RESUMO
Because of the central role ribosomes play for protein translation and ribosome-mediated mRNA and protein quality control (RQC), the ribosome pool is surveyed and dysfunctional ribosomes degraded both during assembly, as well as the functional cycle. Oxidative stress downregulates translation and damages mRNAs and ribosomal proteins (RPs). Although damaged mRNAs are detected and degraded via RQC, how cells mitigate damage to RPs is not known. Here, we show that cysteines in Rps26 and Rpl10 are readily oxidized, rendering the proteins non-functional. Oxidized Rps26 and Rpl10 are released from ribosomes by their chaperones, Tsr2 and Sqt1, and the damaged ribosomes are subsequently repaired with newly made proteins. Ablation of this pathway impairs growth, which is exacerbated under oxidative stress. These findings reveal an unanticipated mechanism for chaperone-mediated ribosome repair, augment our understanding of ribosome quality control, and explain previous observations of protein exchange in ribosomes from dendrites, with broad implications for aging and health.
Assuntos
Proteínas Ribossômicas , Ribossomos , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Biossíntese de ProteínasRESUMO
Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.
Assuntos
Endorribonucleases , MicroRNAs , RNA Mensageiro , Humanos , Genes jun/genética , Genes myc/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Endorribonucleases/química , Endorribonucleases/metabolismo , TranscriptomaRESUMO
α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.
Assuntos
Proteínas Intrinsicamente Desordenadas , Doença de Parkinson , Humanos , alfa-Sinucleína/genética , RNA Mensageiro/genética , Proteínas Intrinsicamente Desordenadas/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Regiões 5' não Traduzidas , RibonucleasesRESUMO
Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated â¼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5'GAG/3'CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target-ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.
Assuntos
DNA/genética , RNA não Traduzido/genética , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Descoberta de Drogas/métodos , Expressão Gênica/genética , Biblioteca Gênica , Humanos , Ligantes , MicroRNAs/genética , Oncogenes/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
As a vital micronutrient, zinc is integral to the structure, function, and signaling networks of diverse proteins. Dysregulated zinc levels, due to either excess intake or deficiency, are associated with a spectrum of health disorders. In this context, understanding zinc-regulated biological processes at the molecular level holds significant relevance to public health and clinical practice. Identifying and characterizing zinc-regulated proteins in their diverse proteoforms, however, remain a difficult task in advancing zinc biology. Herein, we address this challenge by developing a quantitative chemical proteomics platform that globally profiles the reactivities of proteinaceous cysteines upon cellular zinc depletion. Exploiting a protein-conjugated resin for the selective removal of Zn2+ from culture media, we identify an array of zinc-sensitive cysteines on proteins with diverse functions based on their increased reactivity upon zinc depletion. Notably, we find that zinc regulates the enzymatic activities, post-translational modifications, and subcellular distributions of selected target proteins such as peroxiredoxin 6 (PRDX6), platelet-activating factor acetylhydrolase IB subunit alpha1 (PAFAH1B3), and phosphoglycerate kinase (PGK1).
Assuntos
Cisteína , Zinco , Cisteína/química , Zinco/metabolismo , Proteínas/químicaRESUMO
Photolipids have emerged as attractive tools for the optical control of lipid functions. They often contain an azobenzene photoswitch that imparts a cis double-bond upon irradiation. Herein, we present the application of photoswitching to a lipidated natural product, the potent proteasome inhibitor cepafungin I. Several azobenzene-containing lipids were attached to the cyclopeptide core, yielding photoswitchable derivatives. Most notably, PhotoCep4 exhibited a 10-fold higher cellular potency in its light-induced cis-form, matching the potency of natural cepafungin I. The length of the photolipid tail and distal positioning of the azobenzene photoswitch with respect to the macrocycle is critical for this activity. In a proteome-wide experiment, light-triggered PhotoCep4 modulation showed high overlap with constitutively active cepafungin I. The mode of action was studied using crystallography and revealed an identical binding of the cyclopeptide in comparison to cepafungin I, suggesting that differences in their cellular activity originate from switching the tail structure. The photopharmacological approach described herein could be applicable to many other natural products as lipid conjugation is common and often necessary for potent activity. Such lipids are often introduced late in synthetic routes, enabling facile chemical modifications.
