RESUMO
Malaria is a parasitic infectious disease caused by Plasmodium sp, which was responsible for about 409 thousand deaths only in 2019. The clinical manifestations in patients with malaria, which may include fever and anemia and that can occasionally lead to the death of the host, are mainly associated to the asexual blood stage of parasite. The discovery of novel compounds active against stages of the intraerythrocytic cell cycle has been the focus of many researches seeking for alternatives to the control of malaria. The antimalarial effect of a native cationic polypeptide from the venom of a South American rattlesnake named crotamine, with ability of targeting and disrupting the acidic compartments of Plasmodium falciparum parasite, was previously described by us. Herein, we extended our previous studies by investigating the internalization and trafficking of crotamine in P. falciparum-infected erythrocytes at different blood-stages of parasites and periods of incubation. In addition, the effects of several pharmacological inhibitors in the uptake of this snake polypeptide with cell-penetrating properties were also assessed, showing that crotamine internalization was dependent on ATP generated via glycolytic pathway. We show here that crotamine uptake is blocked by the glycolysis inhibitor 2-deoxy-D-glucose, and the most efficient internalization is observed at trophozoite stage of parasite after at least 30 min of incubation. The present data provide important insights into biochemical pathway and cellular features determined by the parasite cycle, which may be underlying the internalization and effects of cationic antimalarials as crotamine.
Assuntos
Venenos de Crotalídeos/química , Eritrócitos , Peptídeos , Plasmodium falciparum , Animais , Crotalus , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Peptídeos/farmacologia , América do SulRESUMO
BACKGROUND AND PURPOSE: The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. KEY RESULTS: PnPP-19 (5, 10 and 20 µg per paw) induced peripheral antinociception in rats. Specific antagonists of µ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 µmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. CONCLUSIONS AND IMPLICATIONS: Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, µ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.
Assuntos
Analgésicos Opioides/farmacologia , Inibidores Enzimáticos/farmacologia , Neprilisina/antagonistas & inibidores , Peptídeos/farmacologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptores Opioides/metabolismo , Venenos de Aranha/química , Animais , Relação Dose-Resposta a Droga , Masculino , Neprilisina/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.
Assuntos
Antimaláricos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Venenos de Crotalídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Venenos de Serpentes/química , Laranja de Acridina/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/isolamento & purificação , Transporte Biológico , Carbocianinas/química , Peptídeos Penetradores de Células/isolamento & purificação , Células Cultivadas , Cloroquina/farmacologia , Venenos de Crotalídeos/isolamento & purificação , Crotalus/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Concentração Inibidora 50 , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Coloração e Rotulagem , Vacúolos/efeitos dos fármacos , Vacúolos/parasitologiaRESUMO
BACKGROUND: There is growing interest in sex differences and RAS components. However, whether gender influences cardiac angiotensin I-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity is still unknown. In the present work, we determined the relationship between ACE and ACE2 activity, left ventricular function and gender in spontaneously hypertensive rats (SHRs). METHODOLOGY/PRINCIPAL FINDINGS: Twelve-week-old female (F) and male (M) SHRs were divided into 2 experimental groups (n = 7 in each group): sham (S) and gonadectomized (G). Fifty days after gonadectomy, we measured positive and negative first derivatives (dP/dt maximum left ventricle (LV) and dP/dt minimum LV, respectively), hypertrophy (morphometric analysis) and ACE and ACE2 catalytic activity (fluorimetrically). Expression of calcium handling proteins was measured by western blot. Male rats exhibited higher cardiac ACE and ACE2 activity as well as hypertrophy compared to female rats. Orchiectomy decreased the activity of these enzymes and hypertrophy, while ovariectomy increased hypertrophy and ACE2, but did not change ACE activity. For cardiac function, the male sham group had a lower +dP/dt than the female sham group. After gonadectomy, the +dP/dt increased in males and reduced in females. The male sham group had a lower -dP/dt than the female group. After gonadectomy, the -dP/dt increased in the male and decreased in the female groups when compared to the sham group. No difference was observed among the groups in SERCA2a protein expression. Gonadectomy increased protein expression of PLB (phospholamban) and the PLB to SERCA2a ratio in female rats, but did not change in male rats. CONCLUSION: Ovariectomy leads to increased cardiac hypertrophy, ACE2 activity, PLB expression and PLB to SERCA2a ratio, and worsening of hemodynamic variables, whereas in males the removal of testosterone has the opposite effects on RAS components.
