RESUMEN
Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 10/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Interferón gamma/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Caspasa 10/química , Caspasa 10/genética , Caspasa 8/química , Caspasa 8/genética , Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Línea Celular , Citocina TWEAK/farmacología , Sinergismo Farmacológico , Células HT29 , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Interferón gamma/genética , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Ácidos Pentanoicos/farmacología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaAsunto(s)
Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , FN-kappa B/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Humanos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556-induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556-induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML.
Asunto(s)
Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Dipéptidos/farmacología , Indoles/farmacología , Ácidos Pentanoicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Péptidos y Proteínas de Señalización Intracelular , Leucemia Mieloide Aguda/metabolismo , Necrosis/metabolismo , Células Tumorales CultivadasRESUMEN
Birinapant/TL32711 (1) is a bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins and was designed to mimic AVPI, the N-terminal tetrapeptide of the second mitochondria-derived activator of caspases (Smac/DIABLO). Birinapant bound to the BIR3 domains of cIAP1, cIAP2, and XIAP with K i values of 1, 36, and 45 nM, respectively. Birinapant-mediated activation of cIAP1 resulted in cIAP1 autoubiquitylation and degradation and correlated with inhibition of TNF-mediated NF-κB activation, induction of tumor cell death in vitro, and tumor regression in vivo. Birinapant is being evaluated in Phase 1/2 trials for the treatment of cancer and hepatitis B virus (HBV) infection. After one year at accelerated storage conditions, a formulation of 1 afforded four degradants in >0.1% abundance by HPLC analysis. The primary degradants (2 and 3) were formed via oxidation of the biindole core, while the secondary degradants (5 and 6) arose via [1,2]-rearrangement of 3 and 2, respectively. Forced degradation conditions were developed, which allowed the isolation of 2 and 3 in multigram quantities. Novel deuterated analogues of 1 were prepared to determine the site of oxidation, and NMR experiments confirmed the chemical structures of 5 and 6. The de novo synthesis of 2, 3, 5, and 6 confirmed these experimental findings.
RESUMEN
Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.
Asunto(s)
Antineoplásicos/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leucemia/tratamiento farmacológico , Proteínas Mitocondriales/fisiología , Imitación Molecular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Humanos , Ratones , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1ß inflammatory responses independent of MLKL and necroptotic cell death.
Asunto(s)
Células de la Médula Ósea/citología , Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Apoptosis , Autoanticuerpos/química , Caspasa 8/metabolismo , Activación Enzimática , Femenino , Inflamación , Proteínas Inhibidoras de la Apoptosis/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/química , Hígado/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Necrosis , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Portadoras/química , Dipéptidos/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , Indoles/farmacología , Proteínas Mitocondriales/química , Imitación Molecular , Neoplasias Experimentales/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Proteínas Reguladoras de la Apoptosis , Dipéptidos/uso terapéutico , Descubrimiento de Drogas , Indoles/uso terapéutico , Ratones , Modelos MolecularesRESUMEN
The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Dipéptidos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Animales , Neoplasias de la Mama/patología , Caspasa 8/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones Desnudos , Proteínas Mitocondriales/metabolismo , Receptores del Factor de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.
Asunto(s)
Ubiquitina/metabolismo , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/fisiología , Secuencia de Aminoácidos , Humanos , Modelos Biológicos , Modelos Moleculares , Fenilalanina/genética , Fenilalanina/fisiología , Unión Proteica/genética , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , Especificidad por Sustrato , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación/genética , Ubiquitinación/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
We describe the structure-activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compounds 13 and 14.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Indoles , Piranos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Sitio Alostérico , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Piranos/síntesis química , Piranos/química , Piranos/farmacología , Relación Estructura-ActividadRESUMEN
The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Biomimética , Dicroismo Circular , Dimerización , Activación Enzimática , Humanos , Lentivirus/genética , Ratones , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Dispersión de Radiación , Transducción de Señal , Ubiquitina/químicaRESUMEN
We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Indoles/química , Piranos/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indoles/síntesis química , Indoles/farmacología , Piranos/síntesis química , Piranos/farmacología , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoAsunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Piranos/síntesis química , Piranos/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Animales , Antivirales/química , Hepacivirus/genética , Hepacivirus/metabolismo , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Concentración 50 Inhibidora , Ratones , Ratones SCID , Ratones Transgénicos , Piranos/química , ARN Viral/sangre , ARN Viral/aislamiento & purificación , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Lisosomas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Inhibidores de Caspasas , Catepsinas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Línea Celular Tumoral , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Lisosomas/efectos de los fármacos , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Receptor de TWEAKRESUMEN
XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.
Asunto(s)
Apoptosis/fisiología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Comunicación Autocrina , Benzoquinonas/metabolismo , Brefeldino A/metabolismo , Caspasa 8/metabolismo , Inhibidores de Caspasas , Línea Celular , Inhibidores Enzimáticos/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lactamas Macrocíclicas/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Imitación Molecular , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Serpinas/metabolismo , Transducción de Señal/fisiología , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Proteínas Virales/metabolismoRESUMEN
Several potent, cell permeable 4-aryl-dihydropyrimidinones have been identified as inhibitors of FATP4. Lipophilic ester substituents at the 5-position and substitution at the para-position (optimal groups being -NO(2) and CF(3)) of the 4-aryl group led to active compounds. In two cases racemates were resolved and the S enantiomers shown to have higher potencies.
Asunto(s)
Proteínas de Transporte de Ácidos Grasos/antagonistas & inhibidores , Pirimidinonas/química , Pirimidinonas/farmacología , Línea Celular , Humanos , Estructura Molecular , Pirimidinonas/síntesis química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of conformationally-restricted analogues of hPTH was prepared, based on the parent peptide agonist, cyclo(Lys(18)-Asp(22))[Ala(1),Nle(8),Lys(18),Asp(22),Leu(27)]hPTH(1-31)NH(2) (2, EC(50)=0.29nM). Truncation of 2 at either the N- or C-termini resulted in peptides with reduced agonist activity as measured by stimulation of adenylate cyclase activity in the rat osteosarcoma cell line (ROS 17/2.8). Alanine- and glycine-scanning at the N-terminus of 2 was consistent with data previously obtained on linear hPTH(1-34). Other locations within the primary sequence of hPTH(1-31)NH(2) were evaluated by the placement of the [i, i+4] lactam constraining element. Ring size and lactam orientations at the 18-22 positions were also examined.
Asunto(s)
Hormona Paratiroidea/química , Fragmentos de Péptidos/química , Adenilil Ciclasas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Humanos , Lactamas/química , Datos de Secuencia Molecular , Hormona Paratiroidea/genética , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Ratas , Receptores de Hormona Paratiroidea/agonistas , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
One common synthetic route creates small-molecule libraries directed toward two functionally distinct target families. The novel structural template 1 can independently display the necessary pharmacophore patterns for inhibition of members of two different biomolecular target families, the matrix metalloproteinases (MMPs) or the phosphodiesterases (PDEs). The incorporation of multiple target family directed design elements into combinatorial library design could help expedite the pharmaceutical lead discovery process. Z=OR' (PDE4), H (MMPs).