Assuntos
Compostos Azo , Lipopeptídeos , Lipopeptídeos/farmacologia , Proteólise , Compostos Azo/química , Peptídeos Cíclicos/farmacologiaRESUMO
Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.
Assuntos
Proteínas Intrinsicamente Desordenadas/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Elementos de Resposta , alfa-Sinucleína/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , RNA Mensageiro/química , RNA Mensageiro/genética , alfa-Sinucleína/metabolismoRESUMO
As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non-oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre-miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre-miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.
Assuntos
Neoplasias da Mama/tratamento farmacológico , MicroRNAs/metabolismo , Ribonucleases/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Humanos , Camundongos , MicroRNAs/química , Estrutura Molecular , Metástase Neoplásica , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ribonucleases/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Ribonuclease targeting chimeras (RiboTACs) induce degradation of an RNA target by facilitating an interaction between an RNA and a ribonuclease (RNase). We describe the screening of a DNA-encoded library (DEL) to identify binders of monomeric RNase L to provide a compound that induced dimerization of RNase L, activating its ribonuclease activity. This compound was incorporated into the design of a next-generation RiboTAC that targeted the microRNA-21 (miR-21) precursor and alleviated a miR-21-associated cellular phenotype in triple-negative breast cancer cells. The RNA-binding module in the RiboTAC is Dovitinib, a known receptor tyrosine kinase (RTK) inhibitor, which was previously identified to bind miR-21 as an off-target. Conversion of Dovitinib into this RiboTAC reprograms the known drug to selectively affect the RNA target. This work demonstrates that DEL can be used to identify compounds that bind and recruit proteins with effector functions in heterobifunctional compounds.
Assuntos
MicroRNAs , Neoplasias de Mama Triplo Negativas , Humanos , Ribonucleases , DNARESUMO
The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound's molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5' untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre , RNA , Alcinos , Sítios de Ligação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , TranscriptomaRESUMO
Liver-derived circulating factors deeply affect the metabolism of distal organs. Herein, we took advantage of the hepatocyte-specific PTEN knockout mice (LPTENKO), a model of hepatic steatosis associated with increased muscle insulin sensitivity and decreased adiposity, to identify potential secreted hepatic factors improving metabolic homeostasis. Our results indicated that protein factors, rather than specific metabolites, released by PTEN-deficient hepatocytes trigger an improved muscle insulin sensitivity and a decreased adiposity in LPTENKO. In this regard, a proteomic analysis of conditioned media from PTEN-deficient primary hepatocytes identified seven hepatokines whose expression/secretion was deregulated. Distinct expression patterns of these hepatokines were observed in hepatic tissues from human/mouse with NAFLD. The expression of specific factors was regulated by the PTEN/PI3K, PPAR or AMPK signaling pathways and/or modulated by classical antidiabetic drugs. Finally, loss-of-function studies identified FGF21 and the triad AHSG, ANGPTL4 and LECT2 as key regulators of insulin sensitivity in muscle cells and in adipocytes biogenesis, respectively. These data indicate that hepatic PTEN deficiency and steatosis alter the expression/secretion of hepatokines regulating insulin sensitivity in muscles and the lipid metabolism in adipose tissue. These hepatokines could represent potential therapeutic targets to treat obesity and insulin resistance.
Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Animais , Homeostase , Fígado/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , ProteômicaRESUMO
For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.
Assuntos
Produtos Biológicos , Produtos Biológicos/química , Ligantes , Proteínas rho de Ligação ao GTP , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.
