Anticalcification effects of DS-1211 in pseudoxanthoma elasticum mouse models and the role of tissue-nonspecific alkaline phosphatase in ABCC6-deficient ectopic calcification.

Pseudoxanthoma elasticum (PXE) is a multisystem, genetic, ectopic mineralization disorder with no effective treatment. Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). This study evaluated the anticalcification effects of DS-1211, an orally administered, potent, and highly selective small molecule TNAP inhibitor, in mouse models of PXE. Calcium content in vibrissae was measured in KK/HlJ and ABCC6-/- mice after DS-1211 administration for 13-14 weeks. Pharmacokinetic and pharmacodynamic effects of DS-1211 were evaluated, including plasma alkaline phosphatase (ALP) activity and biomarker changes in PPi and pyridoxal-phosphate (PLP). Anticalcification effects of DS-1211 through TNAP inhibition were further evaluated in ABCC6-/- mice with genetically reduced TNAP activity, ABCC6-/-/TNAP+/+ and ABCC6-/-/TNAP+/-. In KK/HlJ and ABCC6-/- mouse models, DS-1211 inhibited plasma ALP activity in a dose-dependent manner and prevented progression of ectopic calcification compared with vehicle-treated mice. Plasma PPi and PLP increased dose-dependently with DS-1211 in ABCC6-/- mice. Mice with ABCC6-/-/TNAP+/- phenotype had significantly less calcification and higher plasma PPi and PLP than ABCC6-/-/TNAP+/+ mice. TNAP plays an active role in pathomechanistic pathways of dysregulated calcification, demonstrated by reduced ectopic calcification in mice with lower TNAP activity. DS-1211 may be a potential therapeutic drug for PXE.

In Vitro and In Vivo Pharmacological Profiles of DS-1211, a Novel Potent, Selective, and Orally Bioavailable Tissue-Nonspecific Alkaline Phosphatase Inhibitor.

Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). DS-1211 is a potent and selective novel small molecule TNAP inhibitor with well-characterized pharmacokinetics (PKs) in rodent and monkey. Herein, we report a comprehensive summary of studies establishing the pharmaceutical profile of DS-1211. In vitro studies characterized the mode of inhibition and inhibitory effects of DS-1211 on three human alkaline phosphatase (ALP) isozymes-TNAP, human intestinal ALP, human placental ALP-and on ALP activity across species in mouse, monkey, and human plasma. In vivo PK and pharmacodynamic (PD) effects of a single oral dose of DS-1211 in mice and monkeys were evaluated, including biomarker changes in PPi and pyridoxal 5'-phosphate (PLP). Oral bioavailability (BA) was determined through administration of DS-1211 at a 0.3-mg/kg dose in monkeys. In vitro experiments demonstrated DS-1211 inhibited ALP activity through an uncompetitive mode of action. DS-1211 exhibited TNAP selectivity and potent inhibition of TNAP across species. In vivo studies in mice and monkeys after single oral administration of DS-1211 showed linear PKs, with dose-dependent inhibition of ALP activity and increases in plasma PPi and PLP. Inhibitory effects of DS-1211 were consistent in both mouse and monkey. Mean absolute oral BA was 73.9%. Overall, in vitro and in vivo studies showed DS-1211 is a potent and selective TNAP inhibitor across species. Further in vivo pharmacology studies in ectopic calcification animal models and clinical investigations of DS-1211 in patient populations are warranted. © 2022 Daiichi Sankyo, Inc. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Lead optimization of pyrido[2,3-d][1]benzazepin-6-one derivatives leading to the discovery of a potent, selective, and orally available human parathyroid hormone receptor 1 (hPTHR1) antagonist (DS69910557).

We report the discovery of a series of novel zwitterionic hPTHR1 antagonists. Optimization of lead compound 2 led to 4-[[1-[4-(2,9-dichloro-5,5-dimethyl-6-oxo-pyrido[2,3-d][1]benzazepin-7-yl)phenyl]-3-fluoro-azetidin-3-yl]methylamino]cyclohexanecarboxylic acid (19e, DS69910557), a compound with excellent potency and selectivity over activity at the human ether-a-go-go-related-gene (hERG) channel. Compound 19e demonstrated in vivo potency to decrease the plasma calcium concentration in rats upon oral administration. 2022 Elsevier Ltd. All rights reserved.

Development of 1,8-naphthalimide dyes for rapid imaging of subcellular compartments in plants.

Here, we report the installation of 1,8-naphthalimide dyes in live cell imaging of plants. We developed a series of 1,8-naphthalimide-based probes that illuminate different subcellular compartments by altering their spectral characteristics. Simple infiltration of the probes into leaves rapidly visualized the structure of chloroplasts or the vacuole. We further demonstrated that these probes are applicable to monitor the organelle behaviors in an autophagy pathway.

