RESUMEN
(1R,2R-diaminocyclohexane)(dihydropyrophosphato) platinum(II), also abbreviated as RRD2, belongs to a class of potent antitumor platinum cytostatics called phosphaplatins. Curiously, several published studies have suggested significant mechanistic differences between phosphaplatins and conventional platinum antitumor drugs. Controversial findings have been published regarding the role of RRD2 binding to DNA in the mechanism of its antiproliferative activity in cancer cells. This prompted us to perform detailed studies to confirm or rule out the role of RRD2 binding to DNA in its antiproliferative effect in cancer cells. Here, we show that RRD2 exhibits excellent antiproliferative activity in various cancer cell lines, with IC50 values in the low micromolar or submicromolar range. Moreover, the results of this study demonstrate that DNA lesions caused by RRD2 contribute to killing cancer cells treated with this phosphaplatin derivative. Additionally, our data indicate that RRD2 accumulates in cancer cells but to a lesser extent than cisplatin. On the other hand, the efficiency of cisplatin and RRD2, after they accumulate in cancer cells, in binding to nuclear DNA is similar. Our results also show that RRD2 in the medium, in which the cells were cultured before RRD2 accumulated inside the cells, remained intact. This result is consistent with the view that RRD2 is activated by releasing free pyrophosphate only in the environment of cancer cells, thereby allowing RRD2 to bind to nuclear DNA.
Asunto(s)
Antineoplásicos , Neoplasias , Oxaliplatino/farmacología , Cisplatino/farmacología , Platino (Metal)/farmacología , Difosfatos/farmacología , Compuestos Organoplatinos/farmacología , Compuestos Organoplatinos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , ADN/metabolismoRESUMEN
BACKGROUND: Citrate is the only anticoagulant currently Food and Drug Administration (FDA)-approved for the long-term storage of blood for transfusion. Citrate inhibits phosphofructokinase and may play a pro-inflammatory role, suggesting that there may be an advantage to using alternative anticoagulants. Here, we examine the use of pyrophosphate as an anticoagulant. STUDY DESIGN AND METHODS: Whole blood samples from healthy donors were anticoagulated either with citrate-phosphate-adenine-dextrose (CPDA-1) or our novel anticoagulant mixture pyrophosphate-phosphate-adenine-dextrose (PPDA-1). Samples were assessed for coagulation capacity by thromboelastography immediately after anticoagulation (T0) with and without recalcification, as well as 5 hours after anticoagulation (T1) with recalcification. Complete blood counts were taken at both timepoints. Flow cytometry to evaluate platelet activation as well as blood smears to evaluate cellular morphology were performed at T1. RESULTS: No clotting was detected in samples anticoagulated with either solution without recalcification. After recalcification, clotting function was restored in both groups. R-Time in recalcified PPDA-1 samples was shorter than in CPDA-1 samples. A reduction in platelet count at T1 compared to T0 was observed in both groups. No significant platelet activation was observed in either group at T1. Blood smear indicated platelet clumping in PPDA-1. CONCLUSION: We have shown initial proof of concept that pyrophosphate functions as an anticoagulant at the dose used in this study, though there is an associated loss of platelets over time that may limit its usefulness for blood storage. Further dose optimization of pyrophosphate may limit or reduce the loss of platelets.
Asunto(s)
Anticoagulantes , Difosfatos , Humanos , Anticoagulantes/farmacología , Difosfatos/farmacología , Citratos/farmacología , Plaquetas , Glucosa/farmacología , Adenina/farmacología , Fosfatos/farmacología , Ácido Cítrico , Conservación de la SangreRESUMEN
The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.
Asunto(s)
Nidovirales/enzimología , Nucleótidos/metabolismo , Dominios Proteicos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/enzimología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Coenzimas/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Difosfatos/farmacología , Difosfonatos/farmacología , Guanosina Trifosfato/metabolismo , Manganeso , Modelos Moleculares , Nidovirales/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Uridina Trifosfato/metabolismoRESUMEN
Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5'-triphosphate (UTP)- but not adenosine 5'-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5'-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5'-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.