Assuntos
Cisteína/química , Compostos Heterocíclicos com 2 Anéis/química , Hidrocarbonetos Fluorados/química , Sondas Moleculares/química , Proteômica/métodos , Alquilação , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Diterpenos/farmacologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Autoantígeno Ku/antagonistas & inibidores , Autoantígeno Ku/químicaRESUMO
The curcusone natural products are complex diterpenes featuring a characteristic [6-7-5] tricyclic carbon skeleton similar to the daphnane and tigliane diterpenes. Among them, curcusones A-D demonstrated potent anticancer activity against a broad spectrum of human cancer cell lines. Prior to this study, no total synthesis of the curcusones was achieved and their anticancer mode of action remained unknown. Herein, we report our synthetic and chemoproteomics studies of the curcusone diterpenes which culminate in the first total synthesis of several curcusone natural products and identification of BRCA1-associated ATM activator 1 (BRAT1) as a cellular target. Our efficient synthesis is highly convergent, builds upon cheap and abundant starting materials, features a thermal [3,3]-sigmatropic rearrangement and a novel FeCl3-promoted cascade reaction to rapidly construct the critical cycloheptadienone core of the curcusones, and led us to complete the first total synthesis of curcusones A and B in only 9 steps, C and D in 10 steps, and dimericursone A in 12 steps. The chemical synthesis of dimericursone A from curcusones C and D provided direct evidence to support the proposed Diels-Alder dimerization and cheletropic elimination biosynthetic pathway. Using an alkyne-tagged probe molecule, BRAT1, an important but previously "undruggable" oncoprotein, was identified as a key cellular target via chemoproteomics. We further demonstrate for the first time that BRAT1 can be inhibited by curcusone D, resulting in impaired DNA damage response, reduced cancer cell migration, potentiated activity of the DNA damaging drug etoposide, and other phenotypes similar to BRAT1 knockdown.
Assuntos
Produtos Biológicos/química , Diterpenos/química , Proteínas Nucleares/análise , Produtos Biológicos/síntese química , Diterpenos/síntese química , Humanos , Conformação Molecular , EstereoisomerismoRESUMO
Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.
Assuntos
Benzimidazóis/farmacologia , MicroRNAs/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Ribonucleases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Benzimidazóis/química , Humanos , MicroRNAs/metabolismo , Estrutura Molecular , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/metabolismo , Inibidores de Proteínas Quinases/química , Quinolonas/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Ribonucleases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.
Assuntos
Diamida/química , Proteínas/química , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Herein, we report arylazopyrazole ureas and sulfones as a novel class of photoswitchable serine hydrolase inhibitors and present a chemoproteomic platform for rapid discovery of optically controlled serine hydrolase targets in complex proteomes. Specifically, we identify highly potent and selective photoswitchable inhibitors of the drug-metabolizing enzymes carboxylesterases 1 and 2 and demonstrate their pharmacological application by optically controlling the metabolism of the immunosuppressant drug mycophenolate mofetil. Collectively, this proof-of-concept study provides a first example of photopharmacological tools to optically control drug metabolism by modulating the activity of a metabolizing enzyme. Our arylazopyrazole ureas and sulfones offer synthetically accessible scaffolds that can be expanded to identify specific photoswitchable inhibitors for other serine hydrolases, including lipases, peptidases, and proteases. Our chemoproteomic platform can be applied to other photoswitches and scaffolds to achieve optical control over diverse protein classes.
