Structural and Functional Analyses of Inhibition of Human Dihydroorotate Dehydrogenase by Antiviral Furocoumavirin.

Natural products are important sources of seed compounds for drug discovery. However, it has become difficult in recent years to discover new compounds with valuable pharmacological activities. On the other hand, among the vast number of natural products that have been isolated so far, a considerable number of compounds with specific biological activities are thought to be overlooked in screening that uses biological activity as an index. Therefore, it is conceivable that such overlooked useful compounds may be found by screening compound libraries that have been amassed previously through specific assays. Previously, NPD723, a member of the Natural Products Depository library comprised of a mixture of natural and non-natural products developed at RIKEN, and its metabolite H-006 were found to inhibit growth of various cancer cells at low nanomolar half-maximal inhibitory concentration. Subsequent analysis revealed that H-006 strongly inhibited human dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine biosynthetic pathway. Here, we elucidated the crystal structure of the DHODH-flavin mononucleotide-orotic acid-H-006 complex at 1.7 Å resolution to determine that furocoumavirin, the S-enantiomer of H-006, was the actual inhibitor. The overall mode of interaction of furocoumavirin with the inhibitor binding pocket was similar to that described for previously reported tight-binding inhibitors. However, the structural information together with kinetic characterizations of site-specific mutants identified key unique features that are considered to contribute to the sub-nanomolar inhibition of DHODH by furocoumavirin. Our finding identified new chemical features that could improve the design of human DHODH inhibitors.

Identification of a dihydroorotate dehydrogenase inhibitor that inhibits cancer cell growth by proteomic profiling.

Dihydroorotate dehydrogenase (DHODH) is a central enzyme of the de novo pyrimidine biosynthesis pathway and is a promising drug target for the treatment of cancer and autoimmune diseases. This study presents the identification of a potent DHODH inhibitor by proteomic profiling. Cell-based screening revealed that NPD723, which is reduced to H-006 in cells, strongly induces myeloid differentiation and inhibits cell growth in HL-60 cells. H-006 also suppressed the growth of various cancer cells. Proteomic profiling of NPD723-treated cells in ChemProteoBase showed that NPD723 was clustered with DHODH inhibitors. H-006 potently inhibited human DHODH activity in vitro, whereas NPD723 was approximately 400 times less active than H-006. H-006-induced cell death was rescued by the addition of the DHODH product orotic acid. Moreover, metabolome analysis revealed that H-006 treatment promotes marked accumulation of the DHODH substrate dihydroorotic acid. These results suggest that NPD723 is reduced in cells to its active metabolite H-006, which then targets DHODH and suppresses cancer cell growth. Thus, H-006-related drugs represent a potentially powerful treatment for cancer and other diseases.

Fungal NRPS-PKS Hybrid Enzymes Biosynthesize New γ-Lactam Compounds, Taslactams A-D, Analogous to Actinomycete Proteasome Inhibitors.

Proteasome inhibitors with γ-lactam structure, such as lactacystin and salinosporamide A, have been isolated from actinomycetes and have attracted attention as lead compounds for anticancer drugs. Previously, we identified a unique enzyme TAS1, which is the first reported fungal NRPS-PKS hybrid enzyme, from the filamentous fungus Pyricularia oryzae for the biosynthesis of a mycotoxin tenuazonic acid, a tetramic acid compound without γ-lactam structure. Homologues of TAS1 have been identified in several fungal genomes and classified into four groups (A-D). Here, we show that the group D TAS1 homologues from two filamentous fungi can biosynthesize γ-lactam compounds, taslactams A-D, with high similarity to actinomycete proteasome inhibitors. One of the γ-lactam compounds, taslactam C, showed potent proteasome inhibitory activity. In contrast to actinomycete γ-lactam compounds which require multiple enzymes for biosynthesis, the TAS1 homologue alone was sufficient for the biosynthesis of the fungal γ-lactam compounds.

Identification of a Small Molecule That Enhances Ferroptosis via Inhibition of Ferroptosis Suppressor Protein 1 (FSP1).

