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1.
Int J Mol Sci ; 23(5)2022 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-35269985

RESUMO

The animal trypanosomiases are infections in a wide range of (domesticated) animals with any species of African trypanosome, such as Trypanosoma brucei, T. evansi, T. congolense, T. equiperdum and T. vivax. Symptoms differ between host and infective species and stage of infection and are treated with a small set of decades-old trypanocides. A complication is that not all trypanosome species are equally sensitive to all drugs and the reasons are at best partially understood. Here, we investigate whether drug transporters, mostly identified in T. b. brucei, determine the different drug sensitivities. We report that homologues of the aminopurine transporter TbAT1 and the aquaporin TbAQP2 are absent in T. congolense, while their introduction greatly sensitises this species to diamidine (pentamidine, diminazene) and melaminophenyl (melarsomine) drugs. Accumulation of these drugs in the transgenic lines was much more rapid. T. congolense is also inherently less sensitive to suramin than T. brucei, despite accumulating it faster. Expression of a proposed suramin transporter, located in T. brucei lysosomes, in T. congolense, did not alter its suramin sensitivity. We conclude that for several of the most important classes of trypanocides the presence of specific transporters, rather than drug targets, is the determining factor of drug efficacy.


Assuntos
Arsenicais , Tripanossomicidas , Trypanosoma congolense , Trypanosoma , Animais , Proteínas de Membrana Transportadoras , Pentamidina/metabolismo , Pentamidina/farmacologia , Suramina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma congolense/metabolismo
2.
Pflugers Arch ; 474(5): 553-565, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35169901

RESUMO

Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.


Assuntos
Anemia Falciforme , Reticulócitos , Trifosfato de Adenosina/metabolismo , Anemia Falciforme/metabolismo , Apirase/metabolismo , Cátions/metabolismo , Células Cultivadas , Eritrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hipóxia/metabolismo , Probenecid/metabolismo , Reticulócitos/metabolismo , Suramina/metabolismo , Suramina/farmacologia
3.
Vet Immunol Immunopathol ; 241: 110336, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34649042

RESUMO

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2-) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2-, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2- generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.


Assuntos
Neutrófilos , Fator de Ativação de Plaquetas , Animais , Cálcio , Cães , Neutrófilos/citologia , Fator de Ativação de Plaquetas/farmacologia , Espécies Reativas de Oxigênio , Receptores Acoplados a Proteínas G , Transdução de Sinais , Suramina/farmacologia
4.
J Chem Inf Model ; 61(9): 4442-4454, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34516120

RESUMO

The emergence of new viral infections and drug-resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets are one potential way in which to accelerate this process. Suramin was initially developed as a successful antiparasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous toward positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure-activity relationship studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramin's target diversity, confirming that suramin preferentially binds to protein pockets that are both positively charged and enriched in aromatic or leucine residues. Further, suramin's high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity.


Assuntos
COVID-19 , Suramina , Antibacterianos/farmacologia , Antivirais/farmacologia , Humanos , RNA Viral , SARS-CoV-2 , Suramina/farmacologia
5.
J Virol Methods ; 298: 114283, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34534610

RESUMO

The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is essential for virus replication, therefore it is a promising drug target. Here we present a surface plasmon resonance approach to study the interaction of RdRp with drugs in real time. We monitored the effect of favipiravir, ribavirin, sofosbuvir triphosphate PSI-7409 and suramin on RdRp binding to RNA immobilized on the chip. Suramin precluded interaction of RdRp with RNA and even displaced RdRp from RNA.


