RESUMEN
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.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Suramina/farmacología , Sitios de Unión/efectos de los fármacos , Células HEK293 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-raf/aislamiento & purificación , Proteínas Proto-Oncogénicas c-raf/metabolismo , Suramina/análogos & derivados , Suramina/químicaRESUMEN
Protein-protein interactions are the basis of many important physiological processes and are currently promising, yet difficult, targets for drug discovery. In this context, inhibitor of apoptosis proteins (IAPs)-mediated interactions are pivotal for cancer cell survival; the interaction of the BIR1 domain of cIAP2 with TRAF2 was shown to lead the recruitment of cIAPs to the TNF receptor, promoting the activation of the NF-κB survival pathway. In this work, using a combined in silico-in vitro approach, we identified a drug-like molecule, NF023, able to disrupt cIAP2 interaction with TRAF2. We demonstrated in vitro its ability to interfere with the assembly of the cIAP2-BIR1/TRAF2 complex and performed a thorough characterization of the compound's mode of action through 248 parallel unbiased molecular dynamics simulations of 300 ns (totaling almost 75 µs of all-atom sampling), which identified multiple binding modes to the BIR1 domain of cIAP2 via clustering and ensemble docking. NF023 is, thus, a promising protein-protein interaction disruptor, representing a starting point to develop modulators of NF-κB-mediated cell survival in cancer. This study represents a model procedure that shows the use of large-scale molecular dynamics methods to typify promiscuous interactors.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis , Suramina , Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B , Suramina/análogos & derivados , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
The objective of this study was to investigate the role of ATP in cholinergic neurotransmission in the urinary bladder of control men and of patients obstructed as a result of benign prostatic hyperplasia (BPH). Human detrusor samples were collected from 41 patients who submitted to transvesical prostatectomy resulting from BPH and 26 male organ donors. The release of [3H]acetylcholine ([3H]ACh) was evoked by electrical field stimulation (10 Hz, 200 pulses) in urothelium-denuded detrusor strips. Myographic recordings were performed to test detrusor strip sensitivity to ACh and ATP. Nerve-evoked [3H]ACh release was 1.5-fold higher in detrusor strips from BPH patients compared with controls. This difference was abolished after desensitization of ionotropic P2X1-3 receptors with an ATP analog, α,ß-methylene ATP (30 µM, applied for 15 minutes). TNP-ATP (10 nM, a preferential P2X2/3 antagonist) and A317491 (100 nM, a selective P2X3 antagonist) were about equipotent in decreasing nerve-evoked [3H]ACh release in control detrusor strips, but the selective P2X1 receptor antagonist NF023 (3 µM) was devoid of effect. The inhibitory effect of TNP-ATP (10 nM) increased from 27% ± 9% to 43% ± 6% in detrusor strips of BPH patients, but the effect of A317491 (100 nM) [3H]ACh release unaltered (20% ± 2% vs. 24% ± 4%). The amplitude of ACh (0.1-100 µM)-induced myographic recordings decreased, whereas sensitivity to ATP (0.01-3 mM) increased in detrusor strips from BPH patients. Besides the well characterized P2X1 receptor-mediated contractile activity of ATP in pathologic human bladders, we show here for the first time that cholinergic hyperactivity in the detrusor of BPH patients is facilitated by activation of ATP-sensitive P2X2/3 heterotrimers. SIGNIFICANCE STATEMENT: Bladder outlet obstruction often leads to detrusor overactivity and reduced bladder compliance in parallel to atropine-resistant increased purinergic tone. Our data show that P2X1 purinoceptors are overexpressed in the detrusor of patients with benign prostatic hyperplasia. Besides the P2X1 receptor-mediated detrusor contractions, ATP favors nerve-evoked acetylcholine release via the activation of prejunctional P2X2/3 excitatory receptors in these patients Thus, our hypothesis is that manipulation of the purinergic tone may be therapeutically useful to counteract cholinergic overstimulation in obstructed patients.