Asunto(s)
Arteria Pulmonar , Vasodilatación , Ratas , Animales , Uridina Trifosfato/farmacología , Difosfatos/farmacología , Adenosina Trifosfato/farmacología , Uridina Difosfato/farmacología , Uridina/farmacología , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2RESUMEN
Pyrophosphate-containing calcium phosphate implants promote osteoinduction and bone regeneration. The role of pyrophosphate for inflammatory cell-mesenchymal stem cell (MSC) cross-talk during osteogenesis is not known. In the present work, the effects of lipopolysaccharide (LPS) and pyrophosphate (PPi) on primary human monocytes and on osteogenic gene expression in human adipose-derived MSCs were evaluated in vitro, using conditioned media transfer as well as direct effect systems. Direct exposure to pyrophosphate increased nonadherent monocyte survival (by 120% without LPS and 235% with LPS) and MSC viability (LDH) (by 16-19% with and without LPS). Conditioned media from LPS-primed monocytes significantly upregulated osteogenic genes (ALP and RUNX2) and downregulated adipogenic (PPAR-γ) and chondrogenic (SOX9) genes in recipient MSCs. Moreover, the inclusion of PPi (250 µM) resulted in a 1.2- to 2-fold significant downregulation of SOX9 in the recipient MSCs, irrespective of LPS stimulation or culture media type. These results indicate that conditioned media from LPS-stimulated inflammatory monocytes potentiates the early MSCs commitment towards the osteogenic lineage and that direct pyrophosphate exposure to MSCs can promote their viability and reduce their chondrogenic gene expression. These results are the first to show that pyrophosphate can act as a survival factor for both human MSCs and primary monocytes and can influence the early MSC gene expression. Graphical abstract.
Asunto(s)
Difosfatos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Monocitos/fisiología , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Regeneración Ósea/efectos de los fármacos , Regeneración Ósea/genética , Regeneración Ósea/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Medios de Cultivo Condicionados , Regulación hacia Abajo/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Ensayo de Materiales , Osteogénesis/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.
Asunto(s)
Citocromo P-450 CYP3A , Uridina Difosfato , Animales , Derivados del Benceno , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2C9/farmacología , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP2E1/farmacología , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/farmacología , Difosfatos/metabolismo , Difosfatos/farmacología , Perros , Glucuronosiltransferasa/metabolismo , Glucuronosiltransferasa/farmacología , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Conejos , Ratas , Especificidad de la Especie , Porcinos , Porcinos Enanos/metabolismo , Uridina/metabolismo , Uridina/farmacología , Uridina Difosfato/metabolismo , Uridina Difosfato/farmacologíaRESUMEN
Pyrophosphate (PPi) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PPi can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg2+ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.
Asunto(s)
Proteínas Arqueales , Thermococcus , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Difosfatos/farmacología , Proteínas Arqueales/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Uridina DifosfatoRESUMEN
OBJECTIVE: The aim: To reveal the effect of pyrophosphates on the tooth germ structure in the mandible of embryos (17th day of pregnancy) gestated by females, kept on a pyrophosphate-rich diet since 30 days before fertilization to gestation. PATIENTS AND METHODS: Materials and methods: The effect of food supplements was studied in «Overload phosphates model¼. Experiments were carried out on white nonlinear outbred mice with mass 25-28g (n= 40). The females from the control group were fed with standard rodent food, whereas the experimental females were fed with pyrophosphate-enriched food. The material for the morphological study were the mandible of 17-day-old mouse embryos (E-17), which were examined under a microscope with subsequent photofixation. RESULTS: Results: The examination of the mandible of 17-day-old mouse embryos, gestated by females on a pyrophosphate-rich diet, showed morphological changes in tooth germs at the dental follicle development stage. CONCLUSION: Conclusions: The experimentation revealed that the pyrophosphate excessive intake during dental follicle development leads to early dentinogenesis and oppression of ectodermal structures of tooth germs.
