RESUMEN
Thioamides, which are fascinating isosteres of amides, have garnered significant attention in drug discovery and medicinal chemistry programs, spanning peptides and small molecule compounds. This review provides an overview of the various applications of thioamides in small molecule therapeutic agents targeting a range of human diseases, including cancer, microbial infections (e.g., tuberculosis, bacteria, and fungi), viral infections, neurodegenerative conditions, analgesia, and others. Particular focus is given to design strategies of biologically active thioamide-containing compounds and their biological targets, such as kinases and histone methyltransferase ASH1L. Additionally, the review discusses the impact of the thioamide moiety on key properties, including potency, target interactions, physicochemical characteristics, and pharmacokinetics profiles. We hope that this work will offer valuable insights to inspire the future development of novel bioactive thioamide-containing compounds, facilitating their effective use in combating a wide array of human diseases.
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Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.
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Proteínas de Complejo Poro Nuclear , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas , Translocación Genética , Humanos , Animales , Proteínas de Complejo Poro Nuclear/genética , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Piperazinas/farmacología , Piperazinas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Línea Celular Tumoral , Compuestos de Anilina , PirazinasRESUMEN
Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100â µM to 4â µM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.
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Complejo Mediador , Activación Transcripcional , Humanos , Complejo Mediador/metabolismo , Complejo Mediador/química , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismo , Unión Proteica , Activación Transcripcional/efectos de los fármacosRESUMEN
The compact and versatile oxetane motifs have gained significant attention in drug discovery and medicinal chemistry campaigns. This review presents an overview of the diverse applications of oxetanes in clinical and preclinical drug candidates targeting various human diseases, including cancer, viral infections, autoimmune disorders, neurodegenerative conditions, metabolic disorders, and others. Special attention is given to biologically active oxetane-containing compounds and their disease-related targets, such as kinases, epigenetic and non-epigenetic enzymes, and receptors. The review also details the effect of the oxetane motif on important properties, including aqueous solubility, lipophilicity, pKa, P-glycoprotein (P-gp) efflux, metabolic stability, conformational preferences, toxicity profiles (e.g., cytochrome P450 (CYP) suppression and human ether-a-go-go related gene (hERG) inhibition), pharmacokinetic (PK) properties, potency, and target selectivity. We anticipate that this work will provide valuable insights that can drive future discoveries of novel bioactive oxetane-containing small molecules, enabling their effective application in combating a wide range of human diseases.
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Química Farmacéutica , Descubrimiento de Drogas , Humanos , Éteres Cíclicos/química , Éteres Cíclicos/metabolismo , Conformación MolecularRESUMEN
Natural products are often uniquely suited to modulate protein-protein interactions (PPIs) due to their architectural and functional group complexity relative to synthetic molecules. Here we demonstrate that the natural product garcinolic acid allosterically blocks the CBP/p300 KIX PPI network and displays excellent selectivity over related GACKIX motifs. It does so via a strong interaction (KD 1â µM) with a non-canonical binding site containing a structurally dynamic loop in CBP/p300 KIX. Garcinolic acid engages full-length CBP in the context of the proteome and in doing so effectively inhibits KIX-dependent transcription in a leukemia model. As the most potent small-molecule KIX inhibitor yet reported, garcinolic acid represents an important step forward in the therapeutic targeting of CBP/p300.
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Proteína de Unión a CREB , Estructura Terciaria de Proteína , Dominios Proteicos , Sitios de Unión , Unión Proteica , Proteína de Unión a CREB/químicaRESUMEN
ASH1L is a histone methyltransferase that regulates gene expression through methylation of histone H3 on lysine K36. While the catalytic SET domain of ASH1L has low intrinsic activity, several studies found that it can be vastly enhanced by the interaction with MRG15 protein and proposed allosteric mechanism of releasing its autoinhibited conformation. Here, we found that full-length MRG15, but not the MRG domain alone, can enhance the activity of the ASH1L SET domain. In addition, we showed that catalytic activity of MRG15-ASH1L depends on nucleosome binding mediated by MRG15 chromodomain. We found that in solution MRG15 binds to ASH1L, but has no impact on the conformation of the SET domain autoinhibitory loop or the S-adenosylmethionine cofactor binding site. Moreover, MRG15 binding did not impair the potency of small molecule inhibitors of ASH1L. These findings suggest that MRG15 functions as an adapter that enhances ASH1L catalytic activity by recruiting nucleosome substrate.
