RESUMO
African swine fever virus (ASFV), as a contagious viral pathogen, is responsible for the occurrence of African swine fever (ASF), a rapidly spreading and highly lethal disease. Since ASFV was introduced into China in 2018, it has been quickly spread to many provinces, which brought great challenges to the pig industry in China. Due to the limited knowledge about the pathogenesis of ASFV, neither vaccines nor antiviral drugs are available. We have found that ASFV infection can induce oxidative stress responses in cells, and DNA repair enzymes play a key role in this process. This study employed RNA interference, RT-qPCR, Western blotting, Hemadsorption (HAD), and flow cytometry to investigate the effects of the inhibitors of DNA repair enzymes OGG1 and MTH1 on ASFV replication and evaluated the anti-ASFV effects of the inhibitors. This study provides reference for the development of anti-viral drugs.
Assuntos
Vírus da Febre Suína Africana , DNA Glicosilases , Monoéster Fosfórico Hidrolases , Replicação Viral , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/efeitos dos fármacos , Animais , Replicação Viral/efeitos dos fármacos , Suínos , DNA Glicosilases/metabolismo , DNA Glicosilases/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Febre Suína Africana/virologia , Antivirais/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Inibidores Enzimáticos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células VeroRESUMO
Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.
Vitamin B6 is an important nutrient for optimal brain function, with deficiencies linked to impaired memory, learning and mood in various mental disorders. In older people, vitamin B6 deficiency is also associated with declining memory and dementia. Although this has been known for years, the precise role of vitamin B6 in these disorders and whether supplements can be used to treat or prevent them remained unclear. This is partly because vitamin B6 is actually an umbrella term for a small number of very similar and interchangeable molecules. Only one of these is 'bioactive', meaning it has a biological role in cells. However, therapeutic strategies aimed at increasing only the bioactive form of vitamin B6 are lacking. Previous work showed that disrupting the gene for an enzyme called pyridoxal phosphatase, which breaks down vitamin B6, improves memory and learning in mice. To investigate whether these effects could be mimicked by drug-like compounds, Brenner, Zink, Witzinger et al. used several biochemical and structural biology approaches to search for molecules that bind to and inhibit pyridoxal phosphatase. The experiments showed that a molecule called 7,8-dihydroxyflavone which was previously found to improve memory and learning in laboratory animals with brain disorders binds to pyridoxal phosphatase and inhibits its activity. This led to increased bioactive vitamin B6 levels in mouse brain cells involved in memory and learning. The findings of Brenner et al. suggest that inhibiting pyridoxal phosphatase to increase vitamin B6 levels in the brain could be used together with supplements. The identification of 7,8-dihydroxyflavone as a promising candidate drug is a first step in the discovery of more efficient pyridoxal phosphatase inhibitors. These will be useful experimental tools to directly study whether increasing the levels of bioactive vitamin B6 in the brain may help those with mental health conditions associated with impaired memory, learning and mood.
Assuntos
Inibidores Enzimáticos , Monoéster Fosfórico Hidrolases , Animais , Camundongos , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfato de Piridoxal/metabolismo , Flavonas/farmacologia , Flavonas/metabolismo , Flavonas/química , Camundongos Endogâmicos C57BLRESUMO
Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). The human MTH1 protein catalyzes the hydrolysis of 8-oxo-dGTP, and cancer cells are dependent on MTH1 for their survival. MTH1 inhibitors are possible candidates for a class of anticancer drugs; however, a reliable screening system using live cells has not been developed. Here we report a visualization method for 8-oxo-dGTP and its related nucleotides in living cells. Escherichia coli MutT, a functional homologue of MTH1, is divided into the N-terminal (1-95) and C-terminal (96-129) parts (Mu95 and 96tT, respectively). Mu95 and 96tT were fused to Ash (assembly helper tag) and hAG (Azami Green), respectively, to visualize the nucleotides as fluorescent foci formed upon the Ash-hAG association. The foci were highly increased when human cells expressing Ash-Mu95 and hAG-96tT were treated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-dGTP. The foci formation by 8-oxo-dG(TP) was strikingly enhanced by the MTH1 knockdown. Moreover, known MTH1 inhibitors and oxidizing reagents also increased foci. This is the first system that visualizes damaged nucleotides in living cells, provides an excellent detection method for the oxidized nucleotides and oxidative stress, and enables high throughput screening for MTH1 inhibitors.
