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
Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.
Assuntos
Enzimas Reparadoras do DNA/fisiologia , Nucleotídeos de Desoxiguanina/química , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Domínio Catalítico , Cristalografia por Raios X , Metilases de Modificação do DNA/química , Enzimas Reparadoras do DNA/química , Cães , Escherichia coli/genética , Células HL-60 , Humanos , Hidrólise , Cinética , Camundongos , Nucleotídeos , Monoéster Fosfórico Hidrolases/química , Pirofosfatases/química , Especificidade da Espécie , Suínos , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/química , Peixe-ZebraRESUMO
Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
Assuntos
Metilenotetra-Hidrofolato Desidrogenase (NADP) , Neoplasias , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , Tetra-HidrofolatosRESUMO
A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead compound 16, a SAR was established, showing a narrow region at the nitro-aromatic R(1) moiety and at the warhead, while the R(2) side had a much wider scope including ureas and carbamates. Compound 16 inhibits Ca(2+)-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.
Assuntos
Amidas/química , Amidas/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Carbamatos/química , Carbamatos/farmacologia , Humanos , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Relação Estrutura-Atividade , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/metabolismo , Ureia/química , Ureia/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
The scope and limitation of the combined ruthenium-lipase induced dynamic kinetic resolution (DKR) through O-acetylation of racemic heteroaromatic secondary alcohols, i.e., 1-heteroaryl substituted ethanols, was investigated. After initial screening of reaction conditions, Candida antarctica lipase B (Novozyme 435, N435) together with 4-chloro-phenylacetate as acetyl-donor for kinetic resolution (KR), in conjunction with the ruthenium-based Shvo catalyst for substrate racemization in toluene at 80 degrees C, enabled DKR with high yields and stereoselectivity of various 1-heteroaryl ethanols, such as oxadiazoles, isoxazoles, 1H-pyrazole, or 1H-imidazole. In addition, DFT calculations based on a simplified catalyst complex model for the catalytic (de)hydrogenation step are in agreement with the previously reported outer sphere mechanism. These results support the further understanding of the mechanistic aspects behind the difference in reactivity of 1-heteroaryl substituted ethanols in comparison to reference substrates, as often referred to in the literature.
Assuntos
Etanol/análogos & derivados , Etanol/química , Compostos Heterocíclicos/química , Lipase/metabolismo , Rutênio/química , Biocatálise , Enzimas Imobilizadas , Etanol/síntese química , Etanol/metabolismo , Proteínas Fúngicas , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/metabolismo , Hidrogenação , Imidazóis/síntese química , Imidazóis/química , Isoxazóis/síntese química , Isoxazóis/química , Cinética , Lipase/química , Simulação de Dinâmica Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Pirazóis/síntese química , Pirazóis/química , EstereoisomerismoRESUMO
Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg2+-dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily.
Assuntos
Galactosefosfatos/metabolismo , Glucofosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Humanos , Dobramento de ProteínaRESUMO
Lipoxins are a group of biologically active eicosanoids typically formed by transcellular lipoxygenase activity. Lipoxin A4 (LXA4) and Lipoxin B4 (LXB4) biosynthesis has been detected in a variety of inflammatory conditions. The native lipoxins LXA4 and LXB4 demonstrate potent antiinflammatory and proresolution bioactions. However, their therapeutic potential is compromised by rapid metabolic inactivation by PG dehydrogenase-mediated oxidation and reduction. Here we report on the stereoselective synthesis of aromatic LXA4 and LXB4 analogues by employing Sharpless epoxidation, Pd-mediated Heck coupling, and diastereoselective reduction as the key transformations. Subsequent biological testing has shown that these analogues display potent biological activities. Phagocytic clearance of apoptotic leukocytes plays a critical role in the resolution of inflammation. Both LXA4 analogues (1R)-3a and (1S)-3a were found to stimulate a significant increase in phagocytosis of apoptotic polymorphonuclear leukocytes (PMN) by macrophages, with comparable efficacy to the effect of native LXA4, albeit greater potency, while the LXB4 analogue also stimulated phagocytosis with a maximum effect observed at 10-11 M. LX-stimulated phagocytosis was associated with rearrangement of the actin cytoskeleton consistent with that reported for native lipoxins. Using zymosan-induced peritonitis as a murine model of acute inflammation (1R)-3a significantly reduced PMN accumulation.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Lipoxinas/síntese química , Actinas/fisiologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose , Adesão Celular , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Laminina/fisiologia , Lipoxinas/química , Lipoxinas/farmacologia , Camundongos , Neutrófilos/citologia , Neutrófilos/imunologia , Peritonite/imunologia , Fagocitose , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In modern high-throughput chemistry, the overall workflow is a crucial factor and much work is devoted to speeding up the process of chemistry development. Since automated microwave-based synthesizers are known to streamline the compound production and to accelerate slow organic transformations, this technology was implemented for Heck reactions with sluggish aryl chlorides. Furthermore, homogeneous palladium-catalyzed Heck vinylations of aryl chlorides can be performed under air under optimized conditions. Based on this finding, controlled microwave heating was utilized to accelerate model reactions down to 30 min employing a mixture of ionic liquid and 1,4-dioxane as solvent.
Assuntos
Química Orgânica/métodos , Cloretos/química , Micro-Ondas , Dioxanos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Temperatura Alta , Íons , Espectroscopia de Ressonância Magnética , Modelos Químicos , Solventes/farmacologia , Temperatura , Fatores de TempoRESUMO
Palladium-catalyzed Heck arylations in the polar and robust ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (bmimPF(6)), have for the first time been accomplished under microwave irradiation. The couplings were efficiently performed in sealed tubes within 5-45 min of heating. Without significant reductions in yield, a phosphine-free ionic catalyst phase could be recycled in five successive 20 min reactions at 180 degrees C. The product was easily removed from the reaction medium by distillation.
RESUMO
Internal ligand-controlled Heck vinylations of enamides were performed with high regioselectivity and delivered moderate to good yields of dienamides. Controlled heating by microwave irradiation accelerated the palladium-catalyzed reactions, and full conversions were achieved after reaction times of only 15-30 min. New bidentate fluorous-tagged 1,3-bis(diphenylphosphino)propane ligands (F-dppp's) were synthesized and examined. The cationic vinylations of the enamides with F-dppp ligands rendered essentially the same alpha-selectivity and catalytic activity as in those vinylations where nonfluorous ligands were employed. After reaction, the fluorous-tagged ligand material was easily removed by convenient solid fluorous phase separation. The high selectivity, simplicity, and generality of the experimental procedure should make this approach to 2-acylamino-1,3-butadienes attractive.