Inhibitor development of MTH1 via high-throughput screening with fragment based library and MTH1 substrate binding cavity.

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.

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid ß-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

Energy adaptive response during parthanatos is enhanced by PD98059 and involves mitochondrial function but not autophagy induction.

Parthanatos is a programmed necrotic demise characteristic of ATP (adenosine triphosphate) consumption due to NAD+ (nicotinamide adenine dinucleotide) depletion by poly(ADP-ribose) polymerase 1 (PARP1)-dependent poly(ADP-ribosyl)ation on target proteins. However, how the bioenergetics is adaptively regulated during parthanatos, especially under the condition of macroautophagy deficiency, remains poorly characterized. Here, we demonstrated that the parthanatic inducer N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggered ATP depletion followed by recovery in mouse embryonic fibroblasts (MEFs). Notably, Atg5-/- MEFs showed great susceptibility to MNNG with disabled ATP-producing capacity. Moreover, the differential energy-adaptive responses in wild-type (WT) and Atg5-/- MEFs were unequivocally worsened by inhibition ofAMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity. Importantly, Atg5-/- MEFs disclosed diminished SIRT1 and mitochondrial activity essential to the energy restoration during parthanatos. Strikingly, however, parthanatos cannot be exasperated by bafilomycin A1 and MNNG neither provokes microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and p62 elimination, suggesting that parthanatos does not induce autophagic flux. Intriguingly, we reported unexpectedly that PD98059, even at low concentration insufficient to inhibit MEK, can promote mitochondrial activity and facilitate energy-restoring process during parthanatos, without modulating DNA damage responses as evidenced by PARP1 activity, p53 expression, and gammaH2AX (H2A histone family, member X (H2AX), phosphorylated on Serine 139) induction. Therefore, we propose that Atg5 deficiency confers an infirmity to overcome the energy crisis during parthanatos and further underscore the deficits in mitochondrial quality control, but not incapability of autophagy induction, that explain the vulnerability in Atg5-deficient cells. Collectively, our results provide a comprehensive energy perspective for an improved treatment to alleviate parthanatos-related tissue necrosis and disease progression and also provide a future direction for drug development on the basis of PD98059 as an efficacious compound against parthanatos.

Homoharringtonine as a PHGDH inhibitor: Unraveling metabolic dependencies and developing a potent therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma, a childhood cancer affecting the sympathetic nervous system, continues to challenge the development of potent treatments due to the limited availability of druggable targets for this aggressive illness. Recent investigations have uncovered that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme for de novo serine synthesis, serves as a non-oncogene dependency in high-risk neuroblastoma. In this study, we show that homoharringtonine (HHT) acts as a PHGDH inhibitor, inducing intricate alterations in cellular metabolism, and thus providing an efficient treatment for neuroblastoma. We have experimentally verified the reliance of neuroblastoma on PHGDH and employed molecular docking, thermodynamic evaluations, and X-ray crystallography techniques to determine the bond interactions between HHT and PHGDH. Administering HHT to treat neuroblastoma resulted in effective cell elimination in vitro and tumor reduction in vivo. Metabolite and functional assessments additionally disclosed that HHT treatment suppressed de novo serine synthesis, initiating intricate metabolic reconfiguration and oxidative stress in neuroblastoma. Collectively, these discoveries highlight the potential of targeting PHGDH using HHT as a potent approach for managing high-risk neuroblastoma.

BI-2536 Promotes Neuroblastoma Cell Death via Minichromosome Maintenance Complex Components 2 and 10.

