RESUMEN
The task of predicting the interactions between drugs and targets plays a key role in the process of drug discovery. There is a need to develop novel and efficient prediction approaches in order to avoid costly and laborious yet not-always-deterministic experiments to determine drug-target interactions (DTIs) by experiments alone. These approaches should be capable of identifying the potential DTIs in a timely manner. In this article, we describe the data required for the task of DTI prediction followed by a comprehensive catalog consisting of machine learning methods and databases, which have been proposed and utilized to predict DTIs. The advantages and disadvantages of each set of methods are also briefly discussed. Lastly, the challenges one may face in prediction of DTI using machine learning approaches are highlighted and we conclude by shedding some lights on important future research directions.
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Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Aprendizaje Automático , Bases de Datos Factuales , HumanosRESUMEN
FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.
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Reparación del ADN por Unión de Extremidades , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli Adenosina Difosfato Ribosa/metabolismo , Factor de Células Madre/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/química , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Fibroblastos/ultraestructura , Rayos gamma , Células HCT116 , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efectos de la radiación , Células Secretoras de Insulina/ultraestructura , Modelos Moleculares , Mutación , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/química , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Factor de Células Madre/metabolismo , Ubiquitinación/efectos de la radiaciónRESUMEN
Herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus, establish latency by modulating or mimicking antiapoptotic Bcl-2 proteins to promote survival of carrier cells. BH3 profiling, which assesses the contribution of Bcl-2 proteins towards cellular survival, was able to globally determine the level of dependence on individual cellular and viral Bcl-2 proteins within latently infected cells. Moreover, BH3 profiling predicted the sensitivity of infected cells to small-molecule inhibitors of Bcl-2 proteins.
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Herpesviridae/fisiología , Herpesviridae/patogenicidad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Apoptosis , Línea Celular , Supervivencia Celular , Citomegalovirus/patogenicidad , Citomegalovirus/fisiología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 4/patogenicidad , Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/patogenicidad , Herpesvirus Humano 8/fisiología , Interacciones Huésped-Patógeno , Humanos , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Virales/metabolismo , Latencia del VirusRESUMEN
We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted using the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirms that the reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro.
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Proteínas Anfibias/química , Histonas/química , Cuerpos de Inclusión/química , Proteínas Recombinantes de Fusión/química , Proteínas Anfibias/genética , Proteínas Anfibias/aislamiento & purificación , Animales , Línea Celular , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Guanidina/química , N-Metiltransferasa de Histona-Lisina/química , Histonas/genética , Histonas/aislamiento & purificación , Humanos , Metiltransferasas/química , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/química , Nucleosomas/química , Nucleosomas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Multimerización de Proteína , Replegamiento Proteico , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Cloruro de Sodio/química , Solubilidad , Xenopus laevis/metabolismoRESUMEN
The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865-874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.
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Transformación Celular Neoplásica/metabolismo , Metiltransferasas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Elongación Transcripcional/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células HEK293 , N-Metiltransferasa de Histona-Lisina , Humanos , Metiltransferasas/química , Metiltransferasas/genética , Mutagénesis Sitio-Dirigida , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Unión Proteica , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genéticaRESUMEN
AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.
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Proteína de la Leucemia Mieloide-Linfoide , Peptidomiméticos , Humanos , Cristalografía por Rayos X , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/antagonistas & inhibidores , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Dominios ProteicosRESUMEN
BACKGROUND: Mitochondrial outer membrane permeabilization (MOMP) is a crucial step leading to apoptotic destruction of cancer cells. Bcl-2 family proteins delicately regulate mitochondrial outer membrane integrity through protein-protein interactions, which makes the mitochondrion an ideal cell-free system for screening molecules targeting the Bcl-2 anti-apoptotic proteins. But assay conditions need to be optimized for more reliable results. In this study, we aimed at establishing a reliable functional assay using mitochondria isolated from breast cancer cells to decipher the mode of action of BH3 peptides derived from BH3-only proteins. In this study, high ionic strength buffer was adopted during the initiation of MOMP. Mitochondria isolated from human breast cancer cell lines with distinct expression patterns of Bcl-2 anti-apoptotic proteins were permeabilized by different BH3 peptides alone or in combination, with or without the presence of recombinant anti-apoptotic Bcl-2 family proteins. Cytochrome C and Smac/Diablo were tested in both supernatants and mitochondrial pellets by Western blotting. RESULTS: Sufficient ionic strength was required for optimal release of Cytochrome C. Bad and Noxa BH3 peptides exhibited their bona fide antagonistic effects against Bcl-2/Bcl-xL and Mcl-1 proteins, respectively, whereas Bim BH3 peptide antagonized all three anti-apoptotic Bcl-2 members. Bad and Noxa peptides synergized with each other in the induction of MOMP when mitochondria were dually protected by both Bcl-2/Bcl-xL and Mcl-1. CONCLUSIONS: This method based on MOMP is a useful screening tool for identifying BH3 mimetics with selective toxicity against breast cancer cell mitochondria protected by the three major Bcl-2 anti-apoptotic proteins.