Assuntos
COVID-19 , RNA Polimerase Dependente de RNA , Antivirais/farmacologia , Interações Medicamentosas , Humanos , RNA Viral , SARS-CoV-2 , Suramina/farmacologia , Ressonância de Plasmônio de Superfície
6.
Am J Sports Med ; 49(11): 3102-3112, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34351815

RESUMO

BACKGROUND: The increasing use of platelet-rich plasma (PRP) to treat muscle injuries raises concerns because transforming growth factor-beta (TGF-ß) in PRP may promote fibrosis in the injured muscle and thus impair muscle regeneration. PURPOSE: To investigate whether suramin (a TGF-ß inhibitor) can reduce muscle fibrosis to improve healing of the injured muscle after PRP treatment and identify the underlying molecular mechanism. STUDY DESIGN: Controlled laboratory study. METHODS: Myoblasts isolated from the gastrocnemius muscle of Sprague Dawley rats were treated with PRP or PRP plus suramin. MTT assays were performed to evaluate cell viability. The expression of fibrosis-associated proteins (such as type I collagen and fibronectin), Smad2, and phosphorylated Smad2 was determined using Western blot analysis and immunofluorescent staining. An anti-TGF-ß antibody was employed to verify the role of TGF-ß in fibronectin expression. Gastrocnemius muscles were injured through a partial transverse incision and then treated using PRP or PRP plus suramin. Hematoxylin and eosin staining was conducted to evaluate the healing process 7 days after the injury. Immunofluorescent staining was performed to evaluate fibronectin expression. Muscle contractile properties-fast-twitch and tetanic strength-were evaluated through electric stimulation. RESULTS: PRP plus 25 µg/mL of suramin promoted myoblast proliferation. PRP induced fibronectin expression in myoblasts, but suramin reduced this upregulation. The anti-TGF-ß antibody also reduced the upregulation of fibronectin expression in the presence of PRP. The upregulation of phosphorylated Smad2 by PRP was reduced by either the anti-TGF-ß antibody or suramin. In the animal study, no significant difference was discovered in muscle healing between the PRP versus PRP plus suramin groups. However, the PRP plus suramin group had reduced fibronectin expression at the injury site. Fast-twitch strength and tetanic strength were significantly higher in the injured muscle treated using PRP or PRP plus suramin. CONCLUSION: Simultaneous PRP and suramin use reduced fibrosis in the injured muscle and promoted healing without negatively affecting the muscle's contractile properties. The underlying molecular mechanism may be associated with the phosphorylated Smad2 pathway. CLINICAL RELEVANCE: Simultaneous PRP and suramin use may reduce muscle fibrosis without compromising muscle contractile properties and thus improve muscle healing.


Assuntos
Músculo Esquelético/lesões , Plasma Rico em Plaquetas , Suramina , Cicatrização , Animais , Ratos , Ratos Sprague-Dawley , Suramina/farmacologia , Suramina/uso terapêutico , Fator de Crescimento Transformador beta1/antagonistas & inibidores
7.
Biomed Pharmacother ; 139: 111683, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243631

RESUMO

Diabetes mellitus causes changes in metabolism of extracellular nucleotides acting through P2 receptors (P2Rs). This affects renal function and may lead to glomerular and tubular disturbances. We measured urinary excretion of nucleotides (ATP, ADP, AMP, UTP, UDP, UMP) in streptozotocin-induced diabetic rats (65 mg/kg, i.p., day 0) and the effects of P2Rs' blockade by suramin (10 mg/kg, i.p., days +7, +14) on glomerular P2×7R expression and urinary excretion of glomerular (albumin, nephrin) and tubular (KIM-1, NGAL) injury markers, electrolytes, and oxidative stress markers (TBARS, 8-OHdG). Concentrations of nucleotides, specific proteins, electrolytes, and oxidative stress markers in 24-h urine samples collected in metabolic cages at days -1, +6 and +20 were measured using ion-paired reversed-phase HPLC, immunoenzymatic and fluorometric methods, and flame photometry, respectively. Expression of KIM-1 and P2×7R was examined by immunohistochemistry or immunoblotting. Diabetes was associated with increased urinary excretion of ATP, ADP, UTP, UDP and glomerular P2×7R expression. Suramin attenuated P2×7R expression but did not affect urinary excretion of nucleotides. Urinary excretion of albumin, nephrin, NGAL, and 8-OHdG were increased in diabetic rats and were not affected by suramin. TBARS was higher in diabetic rats and suramin attenuated the excretion dynamics in this group. KIM-1 excretion was higher in diabetic rats and suramin further increased excretion of KIM-1 in both diabetic and non-diabetic rats. Furthermore, suramin attenuated the diabetes-induced natriuresis and kaliuresis. It is possible that suramin affects both glomerular and tubular functions in diabetic rats.


