RESUMEN
Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.
Asunto(s)
Artritis Psoriásica , Espondiloartritis Axial , Interleucina-17 , Psoriasis , Citocinas , Diseño de Fármacos , Humanos , Interleucina-17/antagonistas & inhibidoresRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Recent studies identified the receptor tyrosine kinase EphA4 as a disease-modifying gene that is critical for the progression of motor neuron degeneration. We report on the design and characterization of a family of EphA4 targeting agents that bind to its ligand binding domain with nanomolar affinity. The molecules exhibit excellent selectivity and display efficacy in a SOD1 mutant mouse model of ALS. Interestingly, the molecules appear to act as agonists for the receptor in certain surrogate cellular assays. While the exact mechanisms responsible for the therapeutic effect of the new agonists remain to be elucidated, we believe that the described agent represents both an invaluable pharmacological tool to further decipher the role of the EphA4 in ALS and potentially other human diseases, and a significant stepping stone for the development of novel treatments.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Receptor EphA4/agonistas , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Sitios de Unión , Células Cultivadas , Modelos Animales de Enfermedad , Diseño de Fármacos , Semivida , Humanos , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Unión Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptor EphA4/química , Receptor EphA4/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is an intractable tumor despite therapeutic advances, principally because of its invasive properties. Radiation is a staple in therapeutic regimens, although cells surviving radiation can become more aggressive and invasive. Subtraction hybridization identified melanoma differentiation-associated gene 9 [MDA-9/Syntenin; syndecan-binding protein (SDCBP)] as a differentially regulated gene associated with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a highly conserved double-PDZ domain-containing scaffolding protein, is robustly expressed in human-derived GBM cell lines and patient samples, with expression increasing with tumor grade and correlating with shorter survival times and poorer response to radiotherapy. Knockdown of MDA-9/Syntenin sensitizes GBM cells to radiation, reducing postradiation invasion gains. Radiation induces Src and EGFRvIII signaling, which is abrogated through MDA-9/Syntenin down-regulation. A specific inhibitor of MDA-9/Syntenin activity, PDZ1i (113B7), identified through NMR-guided fragment-based drug design, inhibited MDA-9/Syntenin binding to EGFRvIII, which increased following radiation. Both genetic (shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin reduced invasion gains in GBM cells following radiation. Although not affecting normal astrocyte survival when combined with radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial GBM signaling involving FAK and mutant EGFR, EGFRvIII, and abrogated gains in secreted proteases, MMP-2 and MMP-9, following radiation. In an in vivo glioma model, PDZ1i resulted in smaller, less invasive tumors and enhanced survival. When combined with radiation, survival gains exceeded radiotherapy alone. MDA-9/Syntenin (SDCBP) provides a direct target for therapy of aggressive cancers such as GBM, and defined small-molecule inhibitors such as PDZ1i hold promise to advance targeted brain cancer therapy.
Asunto(s)
Glioblastoma/genética , Invasividad Neoplásica/genética , Sinteninas/genética , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Melanoma/genética , Ratones , Ratones Desnudos , Dominios PDZ/genética , Transducción de Señal/genética , Familia-src Quinasas/genéticaRESUMEN
Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.