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Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteína bcl-X/antagonistas & inhibidores , Apoptosis/genética , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Sistema Libre de Células , Citocromos c/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reproducibilidad de los Resultados , Resonancia por Plasmón de Superficie , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Peripheral blood monocytes are the cells predominantly responsible for systemic dissemination of human cytomegalovirus (HCMV) and a significant cause of morbidity and mortality in immunocompromised patients. HCMV establishes a silent/quiescent infection in monocytes, which is defined by the lack of viral replication and lytic gene expression. The absence of replication shields the virus within infected monocytes from the current available antiviral drugs that are designed to suppress active replication. Our previous work has shown that HCMV stimulates a noncanonical phosphorylation of Akt and the subsequent upregulation of a distinct subset of prosurvival proteins in normally short-lived monocytes. In this study, we found that SIRT2 activity is required for the unique activation profile of Akt induced within HCMV-infected monocytes. Importantly, both therapeutic and prophylactic treatment with a novel SIRT2 inhibitor, FLS-379, promoted death of infected monocytes via both the apoptotic and necroptotic cell death pathways. Mechanistically, SIRT2 inhibition reduced expression of Mcl-1, an Akt-dependent antiapoptotic Bcl-2 family member, and enhanced activation of MLKL, the executioner kinase of necroptosis. We have previously reported HCMV to block necroptosis by stimulating cellular autophagy. Here, we additionally demonstrate that inhibition of SIRT2 suppressed Akt-dependent HCMV-induced autophagy leading to necroptosis of infected monocytes. Overall, our data show that SIRT2 inhibition can simultaneously promote death of quiescently infected monocytes by two distinct death pathways, apoptosis and necroptosis, which may be vital for limiting viral dissemination to peripheral organs in immunosuppressed patients.
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Citomegalovirus , Monocitos , Humanos , Monocitos/metabolismo , Citomegalovirus/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Necroptosis , Sirtuina 2/metabolismo , Apoptosis , Células CultivadasRESUMEN
MLL (KMT2a) translocations are found in ~10% of acute leukemia patients, giving rise to oncogenic MLL-fusion proteins. A common MLL translocation partner is ENL and associated with a poor prognosis in t(11;19) patients. ENL contains a highly conserved N-terminal YEATS domain that binds acetylated histones and interacts with the PAF1c, an epigenetic regulator protein complex essential for MLL-fusion leukemogenesis. Recently, wild-type ENL, and specifically the YEATS domain, was shown to be essential for leukemic cell growth. However, the inclusion and importance of the YEATS domain in MLL-ENL-mediated leukemogenesis remains unexplored. We found the YEATS domain is retained in 84.1% of MLL-ENL patients and crucial for MLL-ENL-mediated leukemogenesis in mouse models. Mechanistically, deletion of the YEATS domain impaired MLL-ENL fusion protein binding and decreased expression of pro-leukemic genes like Eya1 and Meis1. Point mutations that disrupt YEATS domain binding to acetylated histones decreased stem cell frequency and increased MLL-ENL-mediated leukemia latency. Therapeutically, YEATS containing MLL-ENL leukemic cells display increased sensitivity to the YEATS inhibitor SGC-iMLLT compared to control AML cells. Our results demonstrate that the YEATS domain is important for MLL-ENL fusion protein-mediated leukemogenesis and exposes an "Achilles heel" that may be therapeutically targeted for treating t(11;19) patients.
