RESUMEN
BACKGROUND: ABI793 (ABI) is a human monoclonal antibody (mAb) specific for human CD154. To assess the suitability of ABI for baboon transplantation studies, we carried out in vitro studies to determine ABI's reactivity with baboon cells expressing CD154, performed in vivo pharmacokinetic studies in two baboons, and tested the effect of ABI administration on elicited antibody production in two baboons undergoing either pig hematopoietic progenitor cell (PBPC) or heterotopic heart transplantation. METHODS: In vitro: Baboon peripheral blood mononuclear cells were activated in vitro to upregulate CD154, and binding of ABI to CD154 was measured by flow cytometry. In vivo: Serum levels of ABI were measured immediately before and 15 min after the intravenous administration of ABI (20 mg/kg) to two baboons over 28 days. Subsequently, ABI (25 mg/kg on days 0, 1, 4 and 7, and then 20 mg/kg every 5 days) was included in the immunosuppressive regimen in two pig-to-baboon transplants (PBPC or heart transplantation). RESULTS: In vitro: ABI was almost non-reactive to baboon T cells before stimulation, but bound to activated T cells. In vivo: In the pharmacokinetic study, trough levels of ABI (before the next dose) ranged between 190 and 580 microg/ml, and the estimated half-life was 10-15 days. There was no apparent toxicity. Following pig PBPC or heart transplantation, no elicited antibody was detected while ABI was being administered or during several weeks of follow-up. CONCLUSIONS: ABI functions in baboons, is well-tolerated, and prevents an elicited antibody response to pig antigens.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ligando de CD40/inmunología , Trasplante de Corazón/inmunología , Papio , Porcinos , Trasplante Heterólogo/inmunología , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Ligando de CD40/sangre , Ligando de CD40/metabolismo , Citometría de Flujo , Expresión Génica , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Papio/inmunología , Porcinos/inmunologíaRESUMEN
A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a 13C,15N-labeled protein. The method uses an adiabatic 13C inversion pulse optimized to an empirically observed relationship between 1 J(CH) and carbon chemical shift to selectively invert the protein protons (attached to 13C). Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively. Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface. The method is demonstrated with an application to the B12-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B12-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B12).
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Sitios de Unión , Isótopos de Carbono , Clostridium/enzimología , Cobamidas/química , Transferasas Intramoleculares/química , Ligandos , Sustancias Macromoleculares , Modelos Moleculares , Isótopos de Nitrógeno , Conformación Proteica , Subunidades de ProteínaRESUMEN
The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.
Asunto(s)
Proteínas Aviares , Proteínas Musculares/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogénicas c-myc/química , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , CodornizRESUMEN
We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.
Asunto(s)
Ciclofilina A/metabolismo , Inmunosupresores/metabolismo , Lactonas/metabolismo , Compuestos de Espiro/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Células Clonales , Ciclofilina A/antagonistas & inhibidores , Citocinas/biosíntesis , Dihidroorotasa/antagonistas & inhibidores , Dihidroorotasa/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , IMP Deshidrogenasa/metabolismo , Inmunosupresores/farmacología , Células Jurkat , Lactonas/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Monocitos/efectos de los fármacos , Monocitos/inmunología , Unión Proteica/inmunología , Compuestos de Espiro/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismoRESUMEN
1H(i)-15 N(i)-13C'(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the 13C,15N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane torsion angles phi and psi taken from the solution structure. 1H(i)-15N(i)-13C'(i) as well as 13C alpha(i)-1H alpha(i)-13C'(i) dipole-CSA cross-correlation rates were subsequently used to improve the geometry of the metal binding site. The optimized dihedral angles of the two zinc-binding sites in qCRP2(LIM2) are in better agreement with values obtained from crystal structures of other zinc-finger proteins and thus establish the utility of this approach to improve the metal-binding site geometry of zinc-finger proteins studied by NMR spectroscopy in solution.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Dedos de Zinc , Sitios de Unión , Isótopos de Carbono , Hidrógeno , Modelos Moleculares , Modelos Teóricos , Isótopos de Nitrógeno , Unión Proteica , Conformación ProteicaAsunto(s)