Assuntos
Nucleotídeos de Desoxiguanina , Pirofosfatases , Humanos , Nucleotídeos de Desoxiguanina/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Nucleotídeos de Guanina/metabolismo , Oxirredução , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/antagonistas & inibidoresRESUMO
Six previously undescribed prenylated indole diketopiperazine alkaloids, talaromyines A-F (1-6), were isolated from the marine-derived fungus Talaromyces purpureogenus SCSIO 41517. Their structures including absolute configurations were elucidated on the basis of comprehensive spectroscopic data including NMR, HR-ESI-MS, and electronic circular dichroism calculations, together with chemical analysis of hydrolysates. Compounds 1-5 represent the first example of spirocyclic indole diketopiperazines biosynthesized from the condensation of L-tryptophan and L-alanine. Compounds 2 and 4-5 showed selective inhibitory activities against phosphatases TCPTP and MEG2 with IC50 value of 17.9-29.7 µM, respectively. Compounds 4-5 exhibited mild cytotoxic activities against two human cancer cell lines H1975 and HepG-2.
Assuntos
Dicetopiperazinas , Talaromyces , Talaromyces/química , Dicetopiperazinas/química , Dicetopiperazinas/farmacologia , Dicetopiperazinas/isolamento & purificação , Humanos , Estrutura Molecular , Prenilação , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/química , Alcaloides Indólicos/farmacologia , Alcaloides/química , Alcaloides/farmacologia , Alcaloides/isolamento & purificação , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Células Hep G2 , Proliferação de Células/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Linhagem Celular TumoralRESUMO
The effectiveness of photodynamic therapy (PDT) has been greatly restricted by the hypoxic tumor microenvironment and the susceptible resistance of monotherapy. Although nanodrugs based on transition metal complexes capable of integrating PDT with photoactivated chemotherapy (PACT) have garnered tremendous attention as promising candidates for overcoming the above limitations, the therapeutic efficacy of these nanodrugs is still hampered by inadequate loading of active pharmaceutical ingredients (APIs) and the inherent ability of cancer cells to repair damaged DNA. Herein, we developed a photoactivated full-API nanodrug, Ru-T FAND, by one-step self-assembly of RuDPB and TH287. By virtue of its 100 wt% API content and favorable stability in water, the Ru-T FAND exhibited improved cellular uptake behavior and intracellular 1O2 generation. Attractively, the Ru-T FAND with triple anti-cancer modalities can photogenerate 1O2, photo-release DPB ligand and inhibit the repair of DNA damage, ultimately enhancing its phototherapeutic effect on cancer cells. Importantly, the uncaged DPB ligand from RuDPB emits red fluorescence, enabling real-time monitoring of the drug's absorption, distribution and efficacy. Collectively, the presented photoactivated Ru-T FANDs with multiple anti-cancer mechanisms will expand new horizons for the development of safe, efficient and synergistic tumor phototherapy strategies.
Assuntos
Antineoplásicos , Complexos de Coordenação , Dano ao DNA , Fotoquimioterapia , Humanos , Dano ao DNA/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Linhagem Celular Tumoral , Nanopartículas/química , Rutênio/química , Rutênio/farmacologia , Elementos de Transição/química , Elementos de Transição/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismoRESUMO
Cervical cancer cells possess high levels of reactive oxygen species (ROS); thus, increasing oxidative stress above the toxicity threshold to induce cell death is a promising chemotherapeutic strategy. However, the underlying mechanisms of cell death are elusive, and efficacy and toxicity issues remain. Within DNA, 8-oxo-7,8-dihydroguanine (8-oxoG) is the most frequent base lesion repaired by 8-oxoguanine glycosylase 1 (OGG1)-initiated base excision repair. Cancer cells also express high levels of MutT homolog 1 (MTH1), which prevents DNA replication-induced incorporation of 8-oxoG into the genome by hydrolyzing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). Here, we revealed that ROS-inducing agents triggered cervical cancer to undergo parthanatos, which was mainly induced by massive DNA strand breaks resulting from overwhelming 8-oxoG excision by OGG1. Furthermore, the MTH1 inhibitor synergized with a relatively low dose of ROS-inducing agents by enhancing 8-oxoG loading in the DNA. In vivo, this drug combination suppressed the growth of tumor xenografts, and this inhibitory effect was significantly decreased in the absence of OGG1. Hence, the present study highlights the roles of base repair enzymes in cell death induction and suggests that the combination of lower doses of ROS-inducing agents with MTH1 inhibitors may be a more selective and safer strategy for cervical cancer chemotherapy.
