
Analgésicos/química , Analgésicos/farmacologia , Canais de Cálcio Tipo N/metabolismo , Conotoxinas/química , Conotoxinas/farmacologia , Receptores de GABA-B/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Descoberta de Drogas , Células HEK293 , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
13.
Mar Drugs ; 16(4)2018 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-29614714

RESUMO

α-Conotoxins (α-CTxs) are small peptides composed of 11 to 20 amino acid residues with two disulfide bridges. Most of them potently and selectively target nicotinic acetylcholine receptor (nAChR) subtypes, and a few were found to inhibit the GABAB receptor (GABABR)-coupled N-type calcium channels (Cav2.2). However, in all of α-CTxs targeting both receptors, the disulfide connectivity arrangement "C¹-C³, C²-C4" is present. In this work, a novel α4/7-CTx named Lt1.3 (GCCSHPACSGNNPYFC-NH2) was cloned from the venom ducts of Conus litteratus (C. litteratus) in the South China Sea. Lt1.3 was then chemically synthesized and two isomers with disulfide bridges "C¹-C³, C²-C4" and "C¹-C4, C²-C³" were found and functionally characterized. Electrophysiological experiments showed that Lt1.3 containing the common disulfide bridges "C¹-C³, C²-C4" potently and selectively inhibited α3ß2 nAChRs and not GABABR-coupled Cav2.2. Surprisingly, but the isomer with the disulfide bridges "C¹-C4, C²-C³" showed exactly the opposite inhibitory activity, inhibiting only GABABR-coupled Cav2.2 and not α3ß2 nAChRs. These findings expand the knowledge of the targets and selectivity of α-CTxs and provide a new structural motif to inhibit the GABABR-coupled Cav2.2.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Conotoxinas/farmacologia , Caramujo Conus/química , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo N/metabolismo , Conotoxinas/química , Células HEK293 , Humanos , Concentração Inibidora 50 , Antagonistas Nicotínicos/química , Oceanos e Mares , Oócitos , Técnicas de Patch-Clamp , Relação Estrutura-Atividade , Xenopus
14.
Eur J Pharm Sci ; 117: 313-320, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29496533

RESUMO

B07 is a small-molecule CCR5 antagonist-based HIV-1 entry inhibitor that is being developed as an anti-HIV microbicide for preventing sexual transmission of HIV. Here we evaluated its spermicidal and contraceptive potential, including sperm motility, plasma membrane integrity, and contraceptive efficacy tested in rabbits. We found that B07 inhibited sperm motility and movement patterns in a concentration- and time-dependent manner. Within 30 min, B07 induced sperm immobilization with the minimum 100% effective concentration and median effective concentration of 640.0 and 64.4 µg/mL, respectively. The hypo-osmotic swelling test showed that plasma membranes of B07-treated sperms exhibited slight disruption, as verified by electron micrographs. In both B07 gel and N-9 gel groups, not a single implantation site or embryo was observed based on the contraceptive efficacy test in rabbits, indicating that B07 could effectively block the potential of sperm to reach and/or fertilize oocytes. The safety profile of B07 in vivo was evaluated by use of an optimized rabbit vaginal irritation test. While the pathological scores of the N-9 gel group was 14.67 ±â€¯1.21, those of the blank control and B07 gel groups were 2.17 ±â€¯0.76 and 4.00 ±â€¯0.89, respectively, which were within the clinically acceptable range (<8). The proportion of inflammatory cells and CD45+ cells in the cervicovaginal lavages of the B07 gel group showed no significant change compared to those of the control group. Therefore, our results confirmed that B07 exhibited significant spermicidal and contraceptive effects, suggesting its potential for development as a microbicidal spermicide for contraception and prevention of HIV sexual transmission.


Assuntos
Antagonistas dos Receptores CCR5/farmacologia , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/prevenção & controle , Piperazinas/farmacologia , Receptores CCR5/efeitos dos fármacos , Espermicidas/farmacologia , Espermatozoides/efeitos dos fármacos , Administração Intravaginal , Animais , Antagonistas dos Receptores CCR5/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Géis , Inibidores da Fusão de HIV/administração & dosagem , Infecções por HIV/transmissão , Infecções por HIV/virologia , Humanos , Masculino , Piperazinas/administração & dosagem , Gravidez , Taxa de Gravidez , Coelhos , Receptores CCR5/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermicidas/administração & dosagem , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Fatores de Tempo
15.
Molecules ; 23(4)2018 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-29570686

RESUMO

Axitinib is an approved kinase inhibitor for the therapy of advanced metastatic renal cell carcinoma (RCC). It prevents angiogenesis, cellular adhesion, and induces apoptosis of cancer cells. Here, nine axitinib derivatives were designed by replacing the C=C moiety with the N=N group, and the substituted benzene or pyrrole analogs were considered to replace the pyridine ring. Biological activity results showed that most of nascent derivatives exhibited favorable VEGFR-2 kinase inhibitory activities, and TM6, 7, 9, and 11 behaved more potent anti-proliferative activities than axitinib. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.


Assuntos
Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Imidazóis/síntese química , Imidazóis/farmacologia , Indazóis/síntese química , Indazóis/farmacologia , Inibidores da Angiogênese/química , Axitinibe , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Humanos , Imidazóis/química , Indazóis/química , Neoplasias Renais/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
16.
Sci Rep ; 8(1): 1004, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343689

RESUMO

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3ß4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5-5.0 µg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.