Assuntos
Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/urina , Glomérulos Renais/efeitos dos fármacos , Suramina/farmacologia , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Glomérulos Renais/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Estreptozocina/farmacologia
8.
Biochem J ; 478(13): 2405-2423, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198322

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Chlorocebus aethiops , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , RNA Helicases/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/metabolismo
9.
Biochem J ; 478(13): 2425-2443, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198323

RESUMO

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.


Assuntos
Antivirais/química , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Holoenzimas/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
10.
Life Sci ; 283: 119850, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34314735

RESUMO

AIMS: Application of exogenous nucleotides can modulate wound healing via the activation of purinergic receptors. However, evidence for the release of endogenous nucleotides and the subsequent activation of purinergic receptors in this process has not been well defined. Therefore, the current study aimed to investigate wound-mediated nucleotide release and autocrine purinergic signalling during HaCaT keratinocyte wound closure following scratch injury. MAIN METHODS: An in vitro scratch wound apparatus was employed to study wound healing over 24-h in the presence of modulators of ATP release, P2 receptors and pathways downstream of P2 receptor activation. KEY FINDINGS: Adenosine 5'-triphosphate (ATP) was released from scratched cells. The ectonucleotidase apyrase and pharmacological inhibition of the nucleotide release hemichannel, pannexin-1, decreased wound closure over time. The non-selective P2Y receptor antagonist suramin and the selective P2Y2 receptor antagonist AR-C118925XX, but not other P2 antagonists, decreased wound closure. AR-C118925XX decreased wound closure in a concentration-dependent fashion. However, exogenous P2Y2 receptor agonists, ATP or uridine 5'-triphosphate, did not enhance wound closure. PCR and immunoblotting confirmed P2Y2 receptor expression in HaCaT cells. U73122, a phospholipase C antagonist, and 2-aminoethoxydiphenylborate, an inositol 1,4,5-trisphosphate receptor-sensitive Ca2+-release channel antagonist, decreased wound closure consistent with P2Y2 receptor activation. Absence of extracellular or intracellular Ca2+ or inhibition of intracellular Ca2+-release also impaired wound closure. SIGNIFICANCE: These data describe a novel autocrine signalling mechanism in which wound-mediated release of endogenous ATP in response to mechanical scratching of HaCaT cells activates P2Y2 receptors to facilitate wound closure.


Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Autócrina , Receptores Purinérgicos P2Y2/metabolismo , Cicatrização , Células HEK293 , Humanos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Suramina/farmacologia
11.
Viruses ; 13(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068686

RESUMO

Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) the WHO reported more than 148 million confirmed cases and 3.1 million losses globally up to now. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide, resulting in a pandemic of unprecedented magnitude. To date, several clinically safe and efficient vaccines (e.g., Pfizer-BioNTech, Moderna, Johnson & Johnson, and AstraZeneca COVID-19 vaccines) as well as drugs for emergency use have been approved. However, increasing numbers of SARS-Cov-2 variants make it imminent to identify an alternative way to treat SARS-CoV-2 infections. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is repurposing clinically developed drugs, e.g., antiparasitic drugs. The results described in this study demonstrated the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules presented a competitive and noncompetitive inhibition mode, respectively, with IC50 values in the low micromolar range. Surface plasmon resonance (SPR) experiments demonstrated that quinacrine and suramin alone possessed a moderate or weak affinity with SARS-CoV-2 3CLpro but suramin binding increased quinacrine interaction by around a factor of eight. Using docking and molecular dynamics simulations, we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin, in combination with quinacrine, showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. We suppose that the identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease and repurposable drug candidates offer fast therapeutic breakthroughs, mainly in a pandemic moment.