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Nucleosomas , Factores de Transcripción , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/química , Metilación , N-Metiltransferasa de Histona-Lisina/química , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismoRESUMEN
Loss of the tumor suppressor protein menin is a critical event underlying the formation of neuroendocrine tumors (NET) in hormone-expressing tissues including gastrinomas. While aberrant expression of menin impairs its tumor suppression, few studies explore the structure-function relationship of clinical multiple endocrine neoplasia, type 1 (MEN1) mutations in the absence of a complete LOH at both loci. Here, we determined whether clinical MEN1 mutations render nuclear menin unstable and lead to its functional inactivation. We studied the structural and functional implications of two clinical MEN1 mutations (R516fs, E235K) and a third variant (A541T) recently identified in 10 patients with gastroenteropancreatic (GEP)-NETs. We evaluated the subcellular localization and half-lives of the mutants and variant in Men1-null mouse embryo fibroblast cells and in hormone-expressing human gastric adenocarcinoma and NET cell lines. Loss of menin function was assessed by cell proliferation and gastrin gene expression assays. Finally, we evaluated the effect of the small-molecule compound MI-503 on stabilizing nuclear menin expression and function in vitro and in a previously reported mouse model of gastric NET development. Both the R516fs and E235K mutants exhibited severe defects in total and subcellular expression of menin, and this was consistent with reduced half-lives of these mutants. Mutated menin proteins exhibited loss of function in suppressing tumor cell proliferation and gastrin expression. Treatment with MI-503 rescued nuclear menin expression and attenuated hypergastrinemia and gastric hyperplasia in NET-bearing mice. Clinically defined MEN1 mutations and a germline variant confer pathogenicity by destabilizing nuclear menin expression. Significance: We examined the function of somatic and germline mutations and a variant of MEN1 sequenced from gastroenteropancreatic NETs. We report that these mutations and variant promote tumor cell growth and gastrin expression by rendering menin protein unstable and prone to increased degradation. We demonstrate that the menin-MLL (mixed lineage leukemia) inhibitor MI-503 restores menin protein expression and function in vitro and in vivo, suggesting a potential novel therapeutic approach to target MEN1 GEP-NETs.
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Neoplasia Endocrina Múltiple Tipo 1 , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Gastrinas/genética , Hormonas , Neoplasia Endocrina Múltiple Tipo 1/genética , Mutación , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genéticaRESUMEN
Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (34913-8) increases the affinity for the coactivator Med25 >10-fold ( Ki >>100 µM to 10 µM). Importantly, the selectivity of 34913-8 for Med25 compared to other coactivators is excellent. 34913-8 engages Med25 through interaction with the H2 face of its Ac tivator I nteraction D omain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, 34913-8 is a useful tool for studying Med25 and the Mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.
RESUMEN
Cancer is one of the major causes of mortality and morbidity worldwide. Substantial research efforts have been made to develop new chemical entities with improved anticancer efficacy. 2-Aminobenzothiazole is an important class of heterocycles containing one sulfur and two nitrogen atoms, which is associated with a broad spectrum of medical and pharmacological activities, including antitumor, antibacterial, antimalarial, anti-inflammatory, and antiviral activities. In recent years, an extraordinary collection of potent and low-toxicity 2-aminobenzothiazole compounds have been discovered as new anticancer agents. Herein, we provide a comprehensive review of this class of compounds based on their activities against tumor-related proteins, including tyrosine kinases (CSF1R, EGFR, VEGFR-2, FAK, and MET), serine/threonine kinases (Aurora, CDK, CK, RAF, and DYRK2), PI3K kinase, BCL-XL, HSP90, mutant p53 protein, DNA topoisomerase, HDAC, NSD1, LSD1, FTO, mPGES-1, SCD, hCA IX/XII, and CXCR. In addition, the anticancer potentials of 2-aminobenzothiazole-derived chelators and metal complexes are also described here. Moreover, the design strategies, mechanism of actions, structure-activity relationships (SAR) and more advanced stages of pre-clinical development of 2-aminobenzothiazoles as new anticancer agents are extensively reviewed in this article. Finally, the examples that 2-aminobenzothiazoles showcase an advantage over other heterocyclic systems are also highlighted.
