Reduction of Amyloid-ß Production without Inhibiting Secretase Activity by MS-275.

Brain amyloid-ß (Aß) governs the pathogenic process of Alzheimer's disease. Clinical trials to assess the disease-modifying effects of inhibitors or modulators of ß- and γ-secretases have not shown clinical benefit and can cause serious adverse events. Previously, we found that the interleukin-like epithelial-to-mesenchymal transition inducer (ILEI, also known as FAM3C) negatively regulates the Aß production through a decrease in Aß immediate precursor, without the inhibition of ß- and γ-secretase activity. Herein, we found that MS-275, a benzamide derivative that is known to inhibit histone deacetylases (HDACs), exhibits ILEI-like activity to reduce Aß production independent of HDAC inhibition. Chronic MS-275 treatment decreased Aß deposition in the cerebral cortex and hippocampus in an Alzheimer's disease mouse model. Overall, our results indicate that MS-275 is a potential therapeutic candidate for efficiently reducing brain Aß accumulation.

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5'-phosphate (PLP) via Schiff base formation.

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 µM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine ß-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells.

Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate D-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses D-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.

Drug cytotoxicity screening using human intestinal organoids propagated with extensive cost-reduction strategies.

Organoids are regarded as physiologically relevant cell models and useful for compound screening for drug development; however, their applications are currently limited because of the high cost of their culture. We previously succeeded in reducing the cost of human intestinal organoid culture using conditioned medium (CM) of L cells co-expressing Wnt3a, R-spondin1, and Noggin. Here, we further reduced the cost by replacing recombinant hepatocyte growth factor with CM. Moreover, we showed that embedding organoids in collagen gel, a more inexpensive matrix than Matrigel, maintains organoid proliferation and marker gene expression similarly when using Matrigel. The combination of these replacements also enabled the organoid-oriented monolayer cell culture. Furthermore, screening thousands of compounds using organoids expanded with the refined method identified several compounds with more selective cytotoxicity against organoid-derived cells than Caco-2 cells. The mechanism of action of one of these compounds, YC-1, was further elucidated. We showed that YC-1 induces apoptosis through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, the mechanism of which was distinct from cell death caused by other hit compounds. Our cost-cutting methodology enables large-scale intestinal organoid culture and subsequent compound screening, which could expand the application of intestinal organoids in various research fields.

Roles of peptidyl prolyl isomerase Pin1 in viral propagation.

Peptidyl-prolyl isomerase (PPIase) is a unique enzyme that promotes cis-trans isomerization of a proline residue of a target protein. Peptidyl-prolyl cis-trans isomerase NIMA (never in mitosis A)-interacting 1 (Pin1) is a PPIase that binds to the pSer/pThr-Pro motif of target proteins and isomerizes their prolines. Pin1 has been reported to be involved in cancer development, obesity, aging, and Alzheimer's disease and has been shown to promote the growth of several viruses including SARS-CoV-2. Pin1 enhances the efficiency of viral infection by promoting uncoating and integration of the human immunodeficiency virus. It has also been shown that Pin1 interacts with hepatitis B virus proteins and participates in viral replication. Furthermore, Pin1 promotes not only viral proliferation but also the progression of virus-induced tumorigenesis. In this review, we focus on the effects of Pin1 on the proliferation of various viruses and discuss the underlying molecular mechanisms.

Dephospho-Coenzyme A Kinase Is an Exploitable Drug Target against Plasmodium falciparum: Identification of Selective Inhibitors by High-Throughput Screening of a Large Chemical Compound Library.

Malaria is a mosquito-borne fatal infectious disease that affects humans and is caused by Plasmodium parasites, primarily Plasmodium falciparum. Widespread drug resistance compels us to discover novel compounds and alternative drug discovery targets. The coenzyme A (CoA) biosynthesis pathway is essential for the malaria parasite P. falciparum. The last enzyme in CoA biosynthesis, dephospho-CoA kinase (DPCK), is essential to the major life cycle development stages but has not yet been exploited as a drug target in antimalarial drug discovery. We performed a high-throughput screen of a 210,000-compound library using recombinant P. falciparum DPCK (PfDPCK). A high-throughput enzymatic assay using a 1,536-well platform was developed to identify potential PfDPCK inhibitors. PfDPCK inhibitors also inhibited parasite growth in a P. falciparum whole-cell asexual blood-stage assay in both drug-sensitive and drug-resistant strains. Hit compounds were selected based on their potency in cell-free (PfDPCK) and whole-cell (Pf3D7 and PfDd2) assays, selectivity over the human orthologue (HsCOASY) and no cytotoxicity (HepG2). The compounds were ranked using a multiparameter optimization (MPO) scoring model, and the specific binding and the mechanism of inhibition were investigated for the most promising compounds.

Small-Molecule-Induced Activation of Cellular Respiration Inhibits Biofilm Formation and Triggers Metabolic Remodeling in Staphylococcus aureus.

