RESUMEN
Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos de Bifenilo/síntesis química , Nitrofenoles/síntesis química , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Linfoma , Ratones , Ratones SCID , Modelos Moleculares , Nitrofenoles/química , Nitrofenoles/farmacología , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/química , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Trasplante Heterólogo , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/químicaRESUMEN
Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.
Asunto(s)
Compuestos de Anilina/síntesis química , Antineoplásicos/síntesis química , Piperidinas/síntesis química , Sulfonamidas/síntesis química , Proteína bcl-X/antagonistas & inhibidores , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Polarización de Fluorescencia , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Ratones SCID , Paclitaxel/farmacología , Piperidinas/química , Piperidinas/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Suero , Albúmina Sérica/química , Estereoisomerismo , Sulfonamidas/química , Sulfonamidas/farmacología , Trasplante Heterólogo , Rayos UltravioletaRESUMEN
Inhibitor of apoptosis (IAP) proteins are overexpressed in many cancers and have been implicated in tumor growth, pathogenesis, and resistance to chemo- or radiotherapy. On the basis of the NMR structure of a SMAC peptide complexed with the BIR3 domain of X-linked IAP (XIAP), a novel series of XIAP antagonists was discovered. The most potent compounds in this series bind to the baculovirus IAP repeat 3 (BIR3) domain of XIAP with single-digit nanomolar affinity and promote cell death in several human cancer cell lines. In a MDA-MB-231 breast cancer mouse xenograft model, these XIAP antagonists inhibited the growth of tumors. Close structural analogues that showed only weak binding to the XIAP-BIR3 domain were inactive in the cellular assays and showed only marginal in vivo activity. Our results are consistent with a mechanism in which ligands for the BIR3 domain of XIAP induce apoptosis by freeing up caspases. The present study validates the BIR3 domain of XIAP as a target and supports the use of small molecule XIAP antagonists as a potential therapy for cancers that overexpress XIAP.
Asunto(s)
Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proteínas Portadoras/química , Proteínas Mitocondriales/química , Fragmentos de Péptidos/uso terapéutico , Proteínas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proteínas Portadoras/uso terapéutico , Caspasas/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Ratones , Proteínas Mitocondriales/uso terapéutico , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Trasplante Heterólogo , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
We have previously reported a novel series of oxalyl-aryl-amino benzoic acid-based, catalytic site-directed, competitive, reversible protein tyrosine phosphatase 1B (PTP1B) inhibitors. With readily access to key intermediates, we utilized a solution phase parallel synthesis approach and rapidly identified a highly potent PTP1B inhibitor (19, K(i)=76 nM) with moderate selectivity (5-fold) over T-cell PTPase (TCPTP) through interacting with a second phosphotyrosine binding site (site 2) in the close proximity to the catalytic site.
Asunto(s)
Benzoatos/química , Benzoatos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Amidas/química , Amidas/farmacología , Sitios de Unión , Dominio Catalítico , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Ligandos , Estructura Molecular , Fosfotirosina/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Relación Estructura-Actividad , Linfocitos T/enzimologíaRESUMEN
A method is described for the NMR-based screening for the discovery of aminoglycoside mimetics that bind to Escherichia coli A-site RNA. Although aminoglycosides are clinically useful, they exhibit high nephrotoxicity and ototoxicity, and their overuse has led to the development of resistance to important microbial pathogens. To identify a new series of aminoglycoside mimetics that could potentially overcome the problems associated with toxicities and resistance development observed with the aminoglycosides, we have prepared large quantities of E. coli 16 S A-site RNA and conducted an NMR-based screening of our compound library in search for small-molecule RNA binders against this RNA target. From these studies, several classes of compounds were identified as initial hits with binding affinities in the range of 70 microM to 3 mM. Lead optimization through synthetic modifications of these initial hits led to the discovery of several small-molecule aminoglycoside mimetics that are structurally very different from the known aminoglycosides. Structural models of the A-site RNA/ligand complexes were prepared and compared to the three-dimensional structures of the RNA/aminoglycoside complexes.