Asunto(s)
Aminopiridinas/química , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Etilenodiaminas/química , Proteínas Mitocondriales/administración & dosificación , Péptidos Cíclicos/administración & dosificación , Aminopiridinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Proteínas Portadoras , Línea Celular Tumoral , Etilenodiaminas/farmacología , Femenino , Humanos , Ligandos , Ratones , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Nanopartículas/químicaRESUMEN
Recently we described a novel approach, named high-throughput screening (HTS) by NMR that allows the identification, from large combinatorial peptide libraries, of potent and selective peptide mimetics against a given target. Here, we deployed the "HTS by NMR" approach for the design of novel peptoid sequences targeting the N-terminal domain of Yersinia outer proteinâ H (YopH-NT), a bacterial toxin essential for the virulence of Yersinia pestis. We aimed at disrupting the protein-protein interactions between YopH-NT and its cellular substrates, with the goal of inhibiting indirectly YopH enzymatic function. These studies resulted in a novel agent of sequence Ac-F-pY-cPG-d-P-NH2 (pY=phosphotyrosine; cPG=cyclopentyl glycine) with a Kd value against YopH-NT of 310â nm. We demonstrated that such a pharmacological inhibitor of YopH-NT results in the inhibition of the dephosphorylation by full-length YopH of a cellular substrate. Hence, potentially this agent represents a valuable stepping stone for the development of novel therapeutics against Yersinia infections. The data reported further demonstrate the utility of the HTS by NMR approach in deriving novel peptide mimetics targeting protein-protein interactions.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Peptoides/farmacología , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Resonancia Magnética Nuclear Biomolecular , Peptoides/síntesis química , Peptoides/química , Peste/tratamiento farmacológico , Unión Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Relación Estructura-Actividad , Yersinia pestis/efectos de los fármacosRESUMEN
The development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this study, we used a combination of nuclear magnetic resonance-guided structure-activity relationships along with biochemical and cellular studies to derive a novel tumor-homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand-binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Paclitaxel/análogos & derivados , Receptor EphA2/agonistas , Secuencia de Aminoácidos , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Masculino , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Ratones , Ratones Desnudos , Modelos Animales , Terapia Molecular Dirigida , Paclitaxel/química , Paclitaxel/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Ratas , Receptor EphA2/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications.
Asunto(s)
Descubrimiento de Drogas , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Receptor EphA4/química , Bibliotecas de Moléculas Pequeñas/química , Proteína Inhibidora de la Apoptosis Ligada a X/química , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Efrina-A5/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Péptidos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Receptor EphA4/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidoresRESUMEN
The emergence of drug-resistant strains of influenza virus makes exploring new classes of inhibitors that target universally conserved viral targets a highly important goal. The influenza A viral genome is made up of eight single-stranded RNA-negative segments. The RNA promoter, consisting of the conserved sequences at the 3' and 5' end of each RNA genomic segment, is universally conserved among influenza A virus strains and in all segments. Previously, we reported on the identification and NMR structure of DPQ (6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) (compound 1) in complex with the RNA promoter. Here, we report on additional screening and SAR studies with compound 1, including ex vivo anti-influenza activity assays, resulted in improved cellular activity against influenza A virus in the micromolar range.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Virus de la Influenza A/genética , Piperazinas/farmacología , Regiones Promotoras Genéticas , Quinazolinas/farmacología , ARN Viral/efectos de los fármacos , Animales , Perros , Evaluación Preclínica de Medicamentos/métodos , Virus de la Influenza A/efectos de los fármacos , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/virología , Espectroscopía de Resonancia Magnética , Estructura Molecular , Biblioteca de Péptidos , Piperazinas/química , Quinazolinas/química , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Proteínas Nucleares/antagonistas & inhibidores , Péptidos/química , Peptidomiméticos/química , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Péptidos/farmacología , Peptidomiméticos/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Fragment-based ligand design (FBLD) approaches have become more widely used in drug discovery projects from both academia and industry, and are even often preferred to traditional high-throughput screening (HTS) of large collection of compounds (>10(5)). A key advantage of FBLD approaches is that these often rely on robust biophysical methods such as NMR spectroscopy for detection of ligand binding, hence are less prone to artifacts that too often plague the results from HTS campaigns. In this article, we introduce a screening strategy that takes advantage of both the robustness of protein NMR spectroscopy as the detection method, and the basic principles of combinatorial chemistry to enable the screening of large libraries of fragments (>10(5) compounds) preassembled on a common backbone. We used the method to identify compounds that target protein-protein interactions.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Diseño de Fármacos , Espectroscopía de Resonancia Magnética/métodos , Receptor EphA4/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Receptor EphA4/química , Relación Estructura-Actividad , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