Assuntos
Cardiomegalia/enzimologia , Cardiomegalia/fisiopatologia , Hormônios Esteroides Gonadais/farmacologia , Hipertensão/enzimologia , Contração Miocárdica/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Peso Corporal/efeitos dos fármacos , Cardiomegalia/complicações , Densitometria , Feminino , Gônadas/efeitos dos fármacos , Gônadas/cirurgia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos Endogâmicos SHR , Sístole/efeitos dos fármacosRESUMO
Sex hormones modulate the action of both cytokines and the renin-angiotensin system. However, the effects of angiotensin I-converting enzyme (ACE) on the proinflammatory and anti-inflammatory cytokine levels in male and female spontaneously hypertensive rats (SHR) are unclear. We determined the relationship between ACE activity, cytokine levels and sex differences in SHR. Female (F) and male (M) SHR were divided into 4 experimental groups each (n = 7): sham + vehicle (SV), sham + enalapril (10 mg/kg body weight by gavage), castrated + vehicle, and castrated + enalapril. Treatment began 21 days after castration and continued for 30 days. Serum cytokine levels (ELISA) and ACE activity (fluorimetry) were measured. Male rats exhibited a higher serum ACE activity than female rats. Castration reduced serum ACE in males but did not affect it in females. Enalapril reduced serum ACE in all groups. IL-10 (FSV = 16.4 ± 1.1 pg/mL; MSV = 12.8 ± 1.2 pg/mL), TNF-α (FSV = 16.6 ± 1.2 pg/mL; MSV = 12.8 ± 1 pg/mL) and IL-6 (FSV = 10.3 ± 0.2 pg/mL; MSV = 7.2 ± 0.2 pg/mL) levels were higher in females than in males. Ovariectomy reduced all cytokine levels and orchiectomy reduced IL-6 but increased IL-10 concentrations in males. Castration eliminated the differences in all inflammatory cytokine levels (IL-6 and TNF-α) between males and females. Enalapril increased IL-10 in all groups and reduced IL-6 in SV rats. In conclusion, serum ACE inhibition by enalapril eliminated the sexual dimorphisms of cytokine levels in SV animals, which suggests that enalapril exerts systemic anti-inflammatory and anti-hypertensive effects.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Enalapril/farmacologia , Hipertensão/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Fator de Necrose Tumoral alfa/sangue , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Feminino , Hipertensão/fisiopatologia , Masculino , Ovariectomia , Ratos Endogâmicos SHR , Fatores SexuaisRESUMO
Sex hormones modulate the action of both cytokines and the renin-angiotensin system. However, the effects of angiotensin I-converting enzyme (ACE) on the proinflammatory and anti-inflammatory cytokine levels in male and female spontaneously hypertensive rats (SHR) are unclear. We determined the relationship between ACE activity, cytokine levels and sex differences in SHR. Female (F) and male (M) SHR were divided into 4 experimental groups each (n = 7): sham + vehicle (SV), sham + enalapril (10 mg/kg body weight by gavage), castrated + vehicle, and castrated + enalapril. Treatment began 21 days after castration and continued for 30 days. Serum cytokine levels (ELISA) and ACE activity (fluorimetry) were measured. Male rats exhibited a higher serum ACE activity than female rats. Castration reduced serum ACE in males but did not affect it in females. Enalapril reduced serum ACE in all groups. IL-10 (FSV = 16.4 ± 1.1 pg/mL; MSV = 12.8 ± 1.2 pg/mL), TNF-α (FSV = 16.6 ± 1.2 pg/mL; MSV = 12.8 ± 1 pg/mL) and IL-6 (FSV = 10.3 ± 0.2 pg/mL; MSV = 7.2 ± 0.2 pg/mL) levels were higher in females than in males. Ovariectomy reduced all cytokine levels and orchiectomy reduced IL-6 but increased IL-10 concentrations in males. Castration eliminated the differences in all inflammatory cytokine levels (IL-6 and TNF-α) between males and females. Enalapril increased IL-10 in all groups and reduced IL-6 in SV rats. In conclusion, serum ACE inhibition by enalapril eliminated the sexual dimorphisms of cytokine levels in SV animals, which suggests that enalapril exerts systemic anti-inflammatory and anti-hypertensive effects.
Assuntos
Animais , Feminino , Masculino , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Enalapril/farmacologia , Hipertensão/sangue , /sangue , /sangue , Fator de Necrose Tumoral alfa/sangue , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Hipertensão/fisiopatologia , Ovariectomia , Ratos Endogâmicos SHR , Fatores SexuaisRESUMO
Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K(m)) of 47 µM(-1)âs(-1). The purified ((HIS))CPXAC displayed maximal catalytic activity at pH 5.5 and 30°C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K(i) values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.
Assuntos
Cisteína Proteases/metabolismo , Proteínas Recombinantes/metabolismo , Xanthomonas/enzimologia , Sequência de Aminoácidos , Biocatálise/efeitos dos fármacos , Biologia Computacional , Meios de Cultura , Cisteína Proteases/química , Ativação Enzimática/efeitos dos fármacos , Ensaios Enzimáticos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Inibidores de Proteases/farmacologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Temperatura , Xanthomonas/efeitos dos fármacos , Xanthomonas/patogenicidadeRESUMO
We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anticonvulsivantes/farmacologia , Carbamazepina/farmacologia , Epilepsia do Lobo Temporal/fisiopatologia , Peptidil Dipeptidase A/fisiologia , Alelos , Animais , Lobectomia Temporal Anterior , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/cirurgia , Genótipo , Humanos , Mutação INDEL , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/genética , Polimorfismo Genético/genética , Lobo Temporal/efeitos dos fármacos , Lobo Temporal/patologiaRESUMO
The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2' of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.
Assuntos
Catepsinas/antagonistas & inibidores , Clorexidina/análogos & derivados , Dentina/enzimologia , Inibidores Enzimáticos/farmacologia , Adulto , Catepsina B/antagonistas & inibidores , Catepsina K/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Clorexidina/farmacologia , Cumarínicos , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Humanos , Hidrólise , Leucina/análogos & derivados , Leucina/farmacologia , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , Espectrometria de Fluorescência , Adulto JovemRESUMO
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.