GFS9 Affects Piecemeal Autophagy of Plastids in Young Seedlings of Arabidopsis thaliana.

Chloroplasts, and plastids in general, contain abundant protein pools that can be major sources of carbon and nitrogen for recycling. We have previously shown that chloroplasts are partially and sequentially degraded by piecemeal autophagy via the Rubisco-containing body. This degradation occurs during plant development and in response to the environment; however, little is known about the fundamental underlying mechanisms. To discover the mechanisms of piecemeal autophagy of chloroplasts/plastids, we conducted a forward-genetics screen following ethyl-methanesulfonate mutagenesis of an Arabidopsis (Arabidopsis thaliana) transgenic line expressing chloroplast-targeted green fluorescent protein (CT-GFP). This screen allowed us to isolate a mutant, gfs9-5, which hyperaccumulated cytoplasmic bodies labeled with CT-GFP of up to 1.0 µm in diameter in the young seedlings. We termed these structures plastid bodies (PBs). The mutant was defective in a membrane-trafficking factor, green fluorescent seed 9 (GFS9), and PB accumulation in gfs9-5 was promoted by darkness and nutrient deficiency. Transmission electron microscopy indicated that gfs9-5 hyperaccumulated structures corresponding to autophagosomes and PBs. gfs9-5 hyperaccumulated membrane-bound endogenous ATG8 proteins, transgenic yellow fluorescent protein (YFP)-ATG8e proteins and autophagosome-like structures labeled with YFP-ATG8e. The YFP-ATG8e signal was associated with the surface of plastids and their protrusions in gfs9-5. Double mutants of gfs9 and autophagy-defective 5 did not accumulate PBs. In gfs9-5, the YFP-ATG8e proteins and PBs could be delivered to the vacuole and autophagic flux was increased. We discuss a possible connection between GFS9 and autophagy and propose a potential use of gfs9-5 as a new tool to study piecemeal plastid autophagy.

Discovery of a Novel Class of ERRα Agonists.

A novel class of estrogen-related receptor α (ERRα) agonists has been discovered. A structure-activity relationship study of high-throughput screening hits 1 and 2 led to the discovery of benzimidazole 3d (DS20362725) and acetophenone analogue 5c (DS45500853). The X-ray crystal structure of the ERRα ligand-binding domain in complex with 5c and PGC-1α coactivator peptide revealed conformational changes in the ligand-binding pocket to accommodate 5c and the key interaction between the protein and ligand. Since both analogues avoided PPARγ transcriptional activity, they can be useful tool compounds for investigating biological ERRα functions.

Mitophagy in plants.

Mitochondria play a central role in primary metabolism in plants as well as in heterotrophic eukaryotes. Plants must control the quality and number of mitochondria in response to a changing environment, across cell types and developmental stages. Mitophagy is defined as the degradation of mitochondria by autophagy, an evolutionarily conserved system for the removal and recycling of intracellular components. Recent studies have highlighted the importance of mitophagy in plant stress responses. This review article summarizes our current knowledge of plant mitophagy and discusses the underlying mechanisms. In plants, chloroplasts cooperate with mitochondria for energy production, and autophagy also targets chloroplasts through a process known as chlorophagy. Advances in plant autophagy studies now allow a comparative analysis of the autophagic turnover of mitochondria and chloroplasts, via the selective degradation of their soluble proteins, fragments, or entire organelles.

RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants.

Inorganic phosphate (Pi) and nitrogen (N) are essential nutrients for plant growth. We found that a five-fold oversupply of nitrate rescues Arabidopsis (Arabidopsis thaliana) plants from Pi-starvation stress. Analyses of transgenic plants that overexpressed GFP-AUTOPHAGY8 showed that an oversupply of nitrate induced autophagy flux under Pi-depleted conditions. Expression of DIN6 and DIN10, the carbon (C) starvation-responsive genes, was upregulated when nitrate was oversupplied under Pi starvation, which suggested that the plants recognized the oversupply of nitrate as C starvation stress because of the reduction in the C/N ratio. Indeed, formation of Rubisco-containing bodies (RCBs), which contain chloroplast stroma and are induced by C starvation, was enhanced when nitrate was oversupplied under Pi starvation. Moreover, autophagy-deficient mutants did not release Pi (unlike wild-type plants), exhibited no RCB accumulation inside vacuoles, and were hypersensitive to Pi starvation, indicating that RCB-mediated chlorophagy is involved in Pi starvation tolerance. Thus, our results showed that the Arabidopsis response to Pi starvation is closely linked with N and C availability and that autophagy is a key factor that controls plant growth under Pi starvation.

Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii.

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

Chlorophagy does not require PLANT U-BOX4-mediated ubiquitination.