PURPOSE: YSA is an EphA2-targeting peptide that effectively delivers anticancer agents to prostate cancer tumors. Here, we report on how we increased the drug-like properties of this delivery system. EXPERIMENTAL DESIGN: By introducing non-natural amino acids, we have designed two new EphA2 targeting peptides: YNH, where norleucine and homoserine replace the two methionine residues of YSA, and dYNH, where a D-tyrosine replaces the L-tyrosine at the first position of the YNH peptide. We describe the details of the synthesis of YNH and dYNH paclitaxel conjugates (YNH-PTX and dYNH-PTX) and their characterization in cells and in vivo. RESULTS: dYNH-PTX showed improved stability in mouse serum and significantly reduced tumor size in a prostate cancer xenograft model and also reduced tumor vasculature in a syngeneic orthotopic allograft mouse model of renal cancer compared with vehicle or paclitaxel treatments. CONCLUSION: This study reveals that targeting EphA2 with dYNH drug conjugates could represent an effective way to deliver anticancer agents to a variety of tumor types.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Neoplasias/genética , Paclitaxel/administración & dosificación , Péptidos , Receptor EphA2/genética , Animales , Antineoplásicos Fitogénicos/química , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Paclitaxel/química , Péptidos/química , Receptor EphA2/metabolismo , Trasplante Homólogo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Successful replication of the influenza A virus requires both viral proteins and host cellular factors. In this study we used a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We used an established reporter assay to assess influenza viral replication by monitoring the activity of co-expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7, which inhibits a novel yet elusive target. Quantitative real-time PCR studies confirmed the dose-dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 is effective in rescuing high-dose influenza infection in an in vivo mouse model. As oseltamivir-resistant influenza strains emerge, compound 7 could be further investigated as a new and potentially suitable scaffold for the development of anti-influenza agents that act on novel targets.
Asunto(s)
Antivirales/química , Antivirales/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Bibliotecas de Moléculas Pequeñas/farmacología , Tetrazoles/química , Tetrazoles/farmacología , Tetrazoles/uso terapéuticoRESUMEN
Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Gosipol/análogos & derivados , Gosipol/farmacología , Neoplasias/tratamiento farmacológico , Animales , Ensayos de Selección de Medicamentos Antitumorales/economía , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias/genética , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.
Asunto(s)
Fragmentos de Péptidos/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas/química , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Relación Estructura-Actividad , Proteína bcl-X/química , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
INTRODUCTION: Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers. AREAS COVERED: Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments. EXPERT OPINION: We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
Asunto(s)
Antineoplásicos/farmacología , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Diseño de Fármacos , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias/metabolismo , Neoplasias/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Eph receptor tyrosine kinases and ephrin ligands control many physiological and pathological processes, and molecules interfering with their interaction are useful probes to elucidate their complex biological functions. Moreover, targeting Eph receptors might enable new strategies to inhibit cancer progression and pathological angiogenesis as well as promote nerve regeneration. Because our previous work suggested the importance of the salicylic acid group in antagonistic small molecules targeting Eph receptors, we screened a series of salicylic acid derivatives to identify novel Eph receptor antagonists. This identified a disalicylic acid-furanyl derivative that inhibits ephrin-A5 binding to EphA4 with an IC(50) of 3 µm in ELISAs. This compound, which appears to bind to the ephrin-binding pocket of EphA4, also targets several other Eph receptors. Furthermore, it inhibits EphA2 and EphA4 tyrosine phosphorylation in cells stimulated with ephrin while not affecting phosphorylation of EphB2, which is not a target receptor. In endothelial cells, the disalicylic acid-furanyl derivative inhibits EphA2 phosphorylation in response to TNFα and capillary-like tube formation on Matrigel, two effects that depend on EphA2 interaction with endogenous ephrin-A1. These findings suggest that salicylic acid derivatives could be used as starting points to design new small molecule antagonists of Eph receptors.