Asunto(s)
Difosfatos , Diente , Dieta , Difosfatos/farmacología , Femenino , Humanos , Embarazo , Germen DentarioRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (PKA) phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing one to three protein segments of the R domain including residues 708-759 (ΔR708-759), R760-783, and R784-835, each of which contains one or two PKA phosphorylation sites. Deletion of R708-759 or R760-783 had little effect on CFTR gating, whereas all mutations lacking R784-835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R784-835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR784-835 had minor impact on gating potentiation by 2'dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809-835 shortened the IBI of ΔR708-835-CFTR. Consistently, ΔR815-835, but not ΔR784-814, enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R784-835 is required for stabilizing the open state of CFTR; however, R815-835, through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708-783-CFTR. Conversely, the IBI of ΔR708-814-CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784-814-mediated autoinhibition that prevents IBI shortening by R815-835 This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Adenosina Trifosfato/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Desoxiadenosinas/farmacología , Difosfatos/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Fosforilación , Dominios ProteicosRESUMEN
Bisphosphonates (BPs) are major anti-bone-resorptive drugs. Among them, the nitrogen-containing BPs (NBPs) exhibit much stronger anti-bone-resorptive activities than non-nitrogen-containing BPs (non-NBPs). However, BP-related osteonecrosis of the jaw (BRONJ) has been increasing without effective strategies for its prevention or treatment. The release of NBPs (but not non-NBPs) from NBP-accumulated jawbones has been supposed to cause BRONJ, even though non-NBPs (such as etidronate (Eti) and clodronate (Clo)) are given at very high doses because of their low anti-bone-resorptive activities. Our murine experiments have demonstrated that NBPs cause inflammation/necrosis at the injection site, and that Eti and Clo can reduce or prevent the inflammatory/necrotic effects of NBPs by inhibiting their entry into soft-tissue cells. In addition, our preliminary clinical studies suggest that Eti may be useful for treating BRONJ. Notably, Eti, when administered together with an NBP, reduces the latter's anti-bone-resorptive effect. Here, on the basis of the above background, we examined and compared in vitro interactions of NBPs, non-NBPs, and related substances with hydroxyapatite (HA), and obtained the following results. (i) NBPs bind rapidly to HA under pH-neutral conditions. (ii) At high concentrations, Eti and Clo inhibit NBP-binding to HA and rapidly expel HA-bound NBPs (potency Eti>>Clo). (iii) Pyrophosphate also inhibits NBP-binding to HA and expels HA-bound NBPs. Based on these results and those reported previously, we discuss (i) possible anti-BRONJ strategies involving the use of Eti and/or Clo to reduce jawbone-accumulated NBPs, and (ii) a possible involvement of pyrophosphate-mediated release of NBPs as a cause of BRONJ.
Asunto(s)
Difosfatos/farmacología , Difosfonatos/metabolismo , Durapatita/metabolismo , Calcio/farmacología , Concentración de Iones de Hidrógeno , Magnesio/farmacología , NitrógenoRESUMEN
Despite playing critical roles in the immune response and having significant potential in immunotherapy, γδ T cells have garnered little of the limelight. One major reason for this paradox is that their antigen recognition mechanisms are largely unknown, limiting our understanding of their biology and our potential to modulate their activity. One of the best-studied γδ subsets is the human Vγ9Vδ2T cell population, which predominates in peripheral blood and can combat both microbial infections and cancers. Although it has been known for decades that Vγ9Vδ2T cells respond to the presence of small pyrophosphate-based metabolites, collectively named phosphoantigens (pAgs), derived from microbial sources or malignant cells, the molecular basis for this response has been unclear. A major breakthrough in this area came with the identification of the Butyrophilin 3A (BTN3A) proteins, members of the Butyrophilin/Butyrophilin-like protein family, as mediators between pAgs and Vγ9Vδ2T cells. In this article, we review the most recent studies regarding pAg activation of human Vγ9Vδ2T cells, mainly focusing on the role of BTN3A as the pAg sensing molecule, as well as its potential impact on downstream events of the activation process.