Asunto(s)
Adenosina Trifosfato/metabolismo , Tono Muscular , Receptores Purinérgicos P2X1/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/metabolismo , Acetilcolina/metabolismo , Adulto , Anciano , Humanos , Masculino , Persona de Mediana Edad , Contracción Muscular , Fenoles/farmacología , Compuestos Policíclicos/farmacología , Multimerización de Proteína , Antagonistas del Receptor Purinérgico P2X/farmacología , Suramina/análogos & derivados , Suramina/farmacología , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatología , Obstrucción del Cuello de la Vejiga Urinaria/fisiopatologíaRESUMEN
Minichromosome maintenance protein 10 (Mcm10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit Mcm10 from binding to DNA. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human Mcm10 internal domain and full-length Xenopus Mcm10, including analogues that are selective for Mcm10 over human RPA. Binding of suramin analogues to Mcm10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human Mcm10 binding affinity (FP Ki 170 nM, SPR KD 460 nM) and cell activity (IC50 38 µM). Suramin and its analogues are the first identified inhibitors of Mcm10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Suramina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cinética , Proteínas de Mantenimiento de Minicromosoma/genética , Estructura Molecular , Unión Proteica , Suramina/análogos & derivados , Suramina/química , XenopusRESUMEN
Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19 µM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250 µM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.
Asunto(s)
Descubrimiento de Drogas , Naftalenos/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Animales , Humanos , Naftalenos/química , Antagonistas del Receptor Purinérgico P2Y/química , Suramina/análogos & derivadosRESUMEN
Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially bacteraemia. The number of sepsis cases has been steadily increasing over the last decades, and there are still no specific, molecular supportive therapies for sepsis to supplement antibiotic treatment. P2X1 receptors are expressed by a number of immune cells including thrombocytes, which presently have been established as an important player in the acute immune response to bacterial infections. P2X1 receptor-deficient mice have been shown to be relatively protected against urosepsis, with markedly reduced levels of circulating proinflammatory cytokines and intravascular coagulation. However, here we show that continuous intravenous infusion with P2X1 receptor antagonist markedly accelerates development of a septic response to induced bacteraemia with uropathogenic E. coli. Mice exposed to the P2X1 receptor antagonists die very early with haematuria, substantially elevated plasma levels of proinflammatory cytokines, massive intravascular coagulation and a concomitant reduction in circulating thrombocytes. Interestingly, infusion of P2X1 receptor antagonists causes a marked acute reduction in circulating thrombocytes and a higher number of bacteria in the blood. These data support the notion that the number of functional thrombocytes is important for the acute defence against bacteria in the circulation and that the P2X1 receptor potentially could be essential for this response.
Asunto(s)
Plaquetas/efectos de los fármacos , Infecciones por Escherichia coli , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X1 , Sepsis , Infecciones Urinarias , Animales , Bencenosulfonatos , Proteínas Hemolisinas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Pielonefritis , Suramina/análogos & derivados , Escherichia coli UropatógenaRESUMEN
Atherosclerosis is the chronic inflammatory disease, and inflammation-elicited endothelial activation is an early event in the development of atherosclerosis. The P2Y11 receptor is a purinergic receptor and a member of the P2 family of G coupled protein which has been shown to modulate vascular function. Progress in the study of purine receptors has been tremendous and these receptors have become pharmacological targets for various diseases. In this study, we show that the P2Y11R antagonist NF157 can mitigate oxidized LDL (ox-LDL)-induced endothelial inflammation. Our study demonstrates that P2Y11R is expressed to a fair degree in human aortic endothelial cells and is induced by treatment with ox-LDL. Blockage of P2Y11R by its selective antagonist NF157 ameliorates ox-LDL-induced adhesion of THP-1 monocytes to endothelial cells. NF157 inhibits ox-LDL-induced expression of adhesion molecules including E-selectin and VCAM-1. NF157 also suppresses ox-LDL-associated ROS production and induction of the NADPH oxidase subunit NOX-4. Moreover, NF157 has an inhibitory effect on the production of major cytokines including IL-6 and TNF-α. Mechanistically, we show that NF157 mitigates ox-LDL-induced phosphorylation of MAPK kinase p38 and NF-κB activation. Our findings indicate that blockage of P2Y11R signalling by its antagonist NF157 may protect endothelial cells from ox-LDL-induced endothelial inflammation. Therefore, NF157 may have therapeutic implications in the modulation of atherosclerosis-associated inflammation.