Proteínas Aviares , Proteínas Portadoras/química , Fibroblastos/metabolismo , Genes myc/genética , Resonancia Magnética Nuclear Biomolecular , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular Transformada , Transformación Celular Viral , Proteínas de Unión a Ácidos Grasos , Lipocalinas , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Recent experimental data have shown that coadministration of microemulsion cyclosporine and the novel immunosuppressant SDZ-RAD potentiates the immunosuppressive efficacies of both drugs to suppress allograft rejection. Our study was designed to assess the potential of delayed SDZ-RAD administration, in addition to cyclosporine maintenance therapy, to reverse acute rejection in an allogeneic rat lung transplant model. METHODS: Unilateral left lung transplantation was performed using Brown-Norway donors implanted into Lewis recipients. An untreated control group and a cyclosporine monotherapy group (7.5 mg/kg) were followed for 7 days. An additional cyclosporine monotherapy group (7.5 mg/kg), and a combined therapy group treated with cyclosporine (7.5 mg/kg) plus SDZ-RAD (2.5 mg/kg), were followed for 21 days. For treatment of ongoing rejection, 7.5 mg/kg cyclosporine was given as maintenance therapy, and SDZ-RAD (2.5 mg/kg) was added on postoperative day 7. Drugs were given orally, and in the combined therapy regimens, administered 6 hours apart. Outcome variables included daily weight, radiographs, and histology. RESULTS: Radiographs on postoperative day 7 showed mild and moderate opacification of the left chest in the cyclosporine monotherapy groups and the untreated control group. Addition of SDZ-RAD to cyclosporine treatment on postoperative day 7 reversed opacification by postoperative days 14 and 21. Monotherapy with microemulsion CsA resulted in mild histological rejection by day 7, which progressed to moderate rejection by day 21. Addition of SDZ-RAD on postoperative day 7 reversed acute rejection, resulting in none or minimal rejection at day 21. CONCLUSIONS: SDZ RAD reverses acute rejection under cyclosporine maintenance therapy in a stringent lung allotransplant model.
Asunto(s)
Rechazo de Injerto/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Trasplante de Pulmón , Sirolimus/análogos & derivados , Enfermedad Aguda , Animales , Peso Corporal , Everolimus , Trasplante de Pulmón/diagnóstico por imagen , Trasplante de Pulmón/patología , Masculino , Radiografía , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Inducción de Remisión , Sirolimus/uso terapéuticoRESUMEN
Whereas the standard immunosuppressive agents foster development of posttransplant lymphoproliferative disorders (PTLDs), the impact of RAD, a macrolide with potent immunosuppressive properties, and other immunosuppressive macrolides on these disorders remains undetermined. We found that RAD had a profound inhibitory effect on in vitro growth of six different PTLD-like Epstein-Barr virus+ lymphoblastoid B cell lines. Similar to normal T cells, RAD blocked cell-cycle progression in PTLD-like B cells in the early (G(0)/G(1)) phase. Furthermore, RAD increased the apoptotic rate in such cells. The drug also had a profound inhibitory effect on the growth of PTLD-like Epstein-Barr virus+ B cells xenotransplanted s.c. into SCID mice. The degree of the RAD effect varied among the three B cell lines tested and was proportional to its effects on the cell lines in vitro. In this in vivo xenotransplant model, RAD markedly delayed growth or induced regression of the established tumors. In one line, it was able to eradicate the tumor in four of eight mice. When RAD treatment was initiated before tumor cell injection, a marked inhibition of tumor growth was seen in all three lines. In two of them, the drug prevented tumor establishment in approximately 50% of mice (5/11 and 5/8). In summary, RAD is a potent inhibitor of PTLD-like cells in vitro and in vivo. These findings indicate that, in contrast to the standard immunosuppressive agents, macrolides such as RAD may be effective in prevention and treatment of PTLDs.