Assuntos
DNA Glicosilases , Enzimas Reparadoras do DNA , Monoéster Fosfórico Hidrolases , Espécies Reativas de Oxigênio , Neoplasias do Colo do Útero , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Humanos , Feminino , Espécies Reativas de Oxigênio/metabolismo , Animais , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , DNA Glicosilases/metabolismo , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/genética , Camundongos , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Guanina/análogos & derivados , Guanina/farmacologia , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Sinergismo Farmacológico , Células HeLa , Estresse Oxidativo/efeitos dos fármacosRESUMO
Neuronal health depends on quality control functions of autophagy, but mechanisms regulating neuronal autophagy are poorly understood. Previously, we showed that in Drosophila starvation-independent quality control autophagy is regulated by acinus (acn) and the Cdk5-dependent phosphorylation of its serine437 (Nandi et al., 2017). Here, we identify the phosphatase that counterbalances this activity and provides for the dynamic nature of acinus-serine437 (acn-S437) phosphorylation. A genetic screen identified six phosphatases that genetically interacted with an acn gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-acn levels. Cdk5-dependent phosphorylation of acn-S437 in nil1 animals elevates neuronal autophagy and reduces the accumulation of polyQ proteins in a Drosophila Huntington's disease model. Consistent with previous findings that Cd2+ inhibits PPM-type phosphatases, Cd2+ exposure elevated acn-S437 phosphorylation which was necessary for increased neuronal autophagy and protection against Cd2+-induced cytotoxicity. Together, our data establish the acn-S437 phosphoswitch as critical integrator of multiple stress signals regulating neuronal autophagy.
Assuntos
Autofagia/genética , Cádmio/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/fisiologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Serina/genética , Estresse Fisiológico/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Cádmio/toxicidade , Intoxicação por Cádmio , Drosophila melanogaster/enzimologia , Feminino , Masculino , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Serina/metabolismoRESUMO
Tumor hypoxia and hypoxic adaptation of cancer cells represent major barriers to successful cancer treatment. We revealed that improved antioxidant capacity contributes to increased radioresistance of cancer cells with tolerance to chronic-cycling severe hypoxia/reoxygenation stress. We hypothesized, that the improved tolerance to oxidative stress will increase the ability of cancer cells to cope with ROS-induced damage to free deoxy-nucleotides (dNTPs) required for DNA replication and may thus contribute to acquired resistance of cancer cells in advanced tumors to antineoplastic agents inhibiting the nucleotide-sanitizing enzyme MutT Homologue-1 (MTH1), ionizing radiation (IR) or both. Therefore, we aimed to explore potential differences in the sensitivity of cancer cells exposed to acute and chronic-cycling hypoxia/reoxygenation stress to the clinically relevant MTH1-inhibitor TH1579 (Karonudib) and to test whether a multi-targeting approach combining the glutathione withdrawer piperlongumine (PLN) and TH1579 may be suited to increase cancer cell sensitivity to TH1579 alone and in combination with IR. Combination of TH1579 treatment with radiotherapy (RT) led to radiosensitization but was not able to counteract increased radioresistance induced by adaptation to chronic-cycling hypoxia/reoxygenation stress. Disruption of redox homeostasis using PLN sensitized anoxia-tolerant cancer cells to MTH1 inhibition by TH1579 under both normoxic and acute hypoxic treatment conditions. Thus, we uncover a glutathione-driven compensatory resistance mechanism towards MTH1-inhibition in form of increased antioxidant capacity as a consequence of microenvironmental or therapeutic stress.
Assuntos
Enzimas Reparadoras do DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Glutationa/deficiência , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Hipóxia Tumoral , Antioxidantes/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Dioxolanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Pirimidinas , Radiação Ionizante , Hipóxia Tumoral/efeitos dos fármacos , Hipóxia Tumoral/genéticaRESUMO
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.