Staphylococcus aureus, a major pathogen of community-acquired and nosocomial-associated infections, forms biofilms consisting of extracellular matrix-embedded cell aggregates. S. aureus biofilm formation on implanted medical devices can cause local and systemic infections due to the dispersion of cells from the biofilms. Usually, conventional antibiotic treatments are not effective against biofilm-related infections, and there is no effective treatment other than removing the contaminated devices. Therefore, the development of new therapeutic agents to combat biofilm-related infections is urgently needed. We conducted high-throughput screening of S. aureus biofilm inhibitors and obtained a small compound, JBD1. JBD1 strongly inhibits biofilm formation of S. aureus, including methicillin-resistant strains. In addition, JBD1 activated the respiratory activity of S. aureus cells and increased the sensitivity to aminoglycosides. Furthermore, it was shown that the metabolic profile of S. aureus was significantly altered in the presence of JBD1 and that metabolic remodeling was induced. Surprisingly, these JBD1-induced phenotypes were blocked by adding an excess amount of the electron carrier menaquinone to suppress respiratory activation. These results indicate that JBD1 induces biofilm inhibition and metabolic remodeling through respiratory activation. This study demonstrates that compounds that enhance the respiratory activity of S. aureus may be potential leads in the development of therapeutic agents for chronic S. aureus-biofilm-related infections. IMPORTANCE Chronic infections caused by Staphylococcus aureus are characterized by biofilm formation, suggesting that methods to control biofilm formation may be of therapeutic value. The small compound JBD1 showed biofilm inhibitory activity and increased sensitivity to aminoglycosides and respiratory activity of S. aureus. Additionally, transcriptomic and metabolomic analyses demonstrated that JBD1 induced metabolic remodeling. All JBD1-induced phenotypes were suppressed by the extracellular addition of an excess amount of menaquinone, indicating that JBD1-mediated respiratory stimulation inhibits biofilm formation and triggers metabolic remodeling in S. aureus. These findings suggest a strategy for developing new therapeutic agents for chronic S. aureus infections.

Organoid-derived intestinal epithelial cells are a suitable model for preclinical toxicology and pharmacokinetic studies.

Intestinal organoids are physiologically relevant tools used for cellular models. However, the suitability of organoids to examine biological functions over existing established cell lines lacks sufficient evidence. Cytochrome P450 3A4 (CYP3A4) induction by pregnane X receptor ligands, glucose uptake via sodium/glucose cotransporter 1, and microsomal triglyceride transfer protein-dependent ApoB-48 secretion, which are critical for human intestinal metabolism, were observed in organoid-derived two-dimensional cells but little in Caco-2 cells. CYP3A4 induction evaluation involved a simplified method of establishing organoids that constitutively expressed a reporter gene. Compound screening identified several anticancer drugs with selective activities toward Caco-2 cells, highlighting their characteristics as cancer cells. Another compound screening revealed a decline in N-(4-hydroxyphenyl)retinamide cytotoxicity upon rifampicin treatment in organoid-derived cells, under CYP3A4-induced conditions. This study shows that organoid-derived intestinal epithelial cells (IECs) possess similar physiological properties as intestinal epithelium and can serve as tools for enhancing the prediction of biological activity in humans.

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti.

BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.

Application of Acoustic Ejection MS System to High-Throughput Screening for SARS-CoV-2 3CL Protease Inhibitors.

MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.

Prolyl isomerase Pin1 plays an essential role in SARS-CoV-2 proliferation, indicating its possibility as a novel therapeutic target.

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC50 below 5 µM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)-a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1-similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19.

Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells.

In this study, we present a live-cell-based fluorometric coupled assay system to identify the compounds that can regulate the targeted metabolic pathways in live cells. The assay is established through targeting specific metabolic pathways and using "input" and "output" metabolite pairs. The changes in the extracellular output that are generated and released into the extracellular media from the input are assessed as the activity of the pathway. The screening for the glycolytic pathway and amino acid metabolism reveals the activities of the present drugs, 6-BIO and regorafenib, that regulate the metabolic fate of tumor cells.

Pathological Role of Pin1 in the Development of DSS-Induced Colitis.

Inflammatory bowel diseases (IBDs) are serious disorders of which the etiologies are not, as yet, fully understood. In this study, Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein was shown to be dramatically upregulated in the colons of dextran sodium sulfate (DSS)-induced ulcerative colitis model mice. Interestingly, Pin1 knockout (KO) mice exhibited significant attenuation of DSS-induced colitis compared to wild-type (WT) mice, based on various parameters, including body weight, colon length, microscopic observation of the intestinal mucosa, inflammatory cytokine expression, and cleaved caspase-3. In addition, a role of Pin1 in inflammation was suggested because the percentage of M1-type macrophages in the colon was decreased in the Pin1 KO mice while that of M2-type macrophages was increased. Moreover, Pin1 KO mice showed downregulation of both Il17 and Il23a expression in the colon, both of which have been implicated in the development of colitis. Finally, oral administration of Pin1 inhibitor partially but significantly prevented DSS-induced colitis in mice, raising the possibility of Pin1 inhibitors serving as therapeutic agents for IBD.

Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library.

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.

Non-steroidal inhibitors of Drosophila melanogaster steroidogenic glutathione S-transferase Noppera-bo.

Insect growth regulators (IGRs) can be developed by elucidating the molecular mechanisms of insect-specific biological events. Because insect molting, and metamorphosis are controlled by ecdysteroids, their biosynthetic pathways can serve as targets for IGR development. The glutathione S-transferase Noppera-bo (Nobo), which is conserved in dipteran and lepidopteran species, plays an essential role in ecdysteroid biosynthesis. Our previous study using 17ß-estradiol as a molecular probe revealed that Asp113 of Drosophila melanogaster Nobo (DmNobo) is essential for its biological function. However, to develop IGRs with a greater Nobo inhibitory activity than 17ß-estradiol, further structural information is warranted. Here, we report five novel non-steroidal DmNobo inhibitors. Analysis of crystal structures of complexes revealed that DmNobo binds these inhibitors in an Asp113-independent manner. Among amino acid residues at the substrate-recognition site, conformation of conserved Phe39 was dynamically altered upon inhibitor binding. Therefore, these inhibitors can serve as seed compounds for IGR development.

Identification of novel compounds that inhibit SnRK2 kinase activity by high-throughput screening.

Abscisic acid (ABA) is a major phytohormone that regulates abiotic stress responses and development. SNF1-rerated protein kinase 2 (SnRK2) is a key regulator of ABA signaling. To isolate compounds which directly affect SnRK2 activity, we optimized a fluorescence-based system for high-throughput screening (HTS) of SnRK2 kinase regulators. Using this system, we screened a chemical library consisting of 16,000 compounds and identified ten compounds (INH1-10) as potential SnRK2 inhibitors. Further characterization of these compounds by in vitro phosphorylation assays confirmed that three of the ten compounds were SnRK2-specific kinase inhibitors. In contrast, seven of ten compounds inhibited ABA-responsive gene expression in Arabidopsis cells. From these results, INH1 was identified as a SnRK2-specific inhibitor in vitro and in vivo. We propose that INH1 could be a lead compound of chemical tools for studying ABA responses in various plant species.

Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library.

Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo.

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

Identification of Potent In Vivo Autotaxin Inhibitors that Bind to Both Hydrophobic Pockets and Channels in the Catalytic Domain.

Autotaxin (ATX, also known as ENPP2) is a predominant lysophosphatidic acid (LPA)-producing enzyme in the body, and LPA regulates various physiological functions, such as angiogenesis and wound healing, as well as pathological functions, including proliferation, metastasis, and fibrosis, via specific LPA receptors. Therefore, the ATX-LPA axis is a promising therapeutic target for dozens of diseases, including cancers, pulmonary and liver fibroses, and neuropathic pain. Previous structural studies revealed that the catalytic domain of ATX has a hydrophobic pocket and a hydrophobic channel; these serve to recognize the substrate, lysophosphatidylcholine (LPC), and deliver generated LPA to LPA receptors on the plasma membrane. Most reported ATX inhibitors bind to either the hydrophobic pocket or the hydrophobic channel. Herein, we present a unique ATX inhibitor that binds mainly to the hydrophobic pocket and also partly to the hydrophobic channel, inhibiting ATX activity with high potency and selectivity in vitro and in vivo. Notably, our inhibitor can rescue the cardia bifida (two hearts) phenotype in ATX-overexpressing zebrafish embryos.

Inhibitors of the protein-protein interaction between phosphorylated p62 and Keap1 attenuate chemoresistance in a human hepatocellular carcinoma cell line.

Resistance to anticancer agents has been an obstacle to developing therapeutics and reducing medical costs. Whereas sorafenib is used for the treatment of human hepatocellular carcinoma (HCC), resistance limits its efficacy. p62, a multifunctional protein, is overexpressed in several HCC cell lines, such as Huh-1 cells. Phosphorylated p62 (p-p62) inhibits the protein-protein interaction (PPI) between Keap1 and Nrf2, resulting in the Nrf2 overactivation that causes drug resistance. We have found a unique Nrf2 inactivator, named K67, that inhibited the PPI between Keap1 and p-p62 and attenuated sorafenib resistance in Huh-1 cells. Herein, we designed and synthesised novel K67 derivatives by modification of the substituent at the 4-position of the two benzenesulfonyl groups of K67. Although these new derivatives inhibited the Keap1-p-p62 PPI to a level comparable to or weaker than that of K67, the isopropoxy derivative enhanced the sensitivity of Huh-1 cells to sorafenib to a greater extent than K67 without any influence on the viability of Huh-7 cells, which is a non-resistant HCC cell line. The isopropoxy derivative also increased the sensitivity of Huh-1 cells to regorafenib, which suggests that this derivative has the potential to be used as an agent to overcome chemoresistance based on Nrf2 inactivation.