Asunto(s)
División Celular/efectos de los fármacos , Herpesvirus Humano 4/efectos de los fármacos , Inmunosupresores/uso terapéutico , Trastornos Linfoproliferativos/tratamiento farmacológico , Trastornos Linfoproliferativos/prevención & control , Sirolimus/análogos & derivados , Trasplante/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Transformada , Everolimus , Humanos , Inmunosupresores/farmacología , Técnicas In Vitro , Ratones , Ratones Endogámicos ICR , Ratones SCID , Trasplante de Neoplasias , Sirolimus/farmacología , Sirolimus/uso terapéuticoRESUMEN
Macrolide immunosuppressive drugs such as tacrolimus (FK506) and sirolimus (rapamycin) are compounds largely used in modern immunosuppressive therapy and considered as powerful immunosuppressive agents. Some of these molecules are still under clinical development as, for example, SDZ-RAD (40-O-(2-hydroxyethyl)rapamycin), an immunosuppressive drug closely related to rapamycin. SDZ-RAD has a molecular mass of 957.57 Da (C53H83NO14) and shares the same common intracellular receptor as tacrolimus, the FK-506 binding protein (FKBP-12). SDZ-RAD exerts its pharmacological effect by binding to a different effector protein, inhibits the S6p 70-kinase and interrupts a different signal transduction pathway than tacrolimus. Both SDZ-RAD and rapamycin are metabolized mainly by the cytochrome P-450 3A4-dependent mixed function oxygenase enzyme system to hydroxylated and demethylated metabolites. We describe here the isolation from pig liver microsomes of a novel SDZ-RAD metabolite identified by electrospray tandam mass spectrometry as a new SDZ-RAD 17,18,19,20,21,22-tris-epoxide metabolite. The in vitro immunosuppressive activity as measured by the mixed lymphocyte reaction is more or less comparable to that of SDZ-RAD, although its pharmacological mode of action may be different from that classically described for rapamycin.
Asunto(s)
Inmunosupresores/aislamiento & purificación , Sirolimus/análogos & derivados , Animales , Cromatografía Líquida de Alta Presión , Compuestos Epoxi/química , Everolimus , Femenino , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Técnicas In Vitro , Prueba de Cultivo Mixto de Linfocitos , Espectrometría de Masas/métodos , Microsomas Hepáticos/química , Sirolimus/química , Sirolimus/aislamiento & purificación , Sirolimus/farmacología , PorcinosRESUMEN
BACKGROUND: In previous studies of cynomolgus monkey lung allograft recipients, we demonstrated significant immunosuppressive efficacy but reduced tolerability after combined treatment with high doses of microemulsion cyclosporine (CsA) and SDZ RAD (40-O-(2-hydroxyethyl)-rapamycin). The current study was designed to compare efficacy and tolerability of a combination of low-dose CsA and high-dose SDZ RAD (CTL group) to triple therapy using the chimeric anti-interleukin-2 (IL-2) receptor (CD25) monoclonal antibody (mAb) basiliximab (anti-IL-2 receptor mAb) for induction therapy (basiliximab: 5 mg intravenously on days 0 and 4) plus low-dose CsA and low-dose SDZ RAD for maintenance immunosuppression (CD25 group). CsA and anti-IL-2 receptor mAb are drugs that reduce cytokine synthesis and block IL-2-mediated lymphocyte stimulation, respectively. SDZ RAD blocks lymphocyte stimulation by other cytokines (e.g., IL-15) that are not inhibited by anti-IL-2 receptor mAb. METHODS: Twelve unilateral lung transplants were performed. Recipients were observed for 49 days by daily weight assessment, hemograms, blood chemistries, radiographs, and lung biopsies. Monkeys were euthanized before day 49 in the event of excessive weight loss (>25%) or organ failure. Target CsA trough levels were 100-200 ng/ml. Target SDZ RAD trough levels in the CTL group (no mAb) were 20-40 ng/ml, and 10-20 ng/ml in the CD25 group. RESULTS: None of the monkeys in the CD25 group needed to be euthanized early due to signs of drug toxicity. In contrast, four monkeys in the CTL group were sacrificed on days 28-35 as a result of excessive weight loss (n=3) and renal functional impairment (n=1). Three recipients in the CD25 group were euthanized on days 36, 38, and 46 as a result of persistent high fever associated with severe rejection. The median animal survival in the CTL group was 32 vs. 46 days in the CD25 group (P<0.04). The only two long-term survivors in the CTL group showed moderate rejection at day 49. The median rejection scores at day 14 (A0) and day 28 (A2) were identical in the two groups, despite the fact that the mean SDZ RAD trough level was significantly lower in the CD25 group (CTL: 38+/-3 ng/ml, CD25: 18+/-2 ng/ml, P<0.0001). After basiliximab levels fell below the minimum therapeutic level (1 mg/ml) on day 28, the median rejection score at day 49 increased to A4 in the CD25 group. CONCLUSION: This is the first study to combine an anti-IL-2 receptor mAb with a drug from the rapamycin class plus CsA. Our study shows that induction therapy with basiliximab enabled SDZ RAD blood levels to be significantly reduced, which led to improved tolerability without the penalty of increased rejection.
