RESUMO
The ultra-rare, pediatric premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS) is caused by mutation of LMNA, encoding the nuclear architectural protein lamin A. Patients develop atherosclerosis and typically die of heart failure in their teens. FDA-approved Zokinvy prevents farnesylation of lamin A, reduces vascular stiffness, and extends survival in HGPS patients. To view this Bench to Bedside, open or download the PDF.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Farnesiltranstransferase/antagonistas & inibidores , Progéria/tratamento farmacológico , Progéria/enzimologia , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/metabolismo , Humanos , Terapia de Alvo MolecularRESUMO
Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Preparações Farmacêuticas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/ultraestrutura , Azepinas/farmacologia , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/farmacologia , Presenilina-1/química , Presenilina-1/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.
Assuntos
Fator de Transcrição Associado à Microftalmia/metabolismo , NADP Trans-Hidrogenases/metabolismo , Pigmentação da Pele/efeitos da radiação , Raios Ultravioleta , Animais , Linhagem Celular , Estudos de Coortes , AMP Cíclico/metabolismo , Dano ao DNA , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Predisposição Genética para Doença , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Melanossomas/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , NADP Trans-Hidrogenases/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Polimorfismo de Nucleotídeo Único/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/genética , Ubiquitina/metabolismo , Peixe-ZebraRESUMO
Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.
Assuntos
Linhagem da Célula , Poliaminas/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Colite/imunologia , Colite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Epigenoma , Histonas/metabolismo , Inflamação/imunologia , Inflamação/patologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ornitina Descarboxilase/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Fatores de Transcrição/metabolismoRESUMO
Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.
Assuntos
Benzamidas/uso terapêutico , Epigênese Genética/genética , Piridonas/uso terapêutico , Sarcoma/tratamento farmacológico , Compostos de Bifenilo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inibidores Enzimáticos/farmacologia , Epigenômica , Terapia Genética/métodos , Humanos , Morfolinas , Proteínas Nucleares/metabolismo , Sarcoma/genética , Fatores de Transcrição/metabolismoRESUMO
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Assuntos
Antibióticos Antituberculose/farmacologia , Defeitos Congênitos da Glicosilação/metabolismo , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/química , Animais , Antibióticos Antituberculose/química , Sítios de Ligação , Defeitos Congênitos da Glicosilação/genética , Inibidores Enzimáticos/química , Feminino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo dos Lipídeos , Camundongos , Simulação de Acoplamento Molecular , Mutação , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Ligação Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacologia , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Proteína Fosfatase 1/antagonistas & inibidores , Animais , Peso Corporal , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Guanidinas/química , Células HeLa , Humanos , Doença de Huntington/metabolismo , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Proteostase , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de SuperfícieRESUMO
The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD154Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers.
Assuntos
Adenocarcinoma de Pulmão , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mutação de Sentido Incorreto , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.
Assuntos
Antígenos CD28/metabolismo , Ativação Linfocitária , Mitocôndrias/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Carnitina O-Palmitoiltransferase , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Humanos , Interleucina-15/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/metabolismo , Estresse Fisiológico , Linfócitos T/metabolismoAssuntos
Doença Celíaca , Proteínas de Ligação ao GTP , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Doença Celíaca/genética , Doença Celíaca/imunologia , Humanos , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Transglutaminases/genética , Transglutaminases/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/farmacologiaRESUMO
All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential â¼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Subunidades Ribossômicas Maiores de Eucariotos/efeitos dos fármacos , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Triazinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Indóis/química , Indóis/isolamento & purificação , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Relação Estrutura-Atividade , Triazinas/química , Triazinas/isolamento & purificaçãoRESUMO
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.
Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMO
KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Pontos de Checagem do Ciclo Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA , Modelos Animais de Doenças , Xenoenxertos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Células Tumorais CultivadasRESUMO
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Assuntos
Regulação Alostérica , Descoberta de Drogas , Inibidores Enzimáticos , Proteômica , Helicase da Síndrome de Werner , Animais , Feminino , Humanos , Masculino , Camundongos , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Cisteína/efeitos dos fármacos , Cisteína/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Instabilidade de Microssatélites , Modelos Moleculares , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Morte Celular/efeitos dos fármacos , Trifosfato de Adenosina/metabolismoRESUMO
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Assuntos
Antineoplásicos , Descoberta de Drogas , Inibidores Enzimáticos , Instabilidade de Microssatélites , Neoplasias , Mutações Sintéticas Letais , Helicase da Síndrome de Werner , Animais , Feminino , Humanos , Camundongos , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Dano ao DNA/efeitos dos fármacos , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Domínios Proteicos , Reprodutibilidade dos Testes , Supressão Genética , Mutações Sintéticas Letais/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.
Assuntos
Química Farmacêutica/métodos , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteômica/métodos , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Humanos , Lactonas/química , Fenótipo , Fotoquímica/métodos , ProteomaRESUMO
Arginine methylation is an influential post-translational modification occurring on histones, RNA binding proteins, and many other cellular proteins, affecting their function by altering their protein-protein and protein-nucleic acid interactions. Recently, a wealth of information has been gathered, implicating protein arginine methyltransferases (PRMTs), enzymes that deposit arginine methylation, in transcription, pre-mRNA splicing, DNA damage signaling, and immune signaling with major implications for cancer therapy, especially immunotherapy. This review summarizes this recent progress and the current state of PRMT inhibitors, some in clinical trials, as promising drug targets for cancer.
Assuntos
Arginina/química , Metilação , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Processamento Alternativo , Animais , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Comunicação Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Histonas , Humanos , Sistema Imunitário , Imunoterapia/métodos , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/química , Splicing de RNA , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/ultraestrutura , Regulação Alostérica , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Heme/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Simulação de Dinâmica Molecular , Palmitoil Coenzima A/metabolismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
KRAS mutant cancer, characterized by the activation of a plethora of phosphorylation signaling pathways, remains a major challenge for cancer therapy. Despite recent advancements, a comprehensive profile of the proteome and phosphoproteome is lacking. This study provides a proteomic and phosphoproteomic landscape of 43 KRAS mutant cancer cell lines across different tissue origins. By integrating transcriptomics, proteomics, and phosphoproteomics, we identify three subsets with distinct biological, clinical, and therapeutic characteristics. The integrative analysis of phosphoproteome and drug sensitivity information facilitates the identification of a set of drug combinations with therapeutic potentials. Among them, we demonstrate that the combination of DOT1L and SHP2 inhibitors is an effective treatment specific for subset 2 of KRAS mutant cancers, corresponding to a set of TCGA clinical tumors with the poorest prognosis. Together, this study provides a resource to better understand KRAS mutant cancer heterogeneity and identify new therapeutic possibilities.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores Enzimáticos/farmacologia , Mutação , Neoplasias/tratamento farmacológico , Fosfoproteínas/metabolismo , Proteoma , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , Bases de Dados Genéticas , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Espectrometria de Massas , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfoproteínas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The modification of eukaryotic proteins by isoprenoid lipids, which is known as prenylation, controls the localization and activity of a range of proteins that have crucial functions in biological regulation. The roles of prenylated proteins in cells are well conserved across species, underscoring the biological and evolutionary importance of this lipid modification pathway. Genetic suppression and pharmacological inhibition of the protein prenylation machinery have provided insights into several cellular processes and into the aetiology of diseases in which prenylation is involved. The functional dependence of prenylation substrates, such as RAS proteins, on this modification and the therapeutic potential of targeting the prenylation process in pathological conditions accentuate the need to fully understand this form of post-translational modification.