The ubiquinone synthesis pathway is a promising drug target for Chagas disease.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi). It was originally a Latin American endemic health problem, but now is expanding worldwide as a result of increasing migration. The currently available drugs for Chagas disease, benznidazole and nifurtimox, provoke severe adverse effects, and thus the development of new drugs is urgently required. Ubiquinone (UQ) is essential for respiratory chain and redox balance in trypanosomatid protozoans, therefore we aimed to provide evidence that inhibitors of the UQ biosynthesis have trypanocidal activities. In this study, inhibitors of the human COQ7, a key enzyme of the UQ synthesis, were tested for their trypanocidal activities because they were expected to cross-react and inhibit trypanosomal COQ7 due to their genetic homology. We show the trypanocidal activity of a newly found human COQ7 inhibitor, an oxazinoquinoline derivative. The structurally similar compounds were selected from the commercially available compounds by 2D and 3D ligand-based similarity searches. Among 38 compounds selected, 12 compounds with the oxazinoquinoline structure inhibited significantly the growth of epimastigotes of T. cruzi. The most effective 3 compounds also showed the significant antitrypanosomal activity against the mammalian stage of T. cruzi at lower concentrations than benznidazole, a commonly used drug today. We found that epimastigotes treated with the inhibitor contained reduced levels of UQ9. Further, the growth of epimastigotes treated with the inhibitors was partially rescued by UQ10 supplementation to the culture medium. These results suggest that the antitrypanosomal mechanism of the oxazinoquinoline derivatives results from inhibition of the trypanosomal UQ synthesis leading to a shortage of the UQ pool. Our data indicate that the UQ synthesis pathway of T. cruzi is a promising drug target for Chagas disease.

Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

Structural Basis of Activin Receptor-Like Kinase 2 (R206H) Inhibition by Bis-heteroaryl Pyrazole-Based Inhibitors for the Treatment of Fibrodysplasia Ossificans Progressiva Identified by the Integration of Ligand-Based and Structure-Based Drug Design Approaches.

Fibrodysplasia ossificans progressiva (FOP) is a rare but severe genetic disorder in which acute inflammation elicits progressive heterotopic ossification in the muscles, tendons, and ligaments. Classic FOP is caused by the R206H mutation in ALK2/ACVR1. While several activin receptor-like kinase 2 (ALK2) inhibitors were found to be efficacious in animal models of FOP, most of the ALK2 (R206H) inhibitors lacked sufficient oral bioavailability for efficacy. Previously, the synthesis of a series of novel bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitors that achieved both substantial potency and an improved ADMET profile was reported. In the present study, the detailed procedure of the in silico approach employed to identify the initial bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitor RK-59638 and the analysis of the ALK2 (R206H) RK-59638 complex structure to guide the synthetic optimization of the chemical series, obtaining RK-71807 showing improved potency and metabolic stability, were described. According to the initial in silico screening, the screening efficiencies and chemical diversity of the hit compounds of both ligand-based and structure-based methods were evaluated. Then, X-ray structures of ALK2 (R206H) and the inhibitors were analyzed to assess the structure-activity relationships of the synthesized compounds. The 3D-RISM analysis indicated the existence of the additional hydrogen bond via water molecules restricting the attachment point in the pyrazole scaffold. The quantum mechanics calculation of the newly determined ALK2 (R206H) RK-71807 complex structure using a fragment molecular orbital method and pair interaction energy decomposition analysis was employed to evaluate the interaction energies between the inhibitor and each of the amino acid residues and decompose them to electrostatic, exchange-repulsion, and charge transfer energies. The pattern of decomposed interaction energies was then compared to that formed by RK-59638 and LDN-193189 to investigate the structural basis of ALK2 (R206H) inhibition.

ALK2: A Therapeutic Target for Fibrodysplasia Ossificans Progressiva and Diffuse Intrinsic Pontine Glioma.

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are diseases that typically manifest in childhood and are associated with severely reduced life expectancy. However, there are currently no effective therapies for these diseases, which remain incurable. Activin receptor-like kinase-2 (ALK2), encoded by the ACVR1 gene, is a bone morphogenetic protein (BMP) type-I receptor subtype that plays an important physiological role in the development of bones, muscles, brain, and other organs. Constitutively active mutants of ALK2 have been identified as causative of FOP and involved in the tumorigenesis of DIPG owing to abnormal activation of BMP signaling, and therefore have emerged as promising treatment targets. Here, we describe these two diseases, along with the link to ALK2 signal transduction, and highlight potential ALK2 inhibitors that are under development to offer new hope for patients with FOP and DIPG.

Identification of small molecule inhibitors of human COQ7.

