RESUMO
Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
Assuntos
Enzimas Reparadoras do DNA/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nucleotídeos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Dano ao DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Terapia de Alvo Molecular , Neoplasias/patologia , Oxirredução/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirofosfatases/antagonistas & inibidores , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto , Nudix HidrolasesRESUMO
Two screening campaigns using commercial (Chembridge DiverSET) and proprietary (Chemical Biology Consortium Sweden, CBCS) compound libraries, revealed a number of pyridone- and pyrimidinone-derived systems as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). In this letter, we present their preliminary structure-activity-relationships (SAR) and ligand efficiency scores (LE and LLE).
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Piridonas/química , Piridonas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Pirofosfatases/antagonistas & inibidores , Humanos , Ligantes , Pirofosfatases/metabolismo , Relação Estrutura-AtividadeRESUMO
A high-throughput screening campaign using a commercial compound library (ChemBridge DiverSET) revealed diverse chemotypes as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). Triazole, triazolopyrimidine, triazinoindole, quinoline hydrazone and arylpiperazine hits were clustered, confirmed by IC50 determinations, and their preliminary structure-activity-relationships (SAR) and ligand efficiency scores are discussed in this letter.
Assuntos
Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Pirofosfatases/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Hidrazonas/farmacologia , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Pirofosfatases/metabolismo , Quinolinas/síntese química , Quinolinas/química , Quinolinas/farmacologia , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Triazóis/farmacologiaRESUMO
The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.
Assuntos
Aminoidrolases , Leucemia Mieloide Aguda , Aminoidrolases/genética , Humanos , Hidrolases , Leucemia Mieloide Aguda/tratamento farmacológico , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , TimidinaRESUMO
Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells. We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.
Assuntos
Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Terapia de Alvo Molecular , Medicina de Precisão , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Bortezomib/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Heterogeneidade Genética , Genoma Humano , Glioblastoma/genética , Humanos , Camundongos Endogâmicos BALB C , Mutação/genética , Inibidores de Proteassoma/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.
Assuntos
Inibidores Enzimáticos/farmacologia , Pirofosfatases/antagonistas & inibidores , Tiadiazóis/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento MolecularRESUMO
The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Família Multigênica , Pirofosfatases/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Filogenia , Pirofosfatases/classificação , Pirofosfatases/metabolismo , Interferência de RNA , Especificidade por Substrato , Nudix HidrolasesRESUMO
Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Estilbenos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Variações do Número de Cópias de DNA , Sinergismo Farmacológico , Feminino , Gefitinibe , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Estilbenos/uso terapêutico , TranscriptomaRESUMO
About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Recidiva Local de Neoplasia , Receptores do Ácido Retinoico/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Especificidade de Órgãos , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Análise de Sobrevida , Tamoxifeno/uso terapêuticoRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults, and despite state-of-the-art treatment, survival remains poor and novel therapeutics are sorely needed. The aim of the present study was to identify new synergistic drug pairs for GBM. In addition, we aimed to explore differences in drug-drug interactions across multiple GBM-derived cell cultures and predict such differences by use of transcriptional biomarkers. METHODS: We performed a screen in which we quantified drug-drug interactions for 465 drug pairs in each of the 5 GBM cell lines U87MG, U343MG, U373MG, A172, and T98G. Selected interactions were further tested using isobole-based analysis and validated in 5 glioma-initiating cell cultures. Furthermore, drug interactions were predicted using microarray-based transcriptional profiling in combination with statistical modeling. RESULTS: Of the 5 × 465 drug pairs, we could define a subset of drug pairs with strong interaction in both standard cell lines and glioma-initiating cell cultures. In particular, a subset of pairs involving the pharmaceutical compounds rimcazole, sertraline, pterostilbene, and gefitinib showed a strong interaction in a majority of the cell cultures tested. Statistical modeling of microarray and interaction data using sparse canonical correlation analysis revealed several predictive biomarkers, which we propose could be of importance in regulating drug pair responses. CONCLUSION: We identify novel candidate drug pairs for GBM and suggest possibilities to prospectively use transcriptional biomarkers to predict drug interactions in individual cases.