Assuntos
Proteases 3C de Coronavírus/efeitos dos fármacos , Quinacrina/farmacologia , Suramina/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Vacinas contra COVID-19/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Inibidores de Proteases/farmacologia , Quinacrina/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Suramina/metabolismo , Proteínas não Estruturais Virais
12.
PLoS One ; 16(6): e0251649, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34106944

RESUMO

Until now, antiviral therapeutic agents are still urgently required for treatment or prevention of SARS-coronavirus 2 (SCoV-2) virus infection. In this study, we established a sensitive SCoV-2 Spike glycoprotein (SP), including an SP mutant D614G, pseudotyped HIV-1-based vector system and tested their ability to infect ACE2-expressing cells. Based on this system, we have demonstrated that an aqueous extract from the Natural herb Prunella vulgaris (NhPV) displayed potent inhibitory effects on SCoV-2 SP (including SPG614 mutant) pseudotyped virus (SCoV-2-SP-PVs) mediated infections. Moreover, we have compared NhPV with another compound, Suramin, for their anti-SARS-CoV-2 activities and the mode of their actions, and found that both NhPV and Suramin are able to directly interrupt SCoV-2-SP binding to its receptor ACE2 and block the viral entry step. Importantly, the inhibitory effects of NhPV and Suramin were confirmed by the wild type SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) virus infection in Vero cells. Furthermore, our results also demonstrated that the combination of NhPV/Suramin with an anti-SARS-CoV-2 neutralizing antibody mediated a more potent blocking effect against SCoV2-SP-PVs. Overall, by using SARS-CoV-2 SP-pseudotyped HIV-1-based entry system, we provide strong evidence that NhPV and Suramin have anti-SARS-CoV-2 activity and may be developed as a novel antiviral approach against SARS-CoV-2 infection.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/virologia , Extratos Vegetais/farmacologia , Prunella/química , SARS-CoV-2/efeitos dos fármacos , Suramina/farmacologia , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , COVID-19/genética , COVID-19/metabolismo , Linhagem Celular , Chlorocebus aethiops , Quimioterapia Combinada , Humanos , Mutação , Ligação Proteica , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Biol Pharm Bull ; 44(3): 458-460, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33642556

RESUMO

The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.


Assuntos
Trifosfato de Adenosina/fisiologia , Retroalimentação Fisiológica , Músculos Papilares/fisiologia , Sistema Nervoso Simpático , Função Ventricular , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Cobaias , Ventrículos do Coração , Masculino , Contração Muscular , Norepinefrina/fisiologia , Prazosina/farmacologia , Propranolol/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Suramina/farmacologia
14.
Molecules ; 26(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670019

RESUMO

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Suramina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-raf/isolamento & purificação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Suramina/análogos & derivados , Suramina/química
15.
Nat Struct Mol Biol ; 28(3): 319-325, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33674802

RESUMO

The COVID-19 pandemic caused by nonstop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report that suramin, a 100-year-old drug, is a potent inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and acts by blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for treatment of COVID-19. The 2.6 Å cryo-electron microscopy structure of the viral RdRp bound to suramin reveals two binding sites. One site directly blocks the binding of the RNA template strand and the other site clashes with the RNA primer strand near the RdRp catalytic site, thus inhibiting RdRp activity. Suramin blocks viral replication in Vero E6 cells, although the reasons underlying this effect are likely various. Our results provide a structural mechanism for a nonnucleotide inhibitor of the SARS-CoV-2 RdRp.