Assuntos
Crise Blástica/tratamento farmacológico , Enzimas Reparadoras do DNA/antagonistas & inibidores , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mitose , Células-Tronco Neoplásicas/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Crise Blástica/genética , Crise Blástica/metabolismo , Crise Blástica/patologia , Proliferação de Células , Citarabina/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent, lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 catalyzes the breakdown of 3'-phosphoadenosine-5'-phosphate, a by-product of glycosaminoglycan (GAG) sulfation. KO of BPNT2 in mice leads to skeletal abnormalities because of impaired GAG sulfation, especially chondroitin-4-sulfation, which is critical for proper extracellular matrix development. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which the loss of BPNT2 impairs sulfation remains unclear. Here, we used mouse embryonic fibroblasts (MEFs) to test the hypothesis that the catalytic activity of BPNT2 is required for GAG sulfation in vitro. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAGs, including decreased chondroitin-4-sulfate, present in Bpnt2-KO MEFs. We also demonstrate that missense mutations in Bpnt2 adjacent to the catalytic site, which are known to cause chondrodysplasia in humans, recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium (a common psychotropic medication) inhibits GAG sulfation and that this effect depends on the presence of BPNT2. Taken together, this work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosaminoglicanos/metabolismo , Lítio/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Catálise , Linhagem Celular , Glicosaminoglicanos/genética , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/genéticaRESUMO
In recent years, the level of interest has been increased in developing the DNA-repair inhibitors, to enhance the cytotoxic effects in the treatment of cancers. Polynucleotide kinase/phosphatase (PNKP) is a critical human DNA repair enzyme that repairs DNA strand breaks by catalyzing the restoration of 5'-phosphate and 3'-hydroxyl termini that are required for subsequent processing by DNA ligases and polymerases. PNKP is the only protein that repairs the 3'-hydroxyl group and 5'-phosphate group, which depicts PNKP as a potential therapeutic target. Besides, PNKP is the only DNA-repair enzyme that contains the 5'-kinase activity, therefore, targeting this kinase domain would motivate the development of novel PNKP-specific inhibitors. However, there are neither crystal structures of human PNKP nor the kinase inhibitors reported so far. Thus, in this present study, a sequential molecular docking-based virtual screening with multiple PNKP conformations integrating homology modeling, molecular dynamics simulation, and binding free energy calculation was developed to discover novel PNKP kinase inhibitors, and the top-scored molecule was finally submitted to molecular dynamics simulation to reveal the binding mechanism between the inhibitor and PNKP. Taken together, the current study could provide some guidance for the molecular docking based-virtual screening of novel PNKP kinase inhibitors.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Polinucleotídeo 5'-Hidroxiquinase/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Monoéster Fosfórico Hidrolases/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Conformação ProteicaRESUMO
Several recent preclinical studies have demonstrated that simultaneously blocking exogenous and endogenous sources of serine in malignant cells mediates superior anticancer effects as compared with limiting either source alone. Here, we critically summarize key developments in targeting serine to treat cancer and discuss persisting challenges for implementing such a therapeutic approach in patients.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Dieta com Restrição de Proteínas , Neoplasias/terapia , Serina/antagonistas & inibidores , Antimetabólitos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Terapia Combinada/métodos , Proteínas Alimentares/efeitos adversos , Proteínas Alimentares/metabolismo , Humanos , Neoplasias/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Serina/biossíntese , Transaminases/antagonistas & inibidores , Transaminases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness generally manifests as a mild disease in young children and immunocompromised adults. It has however emerged as a significant public health threat in recent years as outbreaks have been occurring regularly, especially in the Asia-Pacific. The disease can result from infections by a wide variety of human enteroviruses, particularly, Enterovirus A71 (EV-A71) has garnered more attention due to its association with severe disease in infected patients. Despite the potential to result severe neurological complications or even fatality, there is currently no effective antiviral for treatment of EV-A71 infections and the only vaccines available are restricted to distribution in China. In this study, we report the in vitro and in vivo evaluation of two candidate antiviral compounds active against EV-A71, a viral capsid inhibitor (G197) and a novel host-targeting phosphatidylinositol 4-kinase III beta inhibitor (N373) which, especially when used in combination, can significantly improve the survival and pathology of infected mice.
Assuntos
Antivirais/farmacologia , Capsídeo , Enterovirus Humano A/fisiologia , Inibidores Enzimáticos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , CamundongosRESUMO
Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.
Assuntos
Acondroplasia/genética , Desenvolvimento Ósseo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Peptídeo Natriurético Tipo C/análogos & derivados , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Piperazinas/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptores do Fator Natriurético Atrial/agonistas , Animais , Doenças do Desenvolvimento Ósseo/genética , Cartilagem/efeitos dos fármacos , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Sinergismo Farmacológico , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Peptídeo Natriurético Tipo C/farmacologia , Tamanho do Órgão , Fosforilação , Cultura Primária de Células , Receptores do Fator Natriurético Atrial/genética , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimentoRESUMO
INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER+ breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER+ breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3ß lysosomal degradation and activation of Wnt/ß-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER+ breast cancer via PI(3,4)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/ß-catenin therapies.