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Lipoproteínas LDL/farmacología , Receptores Purinérgicos P2/metabolismo , Suramina/análogos & derivados , Adhesión Celular/efectos de los fármacos , Selectina E/genética , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamación/tratamiento farmacológico , Interleucina-6/biosíntesis , Monocitos/citología , Monocitos/efectos de los fármacos , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Suramina/farmacología , Suramina/uso terapéutico , Factor de Necrosis Tumoral alfa/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Heterotrimeric G proteins are usually activated by the guanine-nucleotide exchange factor (GEF) activity of GPCRs. However, some non-receptor proteins are also GEFs. GIV (a.k.a Girdin) was the first non-receptor protein for which the GEF activity was ascribed to a well-defined protein sequence that directly binds Gαi. GIV expression promotes metastasis and disruption of its binding to Gαi blunts the pro-metastatic behavior of cancer cells. Although this suggests that inhibition of the Gαi-GIV interaction is a promising therapeutic strategy, protein-protein interactions (PPIs) are considered poorly "druggable" targets requiring case-by-case validation. Here, we set out to investigate whether Gαi-GIV is a druggable PPI. We tested a collection of >1,000 compounds on the Gαi-GIV PPI by in silico ligand screening and separately by a chemical high-throughput screening (HTS) assay. Two hits, ATA and NF023, obtained in both screens were confirmed in secondary HTS and low-throughput assays. The binding site of NF023, identified by NMR spectroscopy and biochemical assays, overlaps with the Gαi-GIV interface. Importantly, NF023 did not disrupt Gαi-Gßγ binding, indicating its specificity toward Gαi-GIV. This work establishes the Gαi-GIV PPI as a druggable target and sets the conceptual and technical framework for the discovery of novel inhibitors of this PPI.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Microfilamentos/metabolismo , Péptidos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Modelos Moleculares , Estructura Molecular , Péptidos/química , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Mapas de Interacción de Proteínas/efectos de los fármacos , Suramina/análogos & derivados , Suramina/química , Suramina/farmacología , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
The transcription factor STAT5a is a potential target for tumor therapy. We present a fluorescence polarization-based, high-throughput screen of chemical libraries containing natural products and known bioactive molecules, for the identification of small-molecule inhibitors of the STAT5a SH2 domain. This screen identified suramin, a drug used to treat African trypanosomiasis, and its analogues NF023 and NF449 as inhibitors of the SH2 domains of STAT5a/b. Our data extend the known in vitro targets of suramin and analogues to include members of the STAT protein family.
Asunto(s)
Bencenosulfonatos/farmacología , Ensayos Analíticos de Alto Rendimiento , Factor de Transcripción STAT5/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Suramina/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Bencenosulfonatos/síntesis química , Bencenosulfonatos/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Suramina/análogos & derivados , Suramina/síntesis químicaRESUMEN
Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Hipertensión Pulmonar/metabolismo , Pulmón/metabolismo , Arteria Pulmonar/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Antihipertensivos/farmacología , Apirasa/deficiencia , Apirasa/genética , Apirasa/farmacología , Presión Arterial , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Hidrólisis , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/genética , Hipertrofia Ventricular Derecha/metabolismo , Hipertrofia Ventricular Derecha/fisiopatología , Hipoxia/complicaciones , Pulmón/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X1/efectos de los fármacos , Índice de Severidad de la Enfermedad , Transducción de Señal , Suramina/análogos & derivados , Suramina/farmacología , Remodelación Vascular , Remodelación VentricularRESUMEN
NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71). In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors--the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan--with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid.
Asunto(s)
Antivirales/farmacología , Bencenosulfonatos/farmacología , Infecciones por Enterovirus/virología , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Acoplamiento Viral/efectos de los fármacos , Sitios de Unión , Cápside/química , Cápside/efectos de los fármacos , Cápside/metabolismo , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/metabolismo , Infecciones por Enterovirus/metabolismo , Células HeLa , Humanos , Células Jurkat , Modelos Moleculares , Datos de Secuencia Molecular , Suramina/análogos & derivadosRESUMEN
UNLABELLED: HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE: Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF279, is due to the blocking of virus interactions with both the CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by the respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and a mechanism of action that are distinct from those of known HIV-1 coreceptor antagonists.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , VIH-1/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Suramina/análogos & derivados , Internalización del Virus/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Femenino , Células HEK293 , Humanos , Masculino , Receptores Purinérgicos P2X1/metabolismo , Suramina/farmacologíaRESUMEN
Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac-mimetics, the most promising IAP-targeting compounds specifically recognize the IAP-BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac-mimetics interfere with the NF-κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto-ubiquitination process. It has been shown that the XIAP-BIR1 (X-BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF-κB survival pathway. Preventing X-BIR1 dimerization abolishes XIAP-mediated NF-κB activation, thus implicating a proximity-induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X-BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X-BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1-specific compounds acting synergistically with existing pro-apoptotic drugs in cancer therapy.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Suramina/análogos & derivados , Cristalización , Descubrimiento de Drogas , Humanos , Simulación del Acoplamiento Molecular , FN-kappa B , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Suramina/química , Suramina/metabolismoRESUMEN