Assuntos
Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Inibidores Enzimáticos/farmacologia , Suramina/farmacologia , Animais , Antivirais/química , Antivirais/metabolismo , Sítios de Ligação , Domínio Catalítico , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Microscopia Crioeletrônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/efeitos dos fármacos , Suramina/química , Suramina/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
16.
Nutrients ; 13(2)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562242

RESUMO

High salt intake has been reported as a risk factor for urinary storage symptoms. However, the association between high salt intake and detrusor muscle contraction is not clear. Therefore, we investigated the effects of high salt intake on the components of detrusor muscle contraction in rats. Six-week-old male Dahl salt-resistant (DR; n = 5) and Dahl salt-sensitive (DS; n = 5) rats were fed a high salt (8% NaCl) diet for one week. The contractile responses of the detrusor muscle to the cumulative administration of carbachol and electrical field stimulation (EFS) with and without suramin and atropine were evaluated via isometric tension study. The concentration-response curves of carbachol were shifted more to the left in the DS group than those in the DR group. Contractile responses to EFS were more enhanced in the DS group than those in the DR group (p < 0.05). Cholinergic component-induced responses were more enhanced in the DS group than those in the DR group (p < 0.05). High salt intake might cause urinary storage symptoms via abnormalities in detrusor muscle contraction and the enhancement of cholinergic signals. Excessive salt intake should be avoided to preserve bladder function.


Assuntos
Contração Isométrica/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cloreto de Sódio na Dieta/efeitos adversos , Cloreto de Sódio na Dieta/farmacologia , Bexiga Urinária/efeitos dos fármacos , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Masculino , Ratos Endogâmicos Dahl , Suramina/farmacologia
17.
Pharmacol Rep ; 73(3): 841-846, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33635529

RESUMO

BACKGROUND: Vascular endothelial growth factor A (VEGF-A) and P2-receptors (P2Rs) are involved in the pathogenesis of diabetic nephropathy. The processing of VEGF-A by matrix metalloproteinases (MMP) regulates its bioavailability. Since the ATP-induced release of MMP-9 is mediated by P2Rs, we investigated the effect of suramin on VEGF-A excretion in urine and the urinary activity of total MMP and MMP-9. METHODS: The effect of suramin (10 mg/kg, ip) on VEGF-A concentration in serum and its excretion in urine was investigated in streptozotocin (STZ)-induced diabetic rats over a 21-day period. The rats received suramin 7 and 14 days after a single STZ injection (65 mg/kg, ip). A 24-h collection of urine was performed on the day preceding the administration of STZ and the first administration of suramin and on the day before the end of the experiment. The VEGF-A in serum and urine, albumin in urine, and total activity of MMP and MMP-9 in urine were measured using immunoassays. RESULTS: Diabetic rats are characterized by a sixfold higher urinary excretion of VEGF-A. Suramin potentiates VEGF-A urinary excretion by 36% (p = 0.046) in non-diabetic and by 75% (p = 0.0322) in diabetic rats but it did not affect VEGF-A concentration in the serum of non-diabetic and diabetic rats. Urinary albumin excretion as well as total MMP and MMP-9 activity was increased in diabetic rats, but these parameters were not affected by suramin. CONCLUSION: Suramin increases the urinary excretion of VEGF-A in normoglycemia and hyperglycaemia, possibly without the involvement of MMP-9. Suramin may be used as a pharmacological tool enhancing VEGF-A urinary secretion.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Suramina/farmacologia , Fator A de Crescimento do Endotélio Vascular/urina , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Nefropatias Diabéticas/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Ratos Wistar , Estreptozocina/farmacologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-33588295