Asunto(s)
Antígenos CD/inmunología , Butirofilinas/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfocitos T/efectos de los fármacos , Animales , Antígenos CD/efectos de los fármacos , Butirofilinas/efectos de los fármacos , Butirofilinas/inmunología , Difosfatos/farmacología , Humanos , Fosforilación/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Coenzima A/farmacología , Difosfatos/farmacología , Diseño de Fármacos , Panteteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Aurora Quinasa A/metabolismo , Coenzima A/síntesis química , Coenzima A/química , Difosfatos/química , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Panteteína/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Farnesol is a sesquiterpene from propolis and citrus fruit that shows promising anti-bacterial activity for caries treatment and prevention, but its hydrophobicity limits the clinical application. We aimed to develop the novel polymeric micelles (PMs) containing a kind of derivative of farnesol and a ligand of pyrophosphate (PPi) that mediated PMs to adhere tightly with the tooth enamel. RESULTS: Farnesal (Far) was derived from farnesol and successfully linked to PEG via an acid-labile hydrazone bond to form PEG-hyd-Far, which was then conjugated to PPi and loaded into PMs to form the aimed novel drug delivery system, PPi-Far-PMs. The in vitro test about the binding of PPi-Far-PMs to hydroxyapatite showed that PPi-Far-PMs could bind rapidly to hydroxyapatite and quickly release Far under the acidic conditions. Results from the mechanical testing and the micro-computed tomography indicated that PPi-Far-PMs could restore the microarchitecture of teeth with caries. Moreover, PPi-Far-PMs diminished the incidence and severity of smooth and sulcal surface caries in rats that were infected with Streptococcus mutans while being fed with a high-sucrose diet. The anti-caries efficacy of free Far can be improved significantly by PPi-Far-PMs through the effective binding of it with tooth enamel via PPi. CONCLUSIONS: This novel drug-delivery system may be useful for the treatment and prevention of dental caries as well as the targeting therapy of anti-bacterial drugs in the oral disease.
Asunto(s)
Cariostáticos , Caries Dental , Durapatita , Farnesol/análogos & derivados , Micelas , Animales , Cariostáticos/química , Cariostáticos/farmacocinética , Cariostáticos/farmacología , Caries Dental/tratamiento farmacológico , Caries Dental/metabolismo , Caries Dental/patología , Difosfatos/química , Difosfatos/farmacocinética , Difosfatos/farmacología , Portadores de Fármacos , Durapatita/química , Durapatita/metabolismo , Farnesol/química , Farnesol/farmacocinética , Farnesol/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Diente Molar/efectos de los fármacos , Diente Molar/ultraestructura , Polietilenglicoles/química , Ratas , Streptococcus mutans/efectos de los fármacosRESUMEN
Immune therapy improves cancer outcomes, yet many patients do not respond. This pre-clinical study investigated whether vascular disrupting agents (VDAs) could convert an immune unresponsive tumor into a responder. CDF1 mice, with 200 mm3 C3H mammary carcinomas in the right rear foot, were intraperitoneally injected with combretastatin A-4 phosphate (CA4P), its A-1 analogue OXi4503, and/or checkpoint inhibitors (anti-PD-1, PD-L1, or CTLA-4 antibodies), administered twice weekly for two weeks. Using the endpoint of tumor growth time (TGT5; time to reach five times the starting volume), we found that none of the checkpoint inhibitors (10 mg/kg) had any effect on TGT5 compared to untreated controls. However, CA4P (100 mg/kg) or OXi4503 (5-50 mg/kg) did significantly increase TGT5. This further significantly increased by combining the VDAs with checkpoint inhibitors, but was dependent on the VDA, drug dose, and inhibitor. For CA4P, a significant increase was found when CA4P (100 mg/kg) was combined with anti-PD-L1, but not with the other two checkpoint inhibitors. With OXi4503 (50 mg/kg), a significant enhancement occurred when combined with anti-PD-L1 or anti-CTLA-4, but not anti-PD-1. We observed no significant improvement with lower OXi4503 doses (5-25 mg/kg) and anti-CTLA-4, although 30% of tumors were controlled at the 25 mg/kg dose. Histological assessment of CD4/CD8 expression actually showed decreased levels up to 10 days after treatment with OXi4503 (50 mg/kg). Thus, the non-immunogenic C3H mammary carcinoma was unresponsive to checkpoint inhibitors, but became responsive in mice treated with VDAs, although the mechanism remains unclear.