Glutathione peroxidase 4 (GPX4) is an intracellular enzyme that oxidizes glutathione while reducing lipid peroxides and is a promising target for cancer therapy. To date, several GPX4 inhibitors have been reported to exhibit cytotoxicity against cancer cells. However, some cancer cells are less sensitive to the known GPX4 inhibitors. This study aimed to explore compounds showing synergistic effects with GPX4 inhibitors. We screened a chemical library and identified a compound named NPD4928, whose cytotoxicity was enhanced in the presence of a GPX4 inhibitor. Furthermore, we identified ferroptosis suppressor protein 1 as its target protein. The results indicate that NPD4928 enhanced the sensitivity of various cancer cells to GPX4 inhibitors, suggesting that the combination might have therapeutic potential via the induction of ferroptosis.

Controlled lipid ß-oxidation and carnitine biosynthesis by a vitamin D metabolite.

Lactone-vitamin D3 is a major metabolite of vitamin D3, a lipophilic vitamin biosynthesized in numerous life forms by sunlight exposure. Although lactone-vitamin D3 was discovered 40 years ago, its biological role remains largely unknown. Chemical biological analysis of its photoaffinity probe identified the hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), a mitochondrial enzyme that catalyzes ß-oxidation of long-chain fatty acids, as its selective binding protein. Intriguingly, the interaction of lactone-vitamin D3 with HADHA does not affect the HADHA enzymatic activity but instead limits biosynthesis of carnitine, an endogenous metabolite required for the transport of fatty acids into the mitochondria for ß-oxidation. Lactone-vitamin D3 dissociates the protein-protein interaction of HADHA with trimethyllysine dioxygenase (TMLD), thereby impairing the TMLD enzyme activity essential in carnitine biosynthesis. These findings suggest a heretofore undescribed role of lactone-vitamin D3 in lipid ß-oxidation and carnitine biosynthesis, and possibly in sunlight-dependent shifts of lipid metabolism in animals.

Identification of Dihydroorotate Dehydrogenase Inhibitors─Indoluidins─That Inhibit Cancer Cell Growth.

Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis and is a promising cancer treatment target. This study reports the identification of indoluidin D and its derivatives as inhibitors of DHODH. Cell-based phenotypic screening revealed that indoluidin D promoted myeloid differentiation and inhibited the proliferation of acute promyelocytic leukemia HL-60 cells. Indoluidin D also suppressed cell growth in various other types of cancer cells. Cancer cell sensitivity profiling with JFCR39 and proteomic profiling with ChemProteoBase revealed that indoluidin D is a DHODH inhibitor. Indoluidin D inhibited human DHODH activity in vitro; the DHODH reaction product orotic acid rescued indoluidin D-induced cell differentiation. We synthesized several indoluidin D diastereomer derivatives and demonstrated that stereochemistry was vital to their molecular activity. The indoluidin D derivative indoluidin E showed similar activity to its parent compound and suppressed tumor growth in a murine lung cancer xenograft model. Hence, indoluidin D and its derivatives selectively inhibit DHODH and suppress cancer cell growth.

Local administration of ReveromycinA ointment suppressed alveolar bone loss in mice.

ReveromycinA (RMA) was developed and is a unique agent for inhibiting osteoclast activity. In a previous study, we experimentally induced periodontal disease in a high-turnover osteoporosis osteoprotegerin-knockout mice (OPG KO) model and found that intraperitoneal administration of RMA inhibited alveolar bone resorption. We prepared a novel RMA-containing ointment for topical non-invasive administration in the oral cavity, in preparation for possible future clinical application. And we investigated whether this ointment can inhibit alveolar bone resorption in an experimental mouse model of periodontal disease. We examined wild-type (WT) and OPG KO mice ligated with wire around contact points on the left first and second molars to cause food impaction and induce experimental periodontal disease. RMA was administered three times a day. Using micro-computed tomography, we measured the volume of alveolar bone loss and also performed histological analysis. Our findings showed that localized administration of RMA containing ointment resulted in suppressed alveolar bone resorption, reduced osteoclast count, and lower immunostaining scores of inflammation sites compared with controls in both OPG KO and WT mice. Localized application of the specific osteoclast suppressor RMA in ointment form in the oral cavity could be a novel treatment for periodontitis that inhibits alveolar bone resorption locally.