Assuntos
Analgésicos/farmacologia , Canais de Cálcio Tipo N/metabolismo , Dor Crônica/tratamento farmacológico , Conotoxinas/farmacologia , Peptídeos/farmacologia , Neuropatia Ciática/tratamento farmacológico , Sequência de Aminoácidos , Analgésicos/síntese química , Analgésicos/isolamento & purificação , Animais , Cálcio/metabolismo , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Conotoxinas/síntese química , Conotoxinas/isolamento & purificação , Caramujo Conus/química , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Injeções Intramusculares , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Técnicas de Síntese em Fase Sólida , Xenopus laevis
17.
J Am Chem Soc ; 139(38): 13483-13486, 2017 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-28885845

RESUMO

Biological tissue exhibits an absorbance minimum in the near-infrared between 700 and 900 nm that permits deep penetration of light. Molecules that undergo photoisomerization in this bio-optical window are highly desirable as core structures for the development of photopharmaceuticals and as components of chemical-biological tools. We report the systematic design, synthesis, and testing of an azobenzene derivative tailored to undergo single-photon photoswitching with near-infrared light under physiological conditions. A fused dioxane ring and a methoxy substituent were used to place oxygen atoms in all four ortho positions, as well as two meta positions, relative to the azobenzene N═N double bond. This substitution pattern, together with a para pyrrolidine group, raises the pKa of the molecule so that it is protonated at physiological pH and absorbs at wavelengths >700 nm. This azobenzene derivative, termed DOM-azo, is stable for months in neutral aqueous solutions, undergoes trans-to-cis photoswitching with 720 nm light, and thermally reverts to the stable trans isomer with a half-life near 1 s.


Assuntos
Compostos Azo/química , Compostos Azo/efeitos da radiação , Raios Infravermelhos , Processos Fotoquímicos/efeitos da radiação , Concentração de Íons de Hidrogênio , Isomerismo , Prótons , Pirrolidinas/química
18.
Toxins (Basel) ; 9(8)2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28788055

RESUMO

α-conotoxin GI, a short peptide toxin in the venom of Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029-0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene-divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent (methanol: acetonitrile = 50:50, v/v). GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18 (50 × 2.1 mm, 5 µm) column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6-66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.


Assuntos
Conotoxinas/sangue , Cromatografia Líquida , Humanos , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
19.
Peptides ; 94: 64-70, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28666820

RESUMO

As a class of peptides with 10 cysteine residues (-C-CC-C-CC-C-C-C-C-), D-superfamily conotoxins (D-conotoxins) can specifically act on nicotinic acetylcholine receptors (nAChRs). According to the conserved signal peptides of D-conotoxins, seven D-conotoxin precursor sequences with a previously undescribed Cys arrangement (-C-C-C-CC-C-C-C-C-C-) were identified by PCR-RACE methodology in the present study. The alignment of sequences revealed that signal peptide regions were same as D-VxXXA from Conus vexillum, and their mature peptides were almost different from the D-conotoxins. Analyses of the evolutionary tree demonstrated that they had low homology to those reported conotoxins with 10 cysteine residues (less than 35%) and lied in a separate branch in the evolutionary tree. Furthermore, a previously undescribed D-superfamily conotoxin Lt28.1 was further expressed in Pichia pastoris and then functionally characterized. The results showed that the recombinant Lt28.1 targeted α9α10 nAChRs but not other nAChRs subtypes. These findings defined a new branch of D-superfamily and expanded our knowledge of targets and potential application of D-conotoxins.


Assuntos
Conotoxinas/metabolismo , Caramujo Conus/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Conotoxinas/genética , Caramujo Conus/química , Filogenia , Pichia/genética , Análise de Sequência de Proteína
20.
Peptides ; 81: 15-20, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27131596

RESUMO

In the present study, we isolated, synthesized and NMR structurally characterized a novel conopeptide Im10A consisting of 11 amino acids (NTICCEGCMCY-NH2) from Conus imperialis. Unlike other conopeptides with four cysteine residues, Im10A had only two residues in loop 1 and one residue in loop 2 (CC-loop1-C-loop2-C), which formed a stable disulfide connectivity "I-IV, II- III" (framework X) with a type I ß-turn. Interestingly, Im10A exhibited 50.7% analgesic activity on rat partial sciatic nerve ligation (PNL) at 2h after Im10A administration. However, 10µM Im10A exhibited no apparent effect on neuronal nicotinic acetylcholine receptor, and it did not target DRG voltage-dependent sodium, potassium and calcium ion channels and opioid receptor. To our knowledge, Im10A had the most concentrated disulfide bridges among conopeptides with four cysteine residues. This finding provided a new motif for the future development of biomimetic compounds.


Assuntos
Analgésicos/química , Analgésicos/farmacologia , Conotoxinas/química , Conotoxinas/farmacologia , Caramujo Conus/química , Sequência de Aminoácidos , Analgésicos/síntese química , Analgésicos/isolamento & purificação , Análise de Variância , Animais , Conotoxinas/síntese química , Conotoxinas/isolamento & purificação , Cisteína/química , Dissulfetos/química , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/metabolismo , Nervo Isquiático/efeitos dos fármacos
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