Asunto(s)
Antígeno B7-H1/inmunología , Bibencilos/farmacología , Antígeno CTLA-4/inmunología , Difosfatos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Mamarias Animales/tratamiento farmacológico , Estilbenos/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno CTLA-4/antagonistas & inhibidores , Progresión de la Enfermedad , Femenino , Inhibidores de Puntos de Control Inmunológico/inmunología , Masculino , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos C3H , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Resultado del TratamientoRESUMEN
BACKGROUND: It is challenging to find an iron compound that combines good bioavailability with minimal sensory changes when added to seasonings or condiments. Ferric pyrophosphate (FePP) is currently used to fortify bouillon cubes, but its bioavailability is generally low. Previously, the addition of a stabilizer, sodium pyrophosphate (NaPP), improved iron bioavailability from a bouillon drink. OBJECTIVE: We assessed whether there is a dose-response effect of added NaPP on iron bioavailability from local meals prepared with intrinsically labeled FePP-fortified bouillon cubes in young Nigerian women using iron stable isotope techniques. METHODS: In a double-blind, randomized, cross-over trial, women (n = 24; aged 18-40 y; mean BMI 20.5 kg/m2) consumed a Nigerian breakfast and lunch for 5 d prepared with bouillon cubes containing 2.5 mg 57Fe (as FePP) and 3 different molar ratios of NaPP: 57Fe (0:1, 3:1, and 6:1). Iron bioavailability was assessed by measuring 57Fe incorporation into erythrocytes 16 d after each 5 d NaPP: 57Fe feeding period. Data were analyzed using a linear regression model of log iron absorption on NaPP ratio, with body weight and baseline body iron stores as covariates and subject as a random intercept. RESULTS: Of the women included, 46% were anemic and 26% were iron deficient. Iron bioavailability was 10.8, 9.8, and 11.0% for the 0:1, 3:1, and 6:1 NaPP:57Fe treatments, respectively. There was no dose-response effect of an increasing NaPP:57Fe ratio (ß ± SE: 0.003 ± 0.028, P = 0.45). CONCLUSIONS: In this study, the addition of NaPP did not increase iron bioavailability from FePP-fortified bouillon cubes. However, iron bioavailability from the Nigerian meals prepared with FePP-fortified bouillon cubes was higher than expected. These results are encouraging for the potential of bouillon cubes as a fortification vehicle. Further studies are needed to assess the effect of FePP-fortified bouillon cubes on improving iron status in low-income populations. This trial was registered at clinicaltrials.gov as NCT02815449.
Asunto(s)
Anemia Ferropénica/prevención & control , Difosfatos/farmacología , Difosfatos/farmacocinética , Alimentos Fortificados , Absorción Intestinal/efectos de los fármacos , Hierro/farmacocinética , Comidas , Adulto , Anemia , Anemia Ferropénica/sangre , Disponibilidad Biológica , Estudios Cruzados , Difosfatos/sangre , Difosfatos/uso terapéutico , Método Doble Ciego , Eritrocitos/metabolismo , Femenino , Humanos , Hierro/sangre , Hierro/uso terapéutico , Isótopos de Hierro/sangre , Nigeria , Adulto JovenRESUMEN
Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi ), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non-calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100-fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi . In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.
Asunto(s)
Calcificación Fisiológica , Espacio Extracelular/metabolismo , Nucleótidos/farmacología , Túnica Media/patología , Calcificación Vascular/patología , Adenosina Trifosfato/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Difosfatos/farmacología , Ratones , Modelos Biológicos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Hidrolasas Diéster Fosfóricas/deficiencia , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/deficiencia , Pirofosfatasas/metabolismo , Receptores Purinérgicos P2/metabolismo , Uridina Trifosfato/farmacologíaRESUMEN
Reverse transcriptase (RT) is responsible for replicating the HIV-1 genome and is a validated therapeutic target for the treatment of HIV infections. During each cycle of the RT-catalyzed DNA polymerization process, inorganic pyrophosphate is released as the by-product of nucleotide incorporation. Small molecules were identified that act as bioisosteres of pyrophosphate and can selectively freeze the catalytic cycle of HIV-1 RT at the pre-translocated stage of the DNA- or RNA-template-primer-enzyme complex.