Assuntos
Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Lisossomos/metabolismo , Camundongos , Mutação , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteólise/efeitos dos fármacos , Proteômica , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Análise Serial de Tecidos , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The concept of liquid-liquid phase separation (LLPS) has emerged as an intriguing mechanism for the organization of membraneless compartments in cells. The alcohol 1,6-hexanediol is widely used as a control to dissolve LLPS assemblies in phase separation studies in diverse fields. However, little is known about potential side effects of 1,6-hexanediol, which could compromise data interpretation and mislead the scientific debate. To examine this issue, we analyzed the effect of 1,6-hexanediol on the activities of various enzymes in vitro. Already at 1% volume concentration, 1,6-hexanediol strongly impaired kinases and phosphatases and partly blocked DNA polymerases, while it had no effect on DNase activity. At concentrations that are usually used to dissolve LLPS droplets (5-10%), both kinases and phosphatases were virtually inactive. Given the widespread function of protein phosphorylation in cells, our data argue for a careful review of 1,6-hexanediol in phase separation studies.
Assuntos
Glicóis/farmacologia , Organelas/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosfotransferases/antagonistas & inibidores , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Glicóis/química , Organelas/genética , Monoéster Fosfórico Hidrolases/química , Fosforilação/efeitos dos fármacos , Fosfotransferases/química , Domínios Proteicos/genéticaRESUMO
OBJECTIVES: Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor. DESIGN AND METHODS: We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin's catalytic activity. RESULTS: Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin's phosphatase activity. CONCLUSIONS: The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.
Assuntos
Inibidores Enzimáticos/química , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/química , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/química , Animais , Bioensaio , Camelídeos Americanos , Cromatografia em Gel , Inibidores Enzimáticos/farmacologia , Mapeamento de Epitopos , Glicogênio/metabolismo , Ouro/química , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Doença de Lafora/enzimologia , Modelos Moleculares , Nitrofenóis/química , Compostos Organometálicos/química , Compostos Organofosforados/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Anticorpos de Domínio Único/isolamento & purificaçãoRESUMO
MutT Homolog 1 (MTH1) has been proven to hydrolyze oxidized nucleotide triphosphates during DNA repair. It can prevent the incorporation of wrong nucleotides during DNA replication and mitigate cell apoptosis. In a cancer cell, abundant reactive oxygen species can lead to substantial DNA damage and DNA mutations by base-pairing mismatch. MTH1 could eliminate oxidized dNTP and prevent cancer cells from entering cell death. Therefore, inhibition of MTH1 activity is considered to be an anti-cancer therapeutic target. In this study, high-throughput screening techniques were combined with a fragment-based library containing 2,313 compounds, which were used to screen for lead compounds with MTH1 inhibitor activity. Four compounds with MTH1 inhibitor ability were selected, and compound MI0639 was found to have the highest effective inhibition. To discover the selectivity and specificity of this action, several derivatives based on the MTH1 and MI0639 complex structure were synthesized. We compared 14 complex structures of MTH1 and the various compounds in combination with enzymatic inhibition and thermodynamic analysis. Nanomolar-range IC50 inhibition abilities by enzyme kinetics and Kd values by thermodynamic analysis were obtained for two compounds, named MI1020 and MI1024. Based on structural information and compound optimization, we aim to provide a strategy for the development of MTH1 inhibitors with high selectivity and specificity.
Assuntos
Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Diaminas/farmacologia , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Diaminas/síntese química , Diaminas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.
Assuntos
Linfoma de Burkitt/tratamento farmacológico , Enzimas Reparadoras do DNA/genética , Linfoma de Células B/tratamento farmacológico , Monoéster Fosfórico Hidrolases/genética , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/biossíntese , Enzimas Reparadoras do DNA/antagonistas & inibidores , Nucleotídeos de Desoxiguanina/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inflammatory diseases, including psoriasis, are characterized by changes in redox regulation. The MTH1 prevents the incorporation of oxidized nucleotides during DNA replication. Using MTH1 small-molecule inhibitors, we found induced apoptosis through 8-oxodeoxyguanosine triphosphate accumulation and DNA double-strand breaks after oxidative stress in normal and malignant keratinocytes. In psoriasis, we detected increased MTH1 expression in lesional skin and PBMCs compared with that in the controls. Using the imiquimod psoriasis mouse model, we found that MTH1 inhibition diminished psoriatic histological characteristics and normalized the levels of neutrophils and T cells in the skin and skin-draining lymph nodes. The inhibition abolished the expression of T helper type 17âassociated cytokines in the skin, which was in line with decreased levels of IL-17-producing γδ T cells in lymph nodes. In human keratinocytes, MTH1 inhibition prevented the upregulation of IL-17âdownstream genes, which was independent of ROS-induced apoptosis. In conclusion, our data support MTH1 inhibition using small molecules suitable for topical application as a promising therapeutic approach to psoriasis.