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Antineoplásicos , Neoplasias , Humanos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Antineoplásicos/química , Proliferación Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Neoplasias/tratamiento farmacológico , Relación Estructura-Actividad , Benzotiazoles/química , Benzotiazoles/farmacologíaRESUMEN
Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes-HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention. SIGNIFICANCE: NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes. See related article by Uckelmann et al., p. 746. This article is highlighted in the In This Issue feature, p. 517.
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Leucemia Mieloide Aguda , Nucleofosmina , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Cromatina/genéticaRESUMEN
Efficient determination of protein ligandability, or the propensity to bind small-molecules, would greatly facilitate drug development for novel targets. Ligandability is currently assessed using computational methods that typically consider the static structural properties of putative binding sites or by experimental fragment screening. Here, we evaluate ligandability of conserved BTB domains from the cancer-relevant proteins LRF, KAISO, and MIZ1. Using fragment screening, we discover that MIZ1 binds multiple ligands. However, no ligands are uncovered for the structurally related KAISO or LRF. To understand the principles governing ligand-binding by BTB domains, we perform comprehensive NMR-based dynamics studies and find that only the MIZ1 BTB domain exhibits backbone µs-ms time scale motions. Interestingly, residues with elevated dynamics correspond to the binding site of fragment hits and recently defined HUWE1 interaction site. Our data argue that examining protein dynamics using NMR can contribute to identification of cryptic binding sites, and may support prediction of the ligandability of novel challenging targets.
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Dominio BTB-POZ , Sitios de Unión , Proteínas/metabolismo , Ligandos , Unión ProteicaRESUMEN
GAS41 is an emerging oncogene overexpressed and implicated in multiple cancers, including non-small cell lung cancer (NSCLC). GAS41 is a dimeric protein that contains the YEATS domain, which is involved in the recognition of lysine-acylated histones. Here, we report the development of GAS41 YEATS inhibitors by employing a fragment-based screening approach. These inhibitors bind to GAS41 YEATS domain in a channel constituting a recognition site for acylated lysine on histone proteins. To enhance inhibitory activity, we developed a dimeric analog with nanomolar activity that blocks interactions of GAS41 with acetylated histone H3. Our lead compound engages GAS41 in cells, blocks proliferation of NSCLC cells, and modulates expression of GAS41-dependent genes, validating on-target mechanism of action. This study demonstrates that disruption of GAS41 protein-protein interactions may represent an attractive approach to target lung cancer cells. This work exemplifies the use of bivalent inhibitors as a general strategy to block challenging protein-protein interactions.
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Amidas/farmacología , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Tiofenos/farmacología , Factores de Transcripción/antagonistas & inhibidores , Amidas/química , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Estructura Molecular , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Tiofenos/química , Factores de Transcripción/metabolismoRESUMEN
Aberrant activation of Wnt/ß-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/ß-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and ß-catenin-mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing ß-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of ß-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of ß-catenin to consensus DNA motifs and the transcription of ß-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for ß-catenin-driven malignancies.
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Neoplasias Colorrectales , beta Catenina , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción TCF/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.
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Complejo Represivo Polycomb 1/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Células K562 , Modelos Moleculares , Estructura Molecular , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Ubiquitinación/efectos de los fármacosRESUMEN
ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.
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Antineoplásicos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Animales , Antineoplásicos/química , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Diseño de Fármacos , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Femenino , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Leucemia/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/genética , Oncogenes , Dominios Proteicos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Ewing sarcomas are driven by EWS-ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS-FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. IMPLICATIONS: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.
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Factor de Transcripción Activador 4/genética , Neoplasias Óseas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteínas Proto-Oncogénicas/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Factor de Transcripción Activador 4/metabolismo , Vías Biosintéticas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/metabolismo , Serina/genética , Serina/metabolismoRESUMEN
Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically "undruggable" targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes.
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Proteínas de Choque Térmico/química , Modelos Químicos , Chaperonas Moleculares/química , Simulación de Dinámica Molecular , Sitios de Unión , Modelos Moleculares , Mutación , Conformación Proteica , Dominios Proteicos , Reproducibilidad de los ResultadosAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Leucemia Mieloide Aguda , Modelos Biológicos , Mutación , Proteínas Proto-Oncogénicas , Tirosina Quinasa 3 Similar a fms , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Masculino , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activatorâ¢coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3â¢Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.
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Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Secuencia de Aminoácidos/genética , Humanos , Complejo Mediador/genética , Complejo Mediador/metabolismo , Modelos Moleculares , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Activación Transcripcional/fisiologíaRESUMEN
The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.