DNA replication is initiated with the recognition of the starting point of multiple replication forks by the origin recognition complex and activation of the minichromosome maintenance complex 10 (MCM10). Subsequently, DNA helicase, consisting of the MCM protein subunits MCM2-7, unwinds double-stranded DNA and DNA synthesis begins. In previous studies, replication factors have been used as clinical targets in cancer therapy. The results showed that MCM2 could be a proliferation marker for numerous types of malignant cancer. We analyzed samples obtained from patients with neuroblastoma, revealing that higher levels of MCM2 and MCM10 mRNA were associated with poor survival rate. Furthermore, we combined the results of the perturbation-induced reversal effects on the expression levels of MCM2 and MCM10 and the sensitivity correlation between perturbations and MCM2 and MCM10 from the Cancer Therapeutics Response Portal database. Small molecule BI-2536, a polo-like kinase 1 (PLK-1) inhibitor, is a candidate for the inhibition of MCM2 and MCM10 expression. To test this hypothesis, we treated neuroblastoma cells with BI-2536. The results showed that the drug decreased cell viability and reduced the expression levels of MCM2 and MCM10. Functional analysis further revealed enrichments of gene sets involved in mitochondria, cell cycle, and DNA replication for BI-2536-perturbed transcriptome. We used cellular assays to demonstrate that BI-2536 promoted mitochondria fusion, G2/M arrest, and apoptosis. In summary, our findings provide a new strategy for neuroblastoma therapy with BI-2536.

Enhancement of the IFN-ß-induced host signature informs repurposed drugs for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent for the outbreak of coronavirus disease 2019 (COVID-19). This global pandemic is now calling for efforts to develop more effective COVID-19 therapies. Here we use a host-directed approach, which focuses on cellular responses to diverse small-molecule treatments, to identify potentially effective drugs for COVID-19. This framework looks at the ability of compounds to elicit a similar transcriptional response to IFN-ß, a type I interferon that fails to be induced at notable levels in response to SARS-CoV-2 infection. By correlating the perturbation profiles of ~3,000 small molecules with a high-quality signature of IFN-ß-responsive genes in primary normal human bronchial epithelial cells, our analysis revealed four candidate COVID-19 compounds, namely homoharringtonine, narciclasine, anisomycin, and emetine. We experimentally confirmed that the predicted compounds significantly inhibited SARS-CoV-2 replication in Vero E6 cells at nanomolar, relatively non-toxic concentrations, with half-maximal inhibitory concentrations of 165.7 nM, 16.5 nM, and 31.4 nM for homoharringtonine, narciclasine, and anisomycin, respectively. Together, our results corroborate a host-centric strategy to inform protective antiviral therapies for COVID-19.

Perturbational Gene-Expression Signatures for Combinatorial Drug Discovery.

Cancer is a complex disease that relies on both oncogenic mutations and non-mutated genes for survival, and therefore coined as oncogene and non-oncogene addictions. The need for more effective combination therapies to overcome drug resistance in oncology has been increasingly recognized, but the identification of potentially synergistic drugs at scale remains challenging. Here we propose a gene-expression-based approach, which uses the recurrent perturbation-transcript regulatory relationships inferred from a large compendium of chemical and genetic perturbation experiments across multiple cell lines, to engender a testable hypothesis for combination therapies. These transcript-level recurrences were distinct from known compound-protein target counterparts, were reproducible in external datasets, and correlated with small-molecule sensitivity. We applied these recurrent relationships to predict synergistic drug pairs for cancer and experimentally confirmed two unexpected drug combinations in vitro. Our results corroborate a gene-expression-based strategy for combinatorial drug screening as a way to target non-mutated genes in complex diseases.

Therapeutic Targeting of Non-oncogene Dependencies in High-risk Neuroblastoma.

PURPOSE: Neuroblastoma is a pediatric malignancy of the sympathetic nervous system with diverse clinical behaviors. Genomic amplification of MYCN oncogene has been shown to drive neuroblastoma pathogenesis and correlate with aggressive disease, but the survival rates for those high-risk tumors carrying no MYCN amplification remain equally dismal. The paucity of mutations and molecular heterogeneity has hindered the development of targeted therapies for most advanced neuroblastomas. We use an alternative method to identify potential drugs that target nononcogene dependencies in high-risk neuroblastoma. EXPERIMENTAL DESIGN: By using a gene expression-based integrative approach, we identified prognostic signatures and potentially effective single agents and drug combinations for high-risk neuroblastoma. RESULTS: Among these predictions, we validated in vitro efficacies of some investigational and marketed drugs, of which niclosamide, an anthelmintic drug approved by the FDA, was further investigated in vivo. We also quantified the proteomic changes during niclosamide treatment to pinpoint nucleoside diphosphate kinase 3 (NME3) downregulation as a potential mechanism for its antitumor activity. CONCLUSIONS: Our results establish a gene expression-based strategy to interrogate cancer biology and inform drug discovery and repositioning for high-risk neuroblastoma.