Given that the associated clinical manifestations of ubiquinone (UQ, or coenzyme Q) deficiency diseases are highly heterogeneous and complicated, effective new research tools for UQ homeostasis studies are awaited. We set out to develop human COQ7 inhibitors that interfere with UQ synthesis. Systematic structure-activity relationship development starting from a screening hit compound led to the identification of highly potent COQ7 inhibitors that did not disturb physiological cell growth of human normal culture cells. These new COQ7 inhibitors may serve as useful tools for studying the balance between UQ supplementation pathways: de novo UQ synthesis and extracellular UQ uptake.

Bis-Heteroaryl Pyrazoles: Identification of Orally Bioavailable Inhibitors of Activin Receptor-Like Kinase-2 (R206H).

Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.

Radiosynthesis and in vivo evaluation of two imidazopyridineacetamides, [(11)C]CB184 and [ (11)C]CB190, as a PET tracer for 18 kDa translocator protein: direct comparison with [ (11)C](R)-PK11195.

OBJECTIVE: We report synthesis of two carbon-11 labeled imidazopyridines TSPO ligands, [(11)C]CB184 and [(11)C]CB190, for PET imaging of inflammatory process along with neurodegeneration, ischemia or brain tumor. Biodistribution of these compounds was compared with that of [(11)C]CB148 and [(11)C](R)-PK11195. METHODS: Both [(11)C]CB184 and [(11)C]CB190 having (11)C-methoxyl group on an aromatic ring were readily prepared using [(11)C]methyl triflate. Biodistribution and metabolism of the compounds were examined with normal mice. An animal PET study using 6-hydroxydopamine treated rats as a model of neurodegeneration was pursued for proper estimation of feasibility of the radioligands to determine neuroinflammation process. RESULTS: [(11)C]CB184 and [(11)C]CB190 were obtained via O-methylation of corresponding desmethyl precursor using [(11)C]methyl triflate in radiochemical yield of 73 % (decay-corrected). In vivo validation as a TSPO radioligand was carried out using normal mice and lesioned rats. In mice, [(11)C]CB184 showed more uptake and specific binding than [(11)C]CB190. Metabolism studies showed that 36 % and 25 % of radioactivity in plasma remained unchanged 30 min after intravenous injection of [(11)C]CB184 and [(11)C]CB190, respectively. In the PET study using rats, lesioned side of the brain showed significantly higher uptake than contralateral side after i.v. injection of either [(11)C]CB184 or [(11)C](R)-PK11195. Indirect Logan plot analysis revealed distribution volume ratio (DVR) between the two sides which might indicate lesion-related elevation of TSPO binding. The DVR was 1.15 ± 0.10 for [(11)C](R)-PK11195 and was 1.15 ± 0.09 for [(11)C]CB184. CONCLUSION: The sensitivity to detect neuroinflammation activity was similar for [(11)C]CB184 and [(11)C](R)-PK11195.

Radiosynthesis and in vivo evaluation of N-[11C]methylated imidazopyridineacetamides as PET tracers for peripheral benzodiazepine receptors.

Imidazopyridineacetoamide 5-8, a series of novel and potentially selective peripheral benzodiazepine receptor (PBR) ligands with affinities comparable to those of known PBR ligands, was investigated. Radiosyntheses of [11C]5, 6, 7 or 8 was accomplished by N-methylation of the corresponding desmethyl precursors with [11C]methyl iodide in the presence of NaH in dimethylformamide (DMF), resulting in 25% to 77% radiochemical yield and specific activitiy of 20 to 150 MBq/nmol. Each of the labeled compounds was injected in ddY mice, and the radioactivity and weight of dissected peripheral organs and brain regions were measured. Organ distribution of [11C]7 was consistent with the known PBR distribution. Moreover, [11C]7 showed the best combination of brain uptake and PBR binding, leading to its high retention in the olfactory bulb and cerebellum, areas where PBR density is high in mouse brain. Coinjection of PK11195 or unlabeled 7 significantly reduced the brain uptake of [11C]7. These results suggest that [11C]7 could be a useful radioligand for positron emission tomography imaging of PBRs.

Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity.

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.

A novel inhibitor of 9-cis-epoxycarotenoid dioxygenase in abscisic acid biosynthesis in higher plants.