RESUMO

Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. The HAT is caused by Trypanosoma brucei rhodesiense (Tbr) parasite in eastern and southern Africa, with suramin as drug of choice for treatment of early stage of the disease. Suramin treatment failures has been observed among HAT patients in Tbr foci in Uganda. In this study, we assessed Tbr parasite strains isolated from HAT patients responsive (Tbr EATRO-232) and non-responsive (Tbr EATRO-734) to suramin treatment in Busoga, Uganda for 1) putative role of suramin resistance in the treatment failure 2) correlation of suramin resistance with Tbr pathogenicity and 3) proteomic pathways underpinning the potential suramin resistance phenotype in vivo. We first assessed suramin response in each isolate by infecting male Swiss white mice followed by treatment using a series of suramin doses. We then assessed relative pathogenicity of the two Tbr isolates by assessing changes pathogenicity indices (prepatent period, survival and mortality). We finally isolated proteins from mice infected by the isolates, and assessed their proteomic profiles using mass spectrometry. We established putative resistance to 2.5 mg/kg suramin in the parasite Tbr EATRO-734. We established that Tbr EATRO-734 proliferated slower and has significantly enriched pathways associated with detoxification and metabolism of energy and drugs relative to Tbr EATRO-232. The Tbr EATRO-734 also has more abundantly expressed mitochondrion proteins and enzymes than Tbr EATRO-232. The suramin treatment failure may be linked to the relatively higher resistance to suramin in Tbr EATRO-734 than Tbr EATRO-232, among other host and parasite specific factors. However, the Tbr EATRO-734 appears to be less pathogenic than Tbr EATRO-232, as evidenced by its lower rate of parasitaemia. The Tbr EATRO-734 putatively surmount suramin challenges through induction of energy metabolism pathways. These cellular and molecular processes may be involved in suramin resistance in Tbr.


Assuntos
Parasitos , Trypanosoma brucei brucei , Tripanossomíase Africana , Animais , Humanos , Masculino , Camundongos , Proteômica , Suramina/farmacologia , Trypanosoma brucei rhodesiense , Tripanossomíase Africana/tratamento farmacológico , Uganda/epidemiologia
19.
J Leukoc Biol ; 110(4): 663-677, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33438260

RESUMO

An important mediator of inflammation is prostaglandin E2 (PGE2 ), whose levels are determined by the activity of the enzyme cyclooxygenase (COX). Of the two isoforms of the enzyme, COX-2 has been shown to be induced in macrophages during inflammation. Although general COX inhibitors, belonging to the class of nonsteroidal anti-inflammatory drugs, or specific COX-2 inhibitors, called coxibs, are useful in the control of acute inflammation, adverse reactions were seen when used chronically in the treatment of rheumatoid arthritis or neurodegenerative diseases. Extracellular ATP (eATP) has been reported as a damage-associated molecular pattern signal. In this report, we show that eATP synergistically increases the levels of COX-2 enzyme and PGE2 in LPS-activated RAW264.7 macrophages and human monocytes. Activation of macrophages also occurred when cultured in media obtained from dying neurons that contained higher levels of ATP. We show that eATP increases the levels of COX-2 protein, which is sustained up to 36 h poststimulation. This is in turn due to sustained levels of phosphorylated, or activated, cyclin-dependent kinase 9 and p38 MAPK in ATP-treated cells compared to LPS-stimulated cells. The eATP-dependent increase in COX-2/PGE2 levels in LPS-activated RAW264.7 cells could be abolished using antagonists for purinergic P2X7 -and P2Y6 receptors. Similarly, the increase in COX-2/PGE2 levels in the peritoneum of LPS-treated mice could be significantly abolished in mice that were preinjected with the nonspecific P2 receptor antagonist, suramin. P2 receptor antagonists, therefore, should be explored in our search for an ideal anti-inflammatory candidate.


Assuntos
Trifosfato de Adenosina/farmacologia , Quinase 9 Dependente de Ciclina/metabolismo , Dinoprostona/metabolismo , Macrófagos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Humanos , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2X/metabolismo , Suramina/farmacologia
20.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445804

RESUMO

Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Antagonistas Purinérgicos/farmacologia , Receptores Purinérgicos/metabolismo , Células-Tronco/efeitos dos fármacos , Suramina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ventrículos Laterais/efeitos dos fármacos , Ventrículos Laterais/metabolismo , Masculino , Células-Tronco Neurais/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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