Assuntos
Carboxilesterase/antagonistas & inibidores , Inibidores Enzimáticos/química , Preparações Farmacêuticas/metabolismo , Raios Ultravioleta , Células CACO-2 , Carboxilesterase/metabolismo , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Hidrólise , Microscopia de Fluorescência , Preparações Farmacêuticas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Estereoisomerismo , Sulfonas/química , Sulfonas/metabolismo , Urease/química , Urease/metabolismoRESUMO
OBJECTIVE: Hepatocellular carcinoma (HCC) development occurs with non-alcoholic fatty liver disease (NAFLD) in the absence of cirrhosis and with an increasing incidence due to the obesity pandemic. Mutations of tumour suppressor (TS) genes and oncogenes (ONC) have been widely characterised in HCC. However, mounting evidence indicates that non-genomic alterations of TS/ONC occur early with NAFLD, thereby potentially promoting hepatocarcinogenesis in an inflammatory/fibrotic context. The aim of this study was to identify and characterise these alterations. DESIGN: The proteome of steatotic liver tissues from mice spontaneously developing HCC was analysed. Alterations of TSs/ONCs were further investigated in various mouse models of NAFLD/HCC and in human samples. The inflammatory, fibrogenic and oncogenic functions of S100A11 were assessed through in vivo, in vitro and ex-vivo analyses. RESULTS: A whole set of TSs/ONCs, respectively, downregulated or upregulated was uncovered in mice and human with NAFLD. Alterations of these TSs/ONCs were preserved or even exacerbated in HCC. Among them, overexpression of S100A11 was associated with high-grade HCC and poor prognosis. S100A11 downregulation in vivo significantly restrains the development of inflammation and fibrosis in mice fed a choline/methionine-deficient diet. Finally, in vitro and ex-vivo analyses revealed that S100A11 is a marker of hepatocyte de-differentiation, secreted by cancer cells, and promoting cell proliferation and migration. CONCLUSION: Cellular stress associated with NAFLD triggers non-genomic alterations of a whole network of TSs/ONCs fostering hepatocarcinogenesis. Among those, overexpression of the oncogenic factor S100A11 promotes inflammation/fibrosis in vivo and is significantly associated with high-grade HCC with poor prognosis.
Assuntos
Carcinogênese , Carcinoma Hepatocelular , Fígado Gorduroso , Neoplasias Hepáticas , Proteínas S100 , Animais , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Carcinogênese/imunologia , Carcinogênese/metabolismo , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular , Progressão da Doença , Descoberta de Drogas , Fígado Gorduroso/imunologia , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica/métodos , Humanos , Inflamação/metabolismo , Fígado/imunologia , Fígado/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Obesidade/imunologia , Prognóstico , Proteínas S100/imunologia , Proteínas S100/metabolismoRESUMO
Many RNAs are processed into biologically active transcripts, the aberrant expression of which can contribute to disease phenotypes. For example, the primary microRNA-17-92 (pri-miR-17-92) cluster contains six microRNAs (miRNAs) that collectively act in several disease settings. Herein, we used sequence-based design of structure-specific ligands to target a common structure in the Dicer processing sites of three miRNAs in the cluster, miR-17, miR-18a, and miR-20a, thereby inhibiting their biogenesis. The compound was optimized to afford a dimeric molecule that binds the Dicer processing site and an adjacent bulge, affording a 100-fold increase in potency. The dimer's mode of action was then extended from simple binding to direct cleavage by conjugation to bleomycin A5 in a manner that imparts RNA-selective cleavage or to indirect cleavage by recruiting an endogenous nuclease, or a ribonuclease targeting chimera (RIBOTAC). Interestingly, the dimer-bleomycin conjugate cleaves the entire pri-miR-17-92 cluster and hence functionally inhibits all six miRNAs emanating from it. The compound selectively reduced levels of the cluster in three disease models: polycystic kidney disease, prostate cancer, and breast cancer, rescuing disease-associated phenotypes in the latter two. Further, the bleomycin conjugate exerted selective effects on the miRNome and proteome in prostate cancer cells. In contrast, the RIBOTAC only depleted levels of pre- and mature miR-17, -18a, and 20a, with no effect on the primary transcript, in accordance with the cocellular localization of RNase L, the pre-miRNA targets, and the compound. These studies demonstrate a strategy to tune RNA structure-targeting compounds to the cellular localization of the target.
Assuntos
Carcinogênese/metabolismo , Ligantes , MicroRNAs/metabolismo , Humanos , Estrutura MolecularRESUMO
Current approaches to introduce terminal alkynes for bioorthogonal reactions into biomolecules still present limitations in terms of either reactivity, selectivity, or adduct stability. We present a method for the ethynylation of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. The acetylene group is directly introduced onto the thiol group of cysteine and can be used for copper-catalyzed alkyne-azide cycloaddition (CuAAC) without further processing. Labeling proceeded with reaction rates comparable to or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab, and high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine reagents. Fine-tuning of the EBX reagents allows optimization of their reactivity and physical properties.