Asunto(s)
Trasplante de Pulmón/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Autopsia , Basiliximab , Biopsia , Peso Corporal , Broncoscopía , Creatinina/sangre , Ciclosporina/administración & dosificación , Ciclosporina/farmacocinética , Ciclosporina/uso terapéutico , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Emulsiones , Everolimus , Rechazo de Injerto/prevención & control , Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Pulmón/patología , Macaca fascicularis , Masculino , Microquímica , Periodo Posoperatorio , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/inmunología , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Donantes de TejidosRESUMEN
BACKGROUND: We studied the efficacy and tolerability of combined immunosuppressive therapy with cyclosporine A microemulsion (Neoral) plus the macrolide SDZ RAD 40-0 (2-hydroxyethyl) rapamycin (RAD) in a stringent cynomolgus monkey lung graft model in comparison with cyclosporine or SDZ RAD monotherapy. METHODS: Thirty-nine cynomolgus monkeys received mixed lymphocyte reaction (MLR) mismatched unilateral lung transplants. Immunosuppressants were administered orally as single daily doses. The observation period was 28 days and follow-up included serial trough blood drug concentrations measured by high performance liquid chromatography/mass spectrometry, blood analyses, chest radiographs, open lung biopsies, as well as tissue drug concentrations and graft histology at necropsy. RESULTS: Graft biopsies in monkeys treated with vehicle (n=4), Neoral (day 1-7: 150 mg/kg/day; day 8-28: 100 mg/kg/day; n=6; mean +/- SE trough level (MTL): 292+/-17 ng/ml) or SDZ RAD monotherapy (1.5 mg/kg/day; n=6; MTL: 15+/-1 ng/ml) showed severe rejection. Coadministration in two transplant monkeys of Neoral (150/100 mg/kg/day) and SDZ RAD (1.5 mg/kg/day) caused their early death. In both animals, SDZ RAD blood levels were more than 5-fold higher than under monotherapy (MTL: 82+/-18 ng/ml). Simultaneous administration (n=6) of Neoral (150/100 mg/kg/day; MTL: 217+/-16 ng/ml) and SDZ RAD (0.3 mg/kg/day; MTL: 24+/-2 ng/ml) improved graft outcome (mild rejection). Side effects included renal failure (n=2) and seizures (n=1). Three monkeys survived to day 28. In this group the MTL for cyclosporin was 143+/-13 and for RAD 38+/-3. Staggered treatment completely prevented rejection in four of six grafts. However, five of six monkeys had moderate to severe diarrhea. In a concentration-controlled trial of simultaneously administered Neoral and SDZ RAD in transplant monkeys (target SDZ RAD MTL: 20-40 ng/ml; cyclosporine MTL: 100-200 ng/ml) all six monkeys survived with improved drug tolerability and an average biopsy score of mild rejection. CONCLUSION: Combination of orally administered SDZ RAD and Neoral showed excellent immunosuppressive efficacy in a stringent lung transplant model. The drug interaction and the narrow therapeutic index of this drug combination required careful dose adjustments to optimize tolerability and efficacy.