Identification of a Small-Molecule Glucose Transporter Inhibitor, Glutipyran, That Inhibits Cancer Cell Growth.

Cancer cells reprogram their metabolism to survive and grow. Small-molecule inhibitors targeting cancer are useful for studying its metabolic pathways and functions and for developing anticancer drugs. Here, we discovered that glutipyran and its derivatives inhibit glycolytic activity and cell growth in human pancreatic cancer cells. According to proteomic profiling of glutipyran-treated cells using our ChemProteoBase, glutipyran was clustered within the group of endoplasmic reticulum (ER) stress inducers that included glycolysis inhibitors. Glutipyran inhibited glucose uptake and suppressed the growth of various cancer cells, including A431 cells that express glucose transporter class I (GLUT1) and DLD-1 GLUT1 knockout cells. When cotreated with the mitochondrial respiration inhibitor metformin, glutipyran exhibited a synergistic antiproliferative effect. Metabolome analysis revealed that glutipyran markedly decreased most metabolites of the glycolytic pathway and the pentose phosphate pathway. Glutipyran significantly suppressed tumor growth in a xenograft mouse model of pancreatic cancer. These results suggest that glutipyran acts as a broad-spectrum GLUT inhibitor and reduces cancer cell growth.

Does local injection of reveromycin A inhibit tooth movement without causing systemic side effects?

OBJECTIVE: To determine the feasibility of local inhibition of osteoclast activity and control of tooth movement with local intraoral reveromycin A (RMA) injection in model mice for experimental tooth movement. MATERIALS AND METHODS: Eight-week-old wild-type mice (n = 6 per group) were divided into four groups consisting of two non-RMA groups that received normal saline for 14 (14-day non-RMA group) or 21 consecutive days (21-day non-RMA group) and 2 RMA groups that received RMA (1.0 mg/kg of weight) for 14 (14-day RMA group) or 21 consecutive days (21-day RMA group). RMA was injected locally into the buccal mucosa of the left first maxillary molar twice daily starting 3 days before placement of the 10-gf Ni-Ti closed coil spring. Tooth movement distance was analysed using micro-computed tomography. The effects on surrounding alveolar bone were evaluated by measuring the ratio of bone surface area to tissue surface area with haematoxylin-eosin-stained sections and counting the number of osteoclasts in periodontal tissue with TRAP-stained sections. Blood tests were performed and bone volume and trabecular separation at the tibial neck were measured to analyse systemic side effects. RESULTS: Local RMA injection inhibited tooth movement by 40.6 per cent, promoted alveolar bone volume maintenance by 37.4 per cent, and inhibited osteoclast activity around the tooth root at 21 days by 40.8 per cent. Systemic effects on osteoclasts or osteoblasts were not observed. CONCLUSION: Local injection of RMA enabled control of tooth movement without systemic side effects in a mouse model.

Alteration of tooth movement by reveromycin A in osteoprotegerin-deficient mice.

