RESUMEN
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.
Asunto(s)
Aminohidrolasas , Leucemia Mieloide Aguda , Aminohidrolasas/genética , Humanos , Hidrolasas , Leucemia Mieloide Aguda/tratamiento farmacológico , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Enzimas Multifuncionales/genética , TimidinaRESUMEN
Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.
Asunto(s)
Peces , Fosfoglucomutasa/metabolismo , Animales , Dominio Catalítico , Glucofosfatos/metabolismo , Fosfoglucomutasa/química , Unión Proteica , Especificidad por SustratoRESUMEN
Botulinum neurotoxins (BoNTs) are exceptionally toxic proteins that cause paralysis but are also extensively used as treatment for various medical conditions. Most BoNTs bind two receptors on neuronal cells, namely, a ganglioside and a protein receptor. Differences in the sequence between the protein receptors from different species can impact the binding affinity and toxicity of the BoNTs. Here we have investigated how BoNT/B, /DC, and /G, all three toxins that utilize synaptotagmin I and II (Syt-I and Syt-II, respectively) as their protein receptors, bind to Syt-I and -II of mouse/rat, bovine, and human origin by isothermal titration calorimetry analysis. BoNT/G had the highest affinity for human Syt-I, and BoNT/DC had the highest affinity for bovine Syt-II. As expected, BoNT/B, /DC, and /G showed very low levels of binding to human Syt-II. Furthermore, we carried out saturation transfer difference (STD) and STD-TOCSY NMR experiments that revealed the region of the Syt peptide in direct contact with BoNT/G, which demonstrate that BoNT/G recognizes the Syt peptide in a model similar to that in the established BoNT/B-Syt-II complex. Our analyses also revealed that regions outside the Syt peptide's toxin-binding region are important for the helicity of the peptide and, therefore, the binding affinity.
Asunto(s)
Toxinas Botulínicas/química , Sinaptotagminas/química , Sinaptotagminas/metabolismo , Sinaptotagminas/ultraestructura , Animales , Sitios de Unión , Fenómenos Biofísicos , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/ultraestructura , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Bovinos , Cristalografía por Rayos X , Gangliósidos/metabolismo , Humanos , Ratones , Modelos Moleculares , Neuronas/metabolismo , Neurotoxinas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , RatasRESUMEN
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.
Asunto(s)
Enzimas Reparadoras del ADN/fisiología , Nucleótidos de Desoxiguanina/química , Monoéster Fosfórico Hidrolasas/fisiología , Animales , Dominio Catalítico , Cristalografía por Rayos X , Metilasas de Modificación del ADN/química , Enzimas Reparadoras del ADN/química , Perros , Escherichia coli/genética , Células HL-60 , Humanos , Hidrólisis , Cinética , Ratones , Nucleótidos , Monoéster Fosfórico Hidrolasas/química , Pirofosfatasas/química , Especificidad de la Especie , Porcinos , Temozolomida/farmacología , Proteínas Supresoras de Tumor/química , Pez CebraRESUMEN
Botulinum neurotoxins (BoNTs) are a family of highly dangerous bacterial toxins, with seven major serotypes (BoNT/A-G). Members of BoNTs, BoNT/A1 and BoNT/B1, have been utilized to treat an increasing number of medical conditions. The clinical trials are ongoing for BoNT/A2, another subtype of BoNT/A, which showed promising therapeutic properties. Both BoNT/A1 and BoNT/A2 utilize three isoforms of synaptic vesicle protein SV2 (SV2A, B, and C) as their protein receptors. We here present a high resolution (2.0 Å) co-crystal structure of the BoNT/A2 receptor-binding domain in complex with the human SV2C luminal domain. The structure is similar to previously reported BoNT/A-SV2C complexes, but a shift of the receptor-binding segment in BoNT/A2 rotates SV2C in two dimensions giving insight into the dynamic behavior of the interaction. Small differences in key residues at the binding interface may influence the binding to different SV2 isoforms, which may contribute to the differences between BoNT/A1 and BoNT/A2 observed in the clinic.
Asunto(s)
Toxinas Botulínicas Tipo A/química , Glicoproteínas de Membrana/química , Proteínas del Tejido Nervioso/química , Sitios de Unión , Toxinas Botulínicas Tipo A/metabolismo , Cristalografía , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to that in nonmalignant cells, making cancer cells more dependent on MTH1 for survival. The possibility of specifically targeting cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. The progression of MTH1 inhibitors into the clinic requires animal studies, and knowledge of species differences in the potency of inhibitors is vitally important. We here show that the human MTH1 inhibitor TH588 is approximately 20-fold less potent with respect to inhibition of mouse MTH1 than the human, rat, pig, and dog MTH1 proteins are. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1 and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant of TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 proteins from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity.
Asunto(s)
Nucleótidos de Desoxiguanina/metabolismo , Perros/metabolismo , Ratones/metabolismo , Monoéster Fosfórico Hidrolasas/química , Pirimidinas/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Concentración 50 Inhibidora , Modelos Moleculares , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Unión Proteica , Conformación Proteica , Pirimidinas/farmacología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por Sustrato , Porcinos , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/químicaRESUMEN
Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clostridium botulinum/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Clostridium botulinum/química , Clostridium botulinum/genética , Cristalografía por Rayos X , Modelos Moleculares , Familia de Multigenes , Fosfatidilinositoles/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 µM. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.
Asunto(s)
Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Simulación por Computador , Cristalografía por Rayos X , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/efectos de los fármacos , Humanos , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/efectos de los fármacos , Unión Proteica , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. Cancer Res; 77(4); 937-48. ©2017 AACR.
Asunto(s)
Inhibidores Enzimáticos/química , Meteniltetrahidrofolato Ciclohidrolasa/química , Metilenotetrahidrofolato Deshidrogenasa (NADP)/química , Mitocondrias/enzimología , Sitios de Unión , Cristalización , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Humanos , Leucovorina/análogos & derivados , Leucovorina/metabolismo , Meteniltetrahidrofolato Ciclohidrolasa/antagonistas & inhibidores , Metilenotetrahidrofolato Deshidrogenasa (NADP)/antagonistas & inhibidores , Antígenos de Histocompatibilidad Menor , NAD/metabolismo , Multimerización de ProteínaRESUMEN
The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD1a can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.
Asunto(s)
Toxinas Botulínicas Tipo A/química , Polisacáridos/química , Receptores de Superficie Celular/química , Toxinas Botulínicas Tipo A/metabolismo , Conformación de Carbohidratos , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance. Cancer Res; 76(8); 2366-75. ©2016 AACR.
Asunto(s)
Hipoxia de la Célula , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Transducción de Señal , Microambiente Tumoral , Animales , Humanos , Oxidación-Reducción , Pez CebraRESUMEN
Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleótidos/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/metabolismo , Pirofosfatasas/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular , Cristalización , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Pirofosfatasas/química , Especificidad por SustratoRESUMEN
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.