Asunto(s)
Ciclosporina/uso terapéutico , Inmunosupresores/uso terapéutico , Trasplante de Pulmón , Sirolimus/análogos & derivados , Animales , Sangre/metabolismo , Broncoscopía , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Everolimus , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Pulmón/patología , Pulmón/fisiopatología , Macaca fascicularis , Sirolimus/sangre , Sirolimus/uso terapéuticoRESUMEN
BACKGROUND: 40-O-(2-Hydroxyethyl)-rapamycin (SDZ RAD) is a novel, potent, macrolide immunosuppressant. Its efficacy in rodent transplantation models provided the rationale for us to evaluate the compound in a more relevant, large animal transplantation model. METHODS: Life-supporting kidney allotransplantation was performed in cynomolgus monkeys: rejection was inferred from a rise in serum creatinine or urea and was subsequently confirmed by histopathology. This model was validated with the microemulsion formulation of cyclosporine (i.e., Neoral). Two studies with a microemulsion formulation of SDZ RAD were performed. First, in a dose-finding study, the SDZ RAD dose was reduced in a stepwise fashion until rejection occurred, either with SDZ RAD as monotherapy, or in combination with a fixed, suboptimal dose of cyclosporine. Second, an efficacy study was performed in which two fixed SDZ RAD doses (0.75 and 1.50 mg/kg/ day) were evaluated in monotherapy and compared with the same doses of rapamycin (sirolimus). All immunosuppressants were administered once daily by gastric gavage. RESULTS: Untreated control animals rejected their grafts between 4 and 8 days after transplantation. Cyclosporine (initially at 150 mg/kg/day, reduced to 100 mg/kg/day 2 weeks after transplantation) yielded long-term (>100 days) rejection-free allograft survival in four of five animals. A 10 mg/kg/day dose of cyclosporine led to rejection between 10 and 27 days after transplantation and was considered suboptimal. In the dose-finding study with SDZ RAD monotherapy, rejection occurred in most of the cases (four of six animals) when a dose level of 0.63 mg/kg/day had been reached. Combined with suboptimal cyclosporine, this threshold SDZ RAD dose was about 2-fold lower. In the efficacy study, median graft survival with histologically proven rejection was 32 days (range 8-91 days, n=6) for 0.75 mg(kg/day SDZ RAD and 59 days (range 28-85 days, n=6) for 1.50 mg/kg/day SDZ RAD. For sirolimus, median graft survival was 43 days (range 5-103 days, n=7) for the 0.75 mg/kg/day dose and 56 days (range 8-103 days, n=8) for the 1.50 mg/kg/day dose. There was no statistically significant difference in efficacy between SDZ RAD and sirolimus. CONCLUSION: SDZ RAD, in the absence of any other immunosuppressant and at doses that do not show any overt toxicity, considerably prolongs rejection-free survival of cynomolgus monkeys after life-supporting kidney allotransplantation.
Asunto(s)
Ciclosporina/administración & dosificación , Inmunosupresores/administración & dosificación , Trasplante de Riñón , Sirolimus/análogos & derivados , Administración Oral , Animales , Ciclosporina/uso terapéutico , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Emulsiones , Everolimus , Supervivencia de Injerto/efectos de los fármacos , Inmunosupresores/uso terapéutico , Macaca fascicularis , Sirolimus/administración & dosificación , Sirolimus/uso terapéutico , Trasplante HomólogoRESUMEN
The structure of the 56 amino acid nucleocapsid protein NCp10 of retrovirus MoMuLV, which contains a single CX(2)CX(4)HX(4)C-type zinc finger, has been determined previously by NMR. The important role of NCp10 (or NCp7 for HIV-1) in the retroviral life cycle seems mainly related to their preferential binding to single-stranded nucleic acids. We report here the structure of the complex formed between the biologically active (14-53)NCp10 and the oligonucleotide d(ACGCC) in aqueous solution determined by 2D (1)H NMR based methods. The aromatic residue Trp(35) of NCp10 directs nucleic acid complexation as shown by its complete fluorescence quenching upon addition of d(ACGCC). (1)H and (31)P NMR studies support the insertion of Trp(35) between the G(3) and C(4) bases. A total of 577 NOE distance restraints, of which 40 were intermolecular, were used for the structure determination. The zinc finger provides a well-defined surface for the binding of d(ACGCC) through hydrophobic interactions and tryptophan stacking on the guanine. This latter interaction was also observed in the NMR-derived structures of the complexes between NCp7, which contains two successive zinc fingers, and single-stranded DNA and RNA, supporting the proposal for a major role played by aromatic residues of NCp proteins in nucleic acid recognition. Upon binding to the nucleotide a new loop in NCp10 that participates in the intermolecular interaction is formed. Additional interactions provided by positively charged residues surrounding the zinc finger appear necessary for tight binding. The structure of the complex NCp10-d(ACGCC) gives a structural explanation for the loss of virus infectivity following point mutations in the finger domain.