Previous studies have suggested that carbon monoxide (CO) poisoning stimulates cAMP production via purine P2Y11-like receptors in the rat striatum, activating cAMP signaling pathways, resulting in hydroxyl radical ((â¢)OH) production. Extracellular ATP was thought likely to trigger the cascade, but the present study has failed to demonstrate a clear increase in the extracellular ATP due to CO poisoning. The CO-induced (â¢)OH production was attenuated by the P2Y11 receptor antagonist NF157, in parallel with its abilities to suppress the CO-induced cAMP production. The (â¢)OH production was more strongly suppressed by a non-selective P2 receptor antagonist, PPADS, which had no effect on cAMP production. More selective antagonists toward the respective P2 receptors susceptible to PPADS, including NF279, had little or no effect on the CO-induced (â¢)OH production. The intrastriatal administration of exogenous ATP dose-dependently stimulated (â¢)OH production, which was dose-dependently antagonized by PPADS and NF279 but not by NF157. Exogenous GTP and CTP dose-dependently stimulated (â¢)OH production, though less potently. The GTP-induced (â¢)OH production was susceptible to both of NF279 and PPADS, but the CTP-induced (â¢)OH production was resistant to PPADS. The mechanism of (â¢)OH production may differ between CO poisoning and exogenous ATP, while multiple P2 receptors could participate in (â¢)OH production. The CO-induced (â¢)OH production was susceptible to the inhibition of NADPH oxidase, but not xanthine oxidase. Also, the NADPH oxidase inhibition suppressed (â¢)OH production induced by forskolin, a stimulator of intracellular cAMP formation. It is likely that (â¢)OH is produced by NADPH oxidase activation via cAMP signaling pathways during CO poisoning.
Asunto(s)
Adenosina Trifosfato/farmacología , Intoxicación por Monóxido de Carbono/fisiopatología , Cuerpo Estriado/metabolismo , Radical Hidroxilo/metabolismo , Antagonistas Purinérgicos/farmacología , Suramina/análogos & derivados , Animales , Cuerpo Estriado/efectos de los fármacos , Masculino , Microdiálisis , Ratas , Ratas Sprague-Dawley , Suramina/farmacologíaRESUMEN
The purine receptor involved in inhibitory responses in the gastrointestinal tract has been recently identified. P2Y1 receptor activation mediates the fast component of the inhibitory junction potential (IJPf) and the non-nitrergic relaxation. The aim of the present work has been to investigate which purinergic agonist better mimics endogenous responses. We used different agonist and antagonist of P2 receptors. Contractility and microelectrode experiments were used to compare the effects of exogenously added purines and electrical field stimulation (EFS)-induced nerve mediated effects in rat and human colonic strips. In rat colon, the IJPf and EFS-induced inhibition of contractions were concentration-dependently inhibited by the P2Y1 antagonist MRS2500 but not by iso-PPADS or NF023 (P2X antagonists) up to 1 µM. In samples from human colon, EFS-induced inhibition of contractions was inhibited by either MRS2500 or apamin (1 µM) but not by iso-PPADS. In both species, α,ß-meATP, a stable analog of ATP, caused inhibition of spontaneous contractions. α,ß-meATP effect was concentration-dependent (EC50: 2.7 µM rat, 4.4 µM human) and was antagonized by either MRS2500 or apamin but unaffected by P2X antagonists. ATP, ADP, ß-NAD and ADP-ribose inhibited spontaneous contractions but did not show the same sensitivity profile to purine receptor antagonists as EFS-induced inhibition of contractions. The effect of α,ß-meATP is due to P2Y1 receptor activation leading the opening of sKca channels. Accordingly, α,ß-meATP mimics the endogenous purinergic mediator. In contrast, exogenously added putative neurotransmitters do not exactly mimic the endogenous mediator. Quick degradation by ecto-nuclease or different distribution of receptors (junctionally vs extrajunctionally) might explain these results.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Colon/efectos de los fármacos , Agonistas Purinérgicos/farmacología , Antagonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2Y1/fisiología , Adenosina Trifosfato/farmacología , Anciano , Animales , Apamina/farmacología , Colon/fisiología , Nucleótidos de Desoxiadenina/farmacología , Estimulación Eléctrica , Femenino , Humanos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Ratas Sprague-Dawley , Suramina/análogos & derivados , Suramina/farmacologíaRESUMEN
We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.
Asunto(s)
Estradiol/metabolismo , Células de la Granulosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oogénesis , Folículo Ovárico/citología , Animales , Bovinos , Tamaño de la Célula/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Femenino , Hormona Folículo Estimulante/farmacología , Fulvestrant , Células de la Granulosa/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oogénesis/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Suramina/análogos & derivados , Suramina/farmacologíaRESUMEN
The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and ß-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating ß-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP.