Asunto(s)
Difosfatos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Biocatálisis , ADN Viral/efectos de los fármacos , ADN Viral/genética , Difosfatos/síntesis química , Difosfatos/química , Relación Dosis-Respuesta a Droga , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Estructura Molecular , Polimerizacion/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The availability of specific stimuli to induce the anticancer cytotoxicity of human TCRVγ9-expressing T lymphocytes has allowed the development of γδ T cell-based cancer immunotherapies. However, the stringent dependence of such strategies on the inherently toxic IL-2 has raised safety concerns for patients, justifying a search for alternative methods for inducing γδ T cell stimulation. IL-33 is a γ-chain receptor-independent cytokine of the IL-1 superfamily that is expressed by endothelial cells from a tumor microenvironment and can sustain Th1 and Th2 immune responses. Therefore, we investigated its ability to support the stimulation of human TCRVγ9(+) γδ T cells. In this study, we report that IL-33 efficiently sustained the in vitro activation of Vγ9 T lymphocytes by synthetic phosphoantigens, zoledronate, and a BTN3A1 Ab in the absence of an exogenous supply of IL-2. IL-33 was as potent as IL-2 in allowing the proliferative amplification of Vγ9 T cells isolated from PBMC following activation by the synthetic phosphoantigen bromohydrin pyrophosphate. IL-33 also induced an identical maturation into TNF-α- and IFN-γ-producing Th1 effector memory cells, and IL-33-stimulated cells showed an equivalent cytotoxicity for various tumor cells in vitro. Finally, we found that the bioactivity of IL-33 on the Vγ9 T cell was indirectly mediated through contact with CD4 T cells and IL-2 production by CD4 T cells and Vγ9 T cells themselves. These data posit IL-33 as an alternative to IL-2 for Vγ9 T cell-based cancer immunotherapies.
Asunto(s)
Interleucina-33/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Células TH1/inmunología , Antígenos CD/inmunología , Butirofilinas , Proliferación Celular , Células Cultivadas , Difosfatos/farmacología , Difosfonatos/farmacología , Células Endoteliales/metabolismo , Humanos , Imidazoles/farmacología , Inmunoterapia , Interferón gamma/biosíntesis , Interleucina-2/inmunología , Interleucina-2/uso terapéutico , Interleucina-33/uso terapéutico , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Ácido ZoledrónicoRESUMEN
Rodents consume solutions of phosphates and pyrophosphates in preference to water. Recently, we found that the preference for trisodium pyrophosphate (Na3HP2O7) was greater in T1R3 knockout (KO) mice than wild-type (WT) controls, suggesting that T1R3 is a pyrophosphate detector. We now show that this heightened Na3HP2O7 preference of T1R3 KO mice extends to disodium phosphate (Na2HPO4), disodium and tetrasodium pyrophosphate (Na2H2PO4 and Na4H2PO4), a tripolyphosphate (Na5P3O10), a non-sodium phosphate [(NH4)2HPO4], and a non-sodium pyrophosphate (K4P2O7) but not to non-P salts with large anions (sodium gluconate, acetate, or propionate). Licking rates for Na3HP2O7 are higher in T1R2 KO mice than WT controls; Na3HP2O7 preference scores are increased even more in T1R2 KO mice and T1R2+T1R3 double KO mice than in T1R3 KO mice; preference scores for Na3HP2O7 are normal in T1R1 KO mice. These results implicate each subunit of the T1R2+T1R3 dimer in the behavioral response to P-containing taste compounds.
Asunto(s)
Difosfatos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Gusto/efectos de los fármacos , Gusto/fisiología , Animales , Difosfatos/administración & dosificación , Preferencias Alimentarias , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Two new Pt(II) derivatives of kiteplatin ([PtCl2(cis-1,4-DACH)]) with pyrophosphate as carrier ligand, one mononuclear (1) and one dinuclear (2), were synthesized with the aim of potentiating the efficacy of kiteplatin. Complex 1 resulted to be remarkably stable at physiological pH, but it undergoes a fast hydrolysis reaction at acidic pH releasing free pyrophosphate and (aquated) kiteplatin. The dinuclear compound 2 resulted to be less stable than 1 at both neutral and acidic pH forming 1 and (aquated) kiteplatin as first step. Both compounds (1 and 2) do not react as such with 5'-GMP, whereas their hydrolysis products readily form adducts with the nucleotide. The in vitro cytotoxicity assays against a panel of six human cancer cell lines showed that complex 2 affects cancer cell viability even at nanomolar concentrations. The cytotoxic activity of 2 is greater (up to 2 orders of magnitude) than that of cisplatin, oxaliplatin, and kiteplatin, whereas the mononuclear complex 1 has shown a cytotoxic activity comparable to that of oxaliplatin and kiteplatin, but higher than cisplatin. The latter result is not surprising, since the presence of two negative charges reduces the uptake of 1 into the tumor cells as compared to the neutral compound 2. The remarkable activity of 2 against the pancreatic cell line BxPC3 (average IC50 = 0.07 µM) deserves further investigation.