Chloroplasts and mitochondria serve as intracellular energy production sites that are powered by the electron transport chain in their membranes. These organelles constantly accumulate damage, as their energetic reactions generate reactive oxygen species. To prevent the accumulation of damaged organelles and perturbation of cellular homeostasis, eukaryotic cells must remove damaged mitochondria and chloroplasts. Autophagy is the main route by which organelles are degraded. A type of mitochondrion-targeted autophagy known as mitophagy removes damaged mitochondria in mammalian cells; dysfunctional mitochondria that lose their membrane potential are marked by protein ubiquitination, becoming targets of selective mitophagy. Studies of the quality control system for chloroplasts in plants revealed the involvement of both autophagy and ubiquitination in the degradation of damaged chloroplasts. We recently assessed the relationship between chloroplast-associated ubiquitination mediated by PLANT U-BOX4 (PUB4) and chloroplast-targeted autophagy (chlorophagy) in the turnover of oxidatively damaged chloroplasts. Multiple assays using an Arabidopsis thaliana mutant revealed that PUB4-associated ubiquitination is dispensable for the induction of chlorophagy. Here, we describe the parallel functions of PUB4 and chlorophagy in chloroplast turnover and plant growth.

Autophagy Contributes to the Quality Control of Leaf Mitochondria.

In autophagy, cytoplasmic components of eukaryotic cells are transported to lysosomes or the vacuole for degradation. Autophagy is involved in plant tolerance to the photooxidative stress caused by ultraviolet B (UVB) radiation, but its roles in plant adaptation to UVB damage have not been fully elucidated. Here, we characterized organellar behavior in UVB-damaged Arabidopsis (Arabidopsis thaliana) leaves and observed the occurrence of autophagic elimination of dysfunctional mitochondria, a process termed mitophagy. Notably, Arabidopsis plants blocked in autophagy displayed increased leaf chlorosis after a 1-h UVB exposure compared to wild-type plants. We visualized autophagosomes by labeling with a fluorescent protein-tagged autophagosome marker, AUTOPHAGY8 (ATG8), and found that a 1-h UVB treatment led to increased formation of autophagosomes and the active transport of mitochondria into the central vacuole. In atg mutant plants, the mitochondrial population increased in UVB-damaged leaves due to the cytoplasmic accumulation of fragmented, depolarized mitochondria. Furthermore, we observed that autophagy was involved in the removal of depolarized mitochondria when mitochondrial function was disrupted by mutation of the FRIENDLY gene, which is required for proper mitochondrial distribution. Therefore, autophagy of mitochondria functions in response to mitochondrion-specific dysfunction as well as UVB damage. Together, these results indicate that autophagy is centrally involved in mitochondrial quality control in Arabidopsis leaves.

Development of potent inhibitors for strigolactone receptor DWARF 14.

Strigolactones (SLs) are plant hormones that suppress shoot branching through perception by their receptor protein DWARF 14 (D14). The artificial regulation of SL signaling has been considered a potent agricultural technique because plant architecture is strongly related to crop yield. In this communication, we describe the development of a small-molecule D14 inhibitor that functions at sub-micromolar levels. This potent inhibitor may be a lead compound for a first-in-class plant growth regulator.

Chloroplast Autophagy and Ubiquitination Combine to Manage Oxidative Damage and Starvation Responses.

Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS1 and PLANT U-BOX4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis (Arabidopsis thaliana). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of reactive oxygen species accumulation, and adaptation to starvation.

Discovery of novel, potent, and orally bioavailable pyrido[2,3-d][1]benzazepin-6-one antagonists for parathyroid hormone receptor 1.

Structural modification of a 1,4-benzodiazepin-2-one-based PTHR1 antagonist 5, a novel type of PTHR1 antagonist previously synthesized in our laboratories, yielded compound 10, which had better chemical stability than compound 5. Successive optimization of the lead 10 improved aqueous solubility, metabolic stability, and animal pharmacokinetics, culminating in the identification of DS37571084 (12). Our study paves the way for the discovery of novel and orally bioavailable PTHR1 antagonists.

Autophagy mitigates high-temperature injury in pollen development of Arabidopsis thaliana.

Autophagy is one of the cellular processes that break down cellular components during senescence, starvation, and stress. The susceptibility of plant pollen development to high-temperature (HT) stress is well known, but the involvement of autophagy in HT injury is yet to be clarified. Here, we found that following transfer to 30 °C, all autophagy-deficient (atg) mutants (atg2-1, 5-1, 7-2, and 10-1) of Arabidopsis thaliana tested displayed visibly impaired pollen development and anther dehiscence. HT-induced male sterility significantly increased in the atg mutants, but the degree of HT-induced obstacles did not change between the wild type (WT) and mutants from the seedling stage to the bolting