INTRODUCTION: Osteoprotegerin-deficient mice develop severe high-turnover osteoporosis with porous low-density trabecular bone from an age-related increase in osteoclast activity and are useful alveolar bone models of osteoporosis or frail periodontal tissue. Bisphosphonate (BP), a first-line drug for osteoporosis, is bone-avid, causing side effects such as brittle and fragile bones and jaw osteonecrosis after tooth extraction. In orthodontics, active movement is precisely controlled by temporarily suppressing and resuming movement. BP impedes such control because of its long half-life of several years in bone. Therefore, we investigated the novel osteoclast-specific inhibitor reveromycin A (RMA), which has a short half-life in bone. We hypothesized that tooth movement could be precisely controlled through temporary discontinuation and re-administration of RMA. METHODS: Osteoprotegerin-deficient mice and wild-type mice were developed as tooth movement models under constant orthodontic force. A constant orthodontic force of 10 g was induced using a nickel-titanium closed coil spring to move the maxillary first molar for 14 days. We administered BP (1.25 mg/kg) or RMA (1.0 mg/kg) continuously and then discontinued it to reveal how the subsequent movement of teeth and surrounding alveolar bone was affected. RESULTS: Continuous BP or RMA administration suppressed osteoclast activity and preserved alveolar bone around the roots, apparently normalizing bone metabolism. Tooth movement remained suppressed after BP discontinuation but resumed at a higher rate after discontinuation of RMA. CONCLUSIONS: RMA appears useful for controlling orthodontic tooth movement because it can be suppressed and resumed through administration and discontinuation, respectively.

Inhibition of mitochondrial complex I by the novel compound FSL0260 enhances high salinity-stress tolerance in Arabidopsis thaliana.

Chemical priming is an attractive and promising approach to improve abiotic stress tolerance in a broad variety of plant species. We screened the RIKEN Natural Products Depository (NPDepo) chemical library and identified a novel compound, FSL0260, enhancing salinity-stress tolerance in Arabidopsis thaliana and rice. Through transcriptome analysis using A. thaliana seedlings, treatment of FSL0260 elevated an alternative respiration pathway in mitochondria that modulates accumulation of reactive oxygen species (ROS). From comparison analysis, we realized that the alternative respiration pathway was induced by treatment of known mitochondrial inhibitors. We confirmed that known inhibitors of mitochondrial complex I, such as rotenone and piericidin A, also enhanced salt-stress tolerance in Arabidopsis. We demonstrated that FSL0260 binds to complex I of the mitochondrial electron transport chain and inhibits its activity, suggesting that inhibition of mitochondrial complex I activates an alternative respiration pathway resulting in reduction of ROS accumulation and enhancement of tolerance to salinity in plants. Furthermore, FSL0260 preferentially inhibited plant mitochondrial complex I rather than a mammalian complex, implying that FSL0260 has a potential to be an agent for improving salt-stress tolerance in agriculture that is low toxicity to humans.

Identification of a Small Compound Targeting PKM2-Regulated Signaling Using 2D Gel Electrophoresis-Based Proteome-wide CETSA.

The cellular thermal shift assay (CETSA) has recently been devised as a label-free method for target validation of small compounds and monitoring the thermal stabilization or destabilization of proteins due to binding with the compound. Herein, we developed a modified method by combining the CETSA and proteomics analysis based on 2D gel electrophoresis, namely 2DE-CETSA, to identify the thermal stability-shifted proteins by binding with a new compound. We applied the 2DE-CETSA for analysis of a target-unknown compound, NPD10084, which exerts anti-proliferative activity against colorectal cancer cells in vitro and in vivo, and identified pyruvate kinase muscle isoform 2 (PKM2) as a candidate target protein. Interestingly, NPD10084 interrupted protein-protein interactions between PKM2 and ß-catenin or STAT3, with subsequent suppression of downstream signaling. We thus demonstrate that our 2DE-CETSA method is applicable for identification of target compounds discovered by phenotypic screening.

Structure and biological activity of Metarhizin C, a stereoisomer of BR-050 from Tolypocladium album RK17-F0007.

Metarhizin C, a stereoisomer of BR-050 was isolated from a fungus Tolypocladium album RK17-F0007 through a screening program to search for new antitumor compounds. A structure of the isomer was determined by spectroscopic methods including detailed analysis of NOESY correlation and mass spectrometry, and found to be identical to that of 3-desacylmetarhizin A with the absolute structure. Previously, it had been isolated by Kikuchi et al and proposed as BR-050 including the stereo-structure. However, detailed analysis for the newly isolated isomer confirmed that 3-desacylmetarhizin A was not identical to BR-050. Therefore, we assigned it metarhizin C as a new BR-050 isomer. Metarhizin C showed selective cytotoxicity against osteosarcoma MG-63 cells in a glucose independent condition with IC50 value of 0.79 µg/ml, while > 30 µg/ml of IC50 value in a normal condition, and inhibited a mitochondrial respiration.