Combinatorial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic strategy.

MYCN-amplified (MNA) neuroblastoma is an aggressive neural crest-derived pediatric cancer. However, MYCN is indispensable for development and transcriptionally regulates extensive network of genes. Integrating anti-MYCN ChIP-seq and gene expression profiles of neuroblastoma patients revealed the metabolic enzymes, MTHFD2 and PAICS, required for one-carbon metabolism and purine biosynthesis were concomitantly upregulated, which were more susceptible to metastatic neuroblastoma. Moreover, we found that MYCN mediated the folate cycle via MTHFD2, which contributed one-carbon unit to enhance purine synthesis, and further regulated nucleotide production by PAICS in response to cancer progression. Dual knockdown of the MYCN-targeted gene pair, MTHFD2 and PAICS, in MNA neuroblastoma cells synergically reduced cell proliferation, colony formation, migration ability, and DNA synthesis. By systematically screening the compound perturbagens, the gene expression levels of MTHFD2 and PAICS were specifically suppressed by anisomycin and apicidin across cell lines, and our co-treatment results also displayed synergistic inhibition of MNA neuroblastoma cell proliferation. Collectively, targeting a combination of MYCN-targeted genes that interrupts the interconnection of metabolic pathways may overcome drug toxicity and improve the efficacy of current therapeutic agents in MNA neuroblastoma.

A Large-Scale Gene Expression Intensity-Based Similarity Metric for Drug Repositioning.

Biological systems often respond to a specific environmental or genetic perturbation without pervasive gene expression changes. Such robustness to perturbations, however, is not reflected on the current computational strategies that utilize gene expression similarity metrics for drug discovery and repositioning. Here we propose a new expression-intensity-based similarity metric that consistently achieved better performance than other state-of-the-art similarity metrics with respect to the gold-standard clustering of drugs with known mechanisms of action. The new metric directly emphasizes the genes exhibiting the greatest changes in expression in response to a perturbation. Using the new framework to systematically compare 3,332 chemical and 3,934 genetic perturbations across 10 cell types representing diverse cellular signatures, we identified thousands of recurrent and cell type-specific connections. We also experimentally validated two drugs identified by the analysis as potential topoisomerase inhibitors. The new framework is a valuable resource for hypothesis generation, functional testing, and drug repositioning.

MicroRNA-mediated networks underlie immune response regulation in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is a common endocrine malignancy with low death rate but increased incidence and recurrence in recent years. MicroRNAs (miRNAs) are small non-coding RNAs with diverse regulatory capacities in eukaryotes and have been frequently implied in human cancer. Despite current progress, however, a panoramic overview concerning miRNA regulatory networks in PTC is still lacking. Here, we analyzed the expression datasets of PTC from The Cancer Genome Atlas (TCGA) Data Portal and demonstrate for the first time that immune responses are significantly enriched and under specific regulation in the direct miRNA--target network among distinctive PTC variants to different extents. Additionally, considering the unconventional properties of miRNAs, we explore the protein-coding competing endogenous RNA (ceRNA) and the modulatory networks in PTC and unexpectedly disclose concerted regulation of immune responses from these networks. Interestingly, miRNAs from these conventional and unconventional networks share general similarities and differences but tend to be disparate as regulatory activities increase, coordinately tuning the immune responses that in part account for PTC tumor biology. Together, our systematic results uncover the intensive regulation of immune responses underlain by miRNA-mediated networks in PTC, opening up new avenues in the management of thyroid cancer.

HMG-CoA reductase inhibitors activate caspase-1 in human monocytes depending on ATP release and P2X7 activation.