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Histonas , Leucemia Mieloide Aguda , Ratones , Animales , Histonas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Leucemia Mieloide Aguda/genética , Translocación Genética , Epigénesis Genética , Células Madre/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismoRESUMEN
Mantle cell lymphoma (MCL) is a rare, incurable hematological malignancy with a heterogeneous presentation and clinical course. A wide variety of chemotherapy-based regimens are currently used in patients who are untreated. Over the last several years, several targeted or small-molecule therapies have shown efficacy in the relapsed/refractory setting and have since been explored in the frontline setting. Lenalidomide plus rituximab was explored in a phase 2 study of 38 patients with MCL who were untreated and ineligible to receive transplantation, in which the combination produced durable remissions. We looked to build upon this regimen by adding venetoclax to the combination. We conducted a multicenter, open-label, nonrandomized, single-arm study to evaluate this combination. We enrolled 28 unselected patients with untreated disease irrespective of age, fitness, or risk factors. Lenalidomide was dosed at 20 mg daily from days 1 to 21 of each 28-day cycle. The dose of venetoclax was determined using the time-to-event continual reassessment method. Rituximab was dosed at 375 mg/m2 weekly, starting on cycle 1, day 1 until cycle 2, day 1. No dose-limiting toxicities were noted. All patients were treated with venetoclax at the maximum tolerated dose of 400 mg daily. The most common adverse events were neutropenia and thrombocytopenia. The overall and complete response rates were 96% and 86%, respectively. In total, 86% of patients achieved minimal residual disease undetectability via next-generation sequencing. The median overall and progression-free survivals were not reached. The combination of lenalidomide, rituximab, and venetoclax is a safe and effective regimen in patients with untreated MCL. This trial was registered at www.clinicaltrials.gov as #NCT03523975.
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Linfoma de Células del Manto , Humanos , Adulto , Rituximab/efectos adversos , Lenalidomida/uso terapéutico , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/efectos adversosRESUMEN
Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-α-l-(2â³,4â³-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC50 10.4 µM). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.
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Eriobotrya/química , Flavonoides/química , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Sitios de Unión , Línea Celular Tumoral , Simulación por Computador , Evaluación Preclínica de Medicamentos , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Hojas de la Planta/química , Unión Proteica , Estructura Terciaria de Proteína , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2-p53 interaction. MI-219 binds to human MDM2 with a K(i) value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2-p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Indoles/farmacología , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Indoles/administración & dosificación , Indoles/sangre , Indoles/química , Ratones , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/sangre , Compuestos de Espiro/química , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9-DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.
RESUMEN
Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.
Asunto(s)
Cromatina/enzimología , Melanoma Experimental/enzimología , Melanoma/enzimología , Sirtuinas/metabolismo , Neoplasias Cutáneas/enzimología , Animales , Cromatina/genética , Melanoma/genética , Melanoma/patología , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sirtuinas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
Disruptor of Telomeric Silencing 1-Like (DOT1L), the sole histone H3 lysine 79 (H3K79) methyltransferase, is required for leukemogenic transformation in a subset of leukemias bearing chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene, as well as other cancers. Thus, DOT1L is an attractive therapeutic target and discovery of small molecule inhibitors remain of high interest. Herein, we are presenting screening results for a unique focused library of 1200 nucleoside analogs originally produced under the aegis of the NIH Pilot Scale Library Program. The complete nucleoside set was screened virtually against DOT1L, resulting in 210 putative hits. In vitro screening of the virtual hits resulted in validation of 11 compounds as DOT1L inhibitors clustered into two distinct chemical classes, adenosine-based inhibitors and a new chemotype that lacks adenosine. Based on the developed DOT1L ligand binding model, a structure-based design strategy was applied and a second-generation of non-nucleoside DOT1L inhibitors was developed. Newly synthesized compound 25 was the most potent DOT1L inhibitor in the new series with an IC50 of 1.0 µM, showing 40-fold improvement in comparison with hit 9 and exhibiting reasonable on target effects in a DOT1L dependent murine cell line. These compounds represent novel chemical probes with a unique non-nucleoside scaffold that bind and compete with the SAM binding site of DOT1L, thus providing foundation for further medicinal chemistry efforts to develop more potent compounds.