Asunto(s)
ADP-Ribosa Cíclica/metabolismo , NADP/análogos & derivados , Receptores Purinérgicos P2Y/metabolismo , ADP-Ribosil Ciclasa/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , AMP Cíclico/metabolismo , Difosfonatos/farmacología , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , Corazón/fisiología , Técnicas In Vitro , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , NADP/metabolismo , NADP/farmacología , Naftalenosulfonatos/farmacología , Ratas , Ratas Wistar , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal/efectos de los fármacos , Suramina/análogos & derivados , Suramina/farmacología , Factores de TiempoRESUMEN
Falcipain-2 is a cysteine protease of the malaria parasite Plasmodium falciparum that plays a key role in the hydrolysis of hemoglobin, a process that is required by intraerythrocytic parasites to obtain amino acids. In this work we show that the polysulfonated napthylurea suramin is capable of binding to falcipain-2, inhibiting its catalytic activity at nanomolar concentrations against both synthetic substrates and the natural substrate hemoglobin. Kinetic measurements suggest that the inhibition occurs through an noncompetitive allosteric mechanism, eliciting substrate inhibition. Smaller suramin analogues and those with substituted methyl groups also showed inhibition within the nanomolar range. Our results identify the suramin family as a potential starting point for the design of falcipain-2 inhibitor antimalarials that act through a novel inhibition mechanism.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Cisteína Endopeptidasas/metabolismo , Plasmodium falciparum/enzimología , Suramina/análogos & derivados , Suramina/farmacología , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Malaria Falciparum/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Plasmodium falciparum/efectos de los fármacosRESUMEN
It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3-24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.
Asunto(s)
Adenosina Trifosfato/metabolismo , Comunicación Autocrina , Exocitosis , Activación de Macrófagos , Macrófagos/inmunología , Receptores Purinérgicos P2/metabolismo , Animales , Líquido Ascítico/efectos de los fármacos , Líquido Ascítico/metabolismo , Comunicación Autocrina/efectos de los fármacos , Línea Celular Tumoral , Exocitosis/efectos de los fármacos , Humanos , Interleucina-6/biosíntesis , Interleucina-6/sangre , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Antagonistas del Receptor Purinérgico P2/farmacología , Antagonistas del Receptor Purinérgico P2/uso terapéutico , Choque Séptico/inducido químicamente , Choque Séptico/tratamiento farmacológico , Choque Séptico/inmunología , Choque Séptico/metabolismo , Suramina/análogos & derivados , Suramina/farmacología , Suramina/uso terapéuticoRESUMEN
BACKGROUND AND OBJECTIVE: Mechanical stress is an important factor in maintaining homeostasis of the periodontium. Interleukin-1beta (IL-1ß) and adenosine triphosphate (ATP) are considered potent inflammatory mediators. In macrophages, ATP-activated P2X7 receptor is involved in IL-1ß processing and release. Our previous works demonstrated mechanical stress-induced expression of osteopontin and RANKL through the ATP/P2Y1 receptor in human periodontal ligament (HPDL) cells. This study was designed to examine the effect of mechanical stress on IL-1ß expression in HPDL cells, as well as the mechanism and involvement of ATP and the P2 purinergic receptor. MATERIAL AND METHODS: Cultured HPDL cells were treated with continuous compressive loading. IL-1ß expression was analyzed at both mRNA and protein levels, using RT-PCR and ELISA, respectively. Cell viability was examined using the MTT assay. ATP was also used to stimulate HPDL cells. Inhibitors, antagonists and the small interfering RNA (siRNA) technique were used to investigate the role of ATP and the specific P2 subtypes responsible for IL-1ß induction along with the intracellular mechanism. RESULTS: Mechanical stress could up-regulate IL-1ß expression through the release of ATP in HPDL cells. ATP alone was also capable of increasing IL-1ß expression. The induction of IL-1ß was markedly inhibited by inhibitors and by siRNA targeting the P2X7 receptor. ATP-stimulated IL-1ß expression was also diminished by intracellular calcium inhibitors. CONCLUSION: Our work clearly indicates the capability of HPDL cells to respond directly to mechanical stimulation. The results signified the important roles of ATP/P2 purinergic receptors, as well as intracellular calcium signaling, in mechanical stress-induced inflammation via up-regulation of the proinflammatory cytokine, IL-1ß, in HPDL cells.