Recent studies have demonstrated the stimulatory effects of HMG-CoA reductase inhibitors, statins, on IL-1ß secretion in monocytes and suggest a crucial role for isoprenoids in the inhibition of caspase-1 activity. In this study, we further elucidated the molecular mechanisms underlying the stimulatory effects of statins on caspase-1. Three commonly recognized mechanistic models for NLRP3 inflammasome activation (i.e., ATP/P2X7/K(+) efflux, ROS production, and lysosomal rupture) were investigated in statin-stimulated human THP-1 monocytes. We found that fluvastatin and lovastatin can synergize with LPS to trigger inflammasome activation. Moreover, statin-induced caspase-1 activation and IL-1ß production in LPS-primed THP-1 cells are dependent on GGPP deficiency and P2X7 activation. In particular, increased ATP release accounts for the action of statins in P2X7 activation. We also provide evidence that statin-induced moderate ROS elevation is involved in this event. Moreover, the cathepsin B inhibitor was shown to reduce statin-induced IL-1ß secretion. Consistently statins can induce cathepsin B activation and lysosomal rupture, as evidenced by LysoTracker staining. Statins also increase intracellular ATP secretion and IL-1ß release in primary human monocytes and murine macrophages. Notably, exogenous ATP-elicited P2X7 activation and consequent IL-1ß release, an index of direct NLRP3 inflammasome activation, were not altered by statins. Taken together, statin-induced enhancement of inflammasome activation in monocytes and macrophages covers multiple mechanisms, including increases in ATP release, ROS production, and lysosomal rupture. These data not only shed new insight into isoprenylation-dependent regulation of caspase-1 but also unmask mechanisms for statin-elicited inflammasome activation.

Anti-atherosclerotic action of Ger-Gen-Chyn-Lian-Tang and AMPK-dependent lipid lowering effect in hepatocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: The Ger-Gen-Chyn-Lian-Tang (GGCLT), an officially standardized mixture of Chinese herbal medicines, consists of Puerariae Radix, Scutellariae Radix, Coptidis Rhizoma and Glycyrrhizae Radix in a ratio of 8:3:3:2. In this study, we evaluated the benefits of GGCLT in atherosclerotic progression. METHODS: The major constituents of GGCLT were analyzed by HPLC. ApoE-/- mice taken 0.15% cholesterol diet were orally given vehicle or GGCLT (2 g/kg/day) for 12 weeks. Serum levels of lipid and glucose were analyzed, and atherosclerosis was examined by histological analyses. Cultures of vascular smooth muscle cells, hepatocytes and bone marrow-derived macrophages were used to investigate the action mechanisms of GGCLT. RESULTS: Our quantitation results indicated that GGCLT contains puerarin, daidzin, daidzein, baicalin, baicalein, wogonin, palmatine, coptisine, berberine and glycyrrhizin. GGCLT decreased serum levels of total cholesterol and LDL, but not TG and HDL in ApoE-/- mice. In parallel, GGCLT treatment reduced atherosclerotic lesions and collagen expression in atheroma plaques. In vascular smooth muscle cells, GGCLT could reduce cell migration, but failed to affect cell viability and proliferation. In hepatocytes, GGCLT can reduce lipid accumulation, and this action was accompanied by the activation of AMPK, upregulation of PPARs, and downregulation of FAS. Pharmacological approach indicated that the latter two events contributing to the anti-lipogenesis is resulting from AMPK pathway, and the lipid lowering effect of GGCLT in hepatocytes is mediated by AMPK and PPARα pathways. Meanwhile, two of the major components of GGCLT, berberine and puerarin, also activated AMPK and decreased lipid accumulation in hepatocytes with berberine of higher efficacy. Besides in hepatocytes, AMPK signaling was also activated by GGCLT in vascular smooth muscle cells and macrophages. CONCLUSIONS: These results demonstrate the anti-atherosclerotic action of Chinese medicine mixture GGCLT in ApoE-/- atherosclerotic mouse model. Mechanistic study suggests that activation of AMPK and PPARα in hepatocytes leading to a decrease of lipid formation contributes to the beneficial action of GGCLT in atherosclerosis treatment.