Asunto(s)
Médula Ósea/efectos de los fármacos , Proliferación Celular , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucemia Experimental/tratamiento farmacológico , Nucleósidos/farmacología , Triazoles/farmacología , Animales , Médula Ósea/enzimología , Simulación por Computador , Inhibidores Enzimáticos/química , Leucemia Experimental/enzimología , Ratones , Nucleósidos/química , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compound 24, which binds both Mcl-1 and Bfl-1 with Ki values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.
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Antineoplásicos/farmacología , Ácido Benzoico/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ácido Benzoico/química , Línea Celular Tumoral , Humanos , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor/metabolismo , Simulación del Acoplamiento Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Aging is the dominant risk factor for most chronic diseases. Development of antiaging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in Caenorhabditis elegans, although not in testing performed in worms and flies with a more restricted set of compounds. Transcriptomic analysis and genetic studies implicated Nrf2/SKN-1 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Small molecules identified in this work may represent attractive tools to elucidate mechanisms of stress resistance in mammalian cells.
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Proteínas de Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , Longevidad/genética , Mamíferos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
The transcriptional activator beta-catenin is the primary mediator of the canonical Wnt signaling pathway and is frequently upregulated in many types of human cancer. Recent studies have suggested that the interaction of beta-catenin and its cofactor, B-cell lymphoma 9 (BCL9), is crucial for its transcriptional activity. Targeting this interaction using small molecules will improve our understanding of the beta-catenin/Wnt signaling pathway and may lead to the development of a new class of anticancer drugs. In this study, we developed a fluorescence polarization (FP)-based BCL9 binding assay. Using our initial FP assay, we performed extensive mutational analysis on four critical hydrophobic residues in the BCL9 peptide and determined the precise region in BCL9 responsible for binding to beta-catenin. These results led to further optimization of our FP assay, making it amenable for high-throughput screening (HTS). We also developed and validated a complementary surface plasmon resonance (SPR)-based binding assay and showed that our synthetic BCL9-based peptides are capable of fully inhibiting the binding of beta-catenin to wild-type BCL9 protein. Our studies provide not only further insight into the interaction between BCL9 and beta-catenin but also quantitative and reliable biochemical binding assays for the discovery of potent and specific small-molecule inhibitors of this interaction.
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Polarización de Fluorescencia , Proteínas de Neoplasias/metabolismo , Resonancia por Plasmón de Superficie , beta Catenina/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Análisis Mutacional de ADN , Polarización de Fluorescencia/métodos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Resonancia por Plasmón de Superficie/métodos , Factores de Transcripción , beta Catenina/químicaRESUMEN
Accurate prediction of the binding affinities of small-molecule ligands to their biological targets is fundamental for structure-based drug design but remains a very challenging task. In this paper, we have performed computational studies to predict the binding models of 31 small-molecule Smac (the second mitochondria-derived activator of caspase) mimetics to their target, the XIAP (X-linked inhibitor of apoptosis) protein, and their binding affinities. Our results showed that computational docking was able to reliably predict the binding models, as confirmed by experimentally determined crystal structures of some Smac mimetics complexed with XIAP. However, all the computational methods we have tested, including an empirical scoring function, two knowledge-based scoring functions, and MM-GBSA (molecular mechanics and generalized Born surface area), yield poor to modest prediction for binding affinities. The linear correlation coefficient (r(2)) value between the predicted affinities and the experimentally determined affinities was found to be between 0.21 and 0.36. Inclusion of ensemble protein-ligand conformations obtained from molecular dynamic simulations did not significantly improve the prediction. However, major improvement was achieved when the free-energy change for ligands between their free- and bound-states, or "ligand-reorganization free energy", was included in the MM-GBSA calculation, and the r(2) value increased from 0.36 to 0.66. The prediction was validated using 10 additional Smac mimetics designed and evaluated by an independent group. This study demonstrates that ligand reorganization free energy plays an important role in the overall binding free energy between Smac mimetics and XIAP. This term should be evaluated for other ligand-protein systems and included in the development of new scoring functions. To our best knowledge, this is the first computational study to demonstrate the importance of ligand reorganization free energy for the prediction of protein-ligand binding free energy.