Asunto(s)
Efrinas/metabolismo , Unión Proteica/efectos de los fármacos , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/metabolismo , Salicilatos/química , Salicilatos/farmacología , Animales , Sitios de Unión , Línea Celular , Haplorrinos , Humanos , Ratones , Modelos Moleculares , Receptores de la Familia Eph/químicaRESUMEN
Overexpression of antiapoptotic members of the Bcl-2 family proteins, such as Bcl-x(L) and Mfl-1, has been shown to be involved in resistance to chemotherapeutic drugs in many forms of cancers. Recent efforts from the Abbott Laboratories resulted in the development of the acylsulfonamide compound and clinical candidate that targets selectively Bcl-2, Bcl-x(L), and Bcl-w while it is not active against Mcl-1 and Bfl-1. However, early clinical and preclinical studies suggest that pan-Bcl-2 antagonists, targeting simultaneously Mcl-1, Bcl-xL, and possibly all other four antiapoptotic Bcl-2 proteins, may result in more efficacious drugs. Here, following an NMR fragment-based approach, SAR by ILOEs, we report on compounds that exhibit nanomolar affinities for both Bcl-x(L) and Mcl-1 in vitro. We believe that these molecules can be used as useful starting point for the development of novel Bcl-2 antagonists, in particular targeting Mcl-1.
Asunto(s)
Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/química , Sulfonamidas/farmacología , Proteína bcl-X/antagonistas & inhibidores , Acilación , Humanos , Espectroscopía de Resonancia Magnética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Proteína bcl-X/metabolismoRESUMEN
Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda-7/IL-24-mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda-7/IL-24-induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda-7/IL-24-mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda-7/IL-24 (Ad.5/3-mda-7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda-7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3-mda-7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda-7/IL-24-mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Terapia Genética/métodos , Gosipol/análogos & derivados , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Gosipol/farmacología , Gosipol/uso terapéutico , Humanos , Interleucinas/administración & dosificación , Masculino , Ratones , Ratones Desnudos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Our focus in the past several years has been on the identification of novel and effective pan-Bcl-2 antagonists. We have recently reported a series of Apogossypolone (ApoG2) derivatives, resulting in the chiral compound (±) BI97D6. We report here the synthesis and evaluation on its optically pure (-) and (+) atropisomers. Compound (-) BI97D6 potently inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 76 ± 5, 31 ± 2, 25 ± 8, and 122 ± 28 nM, respectively. In a cellular assay, compound (-) BI97D6 effectively inhibits cell growth in the PC-3 human prostate cancer and H23 human lung cancer cell lines with EC(50) values of 0.22 ± 0.08 and 0.14 ± 0.02 µM, respectively. Similarly, compound (-) BI97D6 effectively induces apoptosis in the BP3 human lymphoma cell line in a dose-dependent manner. The compound also shows little cytotoxicity against bax(-/-)/bak(-/-) cells, suggesting that it kills cancers cells predominantly via a Bcl-2 pathway. Moreover, compound (-) BI97D6 displays in vivo efficacy in both a Bcl-2-transgenic mouse model and in a prostate cancer xenograft model in mice. Therefore, compound (-) BI97D6 represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.
RESUMEN
Overexpression of antiapoptotic Bcl-2 family proteins is commonly related with tumor maintenance, progression, and chemoresistance. Inhibition of these antiapoptotic proteins is an attractive approach for cancer therapy. Guided by nuclear magnetic resonance (NMR) binding assays, a series of 5,5' substituted compound 6a (Apogossypolone) derivatives was synthesized and identified pan-active antagonists of antiapoptotic Bcl-2 family proteins, with binding potency in the low micromolar to nanomolar range. Compound 6f inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, and Mcl-1 with IC(50) values of 3.10, 3.12, and 2.05 µM, respectively. In a cellular assay, 6f potently inhibits cell growth in several human cancer cell lines in a dose-dependent manner. Compound 6f further displays in vivo efficacy in transgenic mice and demonstrated superior single-agent antitumor efficacy in a PPC-1 mouse xenograft model. Together with its negligible toxicity, compound 6f represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.