Asunto(s)
Proteínas de la Cápside , Cápside/química , Productos del Gen gag/química , VIH-1/química , Virus de la Leucemia Murina de Moloney/química , Oligonucleótidos/química , Proteínas del Núcleo Viral/química , Proteínas Virales , Dedos de Zinc , Secuencia de Aminoácidos , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Fósforo , Conformación Proteica , Protones , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
A novel class of macrolides for which the name sanglifehrins is proposed, has been discovered from actinomycete strains based on their high affinity binding for cyclophilin A (CypA), an immunophilin originally identified as a cytosolic protein binding cyclosporin A (CsA). The sanglifehrins were produced by Streptomyces sp. A92-308110. They were isolated and purified by extraction and several chromatographic, activity-guided steps. Sanglifehrins A and B exhibit a 10 to approximately 20 fold higher affinity for CypA than CsA, whereas the affinity of sanglifehrins C and D for CypA is comparable to that of CsA. Sanglifehrins exhibit a lower immunosuppressive activity than CsA when tested in the mixed lymphocyte reaction. Their in vitro activity indicates that they belong to a novel class of immunosuppressants.
Asunto(s)
Antibacterianos/farmacología , Inmunosupresores/farmacología , Streptomyces/metabolismo , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Células de la Médula Ósea/fisiología , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Ciclosporina/metabolismo , Inmunofilinas/metabolismo , Inmunosupresores/química , Inmunosupresores/aislamiento & purificación , Inmunosupresores/metabolismo , Lactonas/química , Lactonas/aislamiento & purificación , Lactonas/metabolismo , Lactonas/farmacología , Prueba de Cultivo Mixto de Linfocitos , Macrólidos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Compuestos de Espiro/química , Compuestos de Espiro/aislamiento & purificación , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacología , Streptomyces/clasificación , Streptomyces/crecimiento & desarrolloRESUMEN
SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.
Asunto(s)
Fármacos Dermatológicos/uso terapéutico , Enfermedades de la Piel/tratamiento farmacológico , Tacrolimus/análogos & derivados , Animales , Calcineurina/metabolismo , División Celular , Células Cultivadas , Citocinas/metabolismo , Fármacos Dermatológicos/metabolismo , Humanos , Inmunofilinas/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Enfermedades de la Piel/patología , Linfocitos T/patología , Linfocitos T Colaboradores-Inductores/patología , Tacrolimus/uso terapéutico , Proteínas de Unión a TacrolimusRESUMEN
The cyclohexyl-subunit of rapamycin was cleaved by a sequence involving a Baeyer-Villiger reaction and acid hydrolysis of the resulting lactone-acetal as key steps. Binding of this new rapamycin derivative to FKBP12 was only slightly reduced by this modification, whereas the loss of antiproliferative and immunosuppressive activity was dramatic. These findings indicate that part of the cyclohexyl-subunit of rapamycin could belong to its effector domain.
Asunto(s)
Inmunosupresores/metabolismo , Sirolimus/metabolismo , Células Clonales , Hidrólisis , Inmunosupresores/química , Inmunosupresores/farmacología , Prueba de Cultivo Mixto de Linfocitos , Sirolimus/química , Sirolimus/farmacologíaAsunto(s)
Aorta Abdominal/trasplante , Inmunosupresores/farmacología , Preservación de Órganos/métodos , Daño por Reperfusión/prevención & control , Sirolimus/análogos & derivados , Trasplante Isogénico/fisiología , Adenosina , Alopurinol , Animales , Aorta Abdominal/patología , Aorta Abdominal/fisiología , Frío , Everolimus , Glutatión , Insulina , Isquemia , Masculino , Soluciones Preservantes de Órganos , Rafinosa , Ratas , Ratas Endogámicas Lew , Sirolimus/farmacologíaRESUMEN
The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.
Asunto(s)
Proteínas de la Cápside , Productos del Gen vpr/química , Productos del Gen vpr/metabolismo , Proteínas Virales , Secuencia de Aminoácidos , Cápside/química , Cápside/metabolismo , Dicroismo Circular , Dimerización , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Productos del Gen vpr/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , ARN Viral/química , ARN Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Levaduras/genética , Dedos de Zinc , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
A series of 32-(O)-acylated and 32-(O)-thioacylated derivatives of the antibiotic ascomycin (1) have been synthesized. These readily accessible analogues exhibit potent immunosuppressive activity in vitro, as measured by an interleukin-2 reporter gene assay and the mixed lymphocyte reaction. Such molecules are expected to have a therapeutic potential in chronic inflammatory diseases of the airways such as asthma.