Abscisic acid (ABA) is a major regulator in the adaptation of plants to environmental stresses, plant growth, and development. In higher plants, the ABA biosynthesis pathway involves the oxidative cleavage of 9-cis-epoxycarotenoids, which may be the key regulatory step in the pathway catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). We developed a new inhibitor of ABA biosynthesis targeting NCED and named it abamine (ABA biosynthesis inhibitor with an amine moiety). Abamine is a competitive inhibitor of NCED, with a Ki of 38.8 microm. In 0.4 m mannitol solution, which mimics the effects of osmotic stress, abamine both inhibited stomatal closure in spinach (Spinacia oleracea) leaves, which was restored by coapplication of ABA, and increased luminescence intensity in transgenic Arabidopsis containing the RD29B promoter-luciferase fusion. The ABA content of plants in 0.4 m mannitol was increased approximately 16-fold as compared with that of controls, whereas 50 to 100 microm abamine inhibited about 50% of this ABA accumulation in both spinach leaves and Arabidopsis. Abamine-treated Arabidopsis was more sensitive to drought stress and showed a significant decrease in drought tolerance than untreated Arabidopsis. These results suggest that abamine is a novel ABA biosynthesis inhibitor that targets the enzyme catalyzing oxidative cleavage of 9-cis-epoxycarotenoids. To test the effect of abamine on plants other than Arabidopsis, it was applied to cress (Lepidium sativum) plants. Abamine enhanced radicle elongation in cress seeds, which could be due to a decrease in the ABA content of abamine-treated plants. Thus, it is possible to think that abamine should enable us to elucidate the functions of ABA in cells or plants and to find new mutants involved in ABA signaling.

The Influence of Chemical Genetics on Plant Science: Shedding Light on Functions and Mechanism of Action of Brassinosteroids Using Biosynthesis Inhibitors.

When exogenous chemicals allow rapid, conditional, reversible, selective, and dose-dependent control of biological functions, they act like conditional mutations, either inducing or suppressing the formation of a specific phenotype of interest. Exploration of the small molecules that induce the brassinosteroid (BR) deficient-like phenotype in Arabidopsis led us to identify brassinazole as the first candidate for a BR biosynthesis inhibitor. Brassinazole treatment reduced BR content in plant cells. Investigation of target site(s) of brassinazole revealed that the compound directly binds to the DWF4 protein, a cytochrome P450 monooxygenase that catalyzes 22-hydroxylation of the side chain of BRs. These results suggest that brassinazole is a BR biosynthesis inhibitor. There are currently at least two BR biosynthesis inhibitors that act like conditional mutations in BR biosynthesis. They allow the investigation of the functions of BRs in a variety of plant species. Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these inhibitors allowed the identification of new components working in BR signal transduction. This method has advantages over mutant screens using BR-deficient mutants as a background. Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological systems in plants and this would be a complement to classical biochemical and genetic methods.

N-benzylideneaniline and N-benzylaniline are potent inhibitors of lignostilbene-alpha,beta-dioxygenase, a key enzyme in oxidative cleavage of the central double bond of lignostilbene.

Lignostilbene-alpha,beta-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and beta-carotene 15,15'-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC50 = 0.3 microM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC50 = 10 microM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice.

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.

Triadimefon, a fungicidal triazole-type P450 inhibitor, induces brassinosteroid deficiency-like phenotypes in plants and binds to DWF4 protein in the brassinosteroid biosynthesis pathway.

Triadimefon (Bayleton), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14 alpha-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the phenotype was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.

A specific and potent inhibitor of brassinosteroid biosynthesis possessing a dioxolane ring.

Screening for brassinosteroid biosynthesis inhibitors was performed to find azole derivatives that induced dwarfism, to resemble brassinosteroid-deficient mutants in Arabidopsis, and which could be rescued by brassinosteroid. Through this screening experiment, propiconazole fungicide was selected as a likely inhibitor of brassinosteroid biosynthesis and, thus, propiconazole derivatives with optimized activity and selectivity were synthesized. The biological activity of these compounds was evaluated by examining cress stem elongation. Among the compounds tested, 2RS,4RS-1-[2-(4-trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole (12) showed the most potent capability to retard cress stem elongation in the light. The compound-induced hypocotyl dwarfism was restored by the coapplication of 10 nM brassinolide but not by 1 microM gibberellin. These results suggest that 12 should affect brassinosteroid biosynthesis. The potency and specificity of 12 were greater than those of brassinazole, a previously reported brassinosteroid biosynthesis inhibitor.

Design and synthesis of lignostilbene-alpha,beta-dioxygenase inhibitors.

Lignostilbene-alpha,beta-dioxygenase cleaves the olefinic double bond of phenolic stilbenes by a mechanism similar to that of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis. Several analogues of stilbene were designed and synthesized, and their efficacy as inhibitors of lignostilbene-alpha,beta-dioxygenase was examined. The compound (Z)-1-(4-hydroxyphenyl)-1-fluoro-2-phenylethene (2) was found to be a potent inhibitor of this enzyme with an IC(50) of 3 microM.