Bioenergetic and proteomic profiling to screen small molecule inhibitors that target cancer metabolisms.

Cancer cells can reprogram their metabolic machinery to survive. This altered metabolism, which is distinct from the metabolism of normal cells, is thought to be a possible target for the development of new cancer therapies. In this study, we constructed a screening system that focuses on bioenergetic profiles (specifically oxygen consumption rate and extracellular acidification rate) and characteristic proteomic changes. Thus, small molecules that target cancer-specific metabolism were investigated. We screened the chemical library of RIKEN Natural Products Depository (NPDepo) and found that unantimycin A, which was recently isolated from the fraction library of microbial metabolites, and NPL40330, which is derived from a chemical library, inhibit mitochondrial respiration. Furthermore, we developed an in vitro reconstitution assay method for mitochondrial electron transport chain using semi-intact cells with specific substrates for each complex of the mitochondrial electron transport chain. Our findings revealed that NPL40330 and unantimycin A target mitochondrial complexes I and III, respectively.

Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K.

Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome-plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases.

Identification and structure-activity relationship of purine derivatives as novel MTH1 inhibitors.

The human mutT homolog-1 (MTH1) protein prevents the incorporation of oxidized nucleotides such as 2-OH-dATP and 8-oxo-dGTP during DNA replication by hydrolyzing them into their corresponding monophosphates. It was found previously that cancer cells could tolerate oxidative stress due to this enzymatic activity of MTH1 and its inhibition could be a promising approach to treat several types of cancer. This finding has been challenged recently with increasing line of evidence suggesting that the cancer cell-killing effects of MTH1 inhibitors may be related to their engagement of off-targets. We have previously reported a few purine-based MTH1 inhibitors that enabled us to elucidate the dispensability of MTH1 in cancer cell survival. Here, we provide a detailed process of the identification of purine-based MTH1 inhibitors. Several new compounds with potency in the submicromolar range are disclosed. Furthermore, the structure-activity relationship and associated binding mode prediction using molecular docking have provided insights for the development of highly potent MTH1 inhibitors.

Proteomic profiling reveals that collismycin A is an iron chelator.

Collismycin A (CMA), a microbial product, has anti-proliferative activity against cancer cells, but the mechanism of its action remains unknown. Here, we report the identification of the molecular target of CMA by ChemProteoBase, a proteome-based approach for drug target identification. ChemProteoBase profiling showed that CMA is closely clustered with di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, an iron chelator. CMA bound to both Fe(II) and Fe(III) ions and formed a 2:1 chelator-iron complex with a redox-inactive center. CMA-induced cell growth inhibition was completely canceled by Fe(II) and Fe(III) ions, but not by other metal ions such as Zn(II) or Cu(II). Proteomic and transcriptomic analyses showed that CMA affects the glycolytic pathway due to the accumulation of HIF-1α. These results suggest that CMA acts as a specific iron chelator, leading to the inhibition of cancer cell growth.

Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival.

Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor-induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival.

Identification of a novel compound that inhibits osteoclastogenesis by suppressing nucleoside transporters.

We screened small-molecule compounds that inhibit osteoclast differentiation to find new anti-osteoporosis agents and found that a novel compound, SUKU-1, suppressed osteoclastogenesis. We also synthesized 38 derivatives of SUKU-1 and discovered that nine of them had inhibitory effects on osteoclastogenesis and that SUKU-33 was the most potent inhibitor. Next, we investigated the mechanisms by which SUKU-33 suppressed osteoclast differentiation. By measuring the uptake of [(3) H]-uridine in cells, we found that SUKU-33 suppressed both equilibrative nucleoside transporters and concentrative nucleoside transporters. These results suggest that SUKU-33 inhibits osteoclast differentiation by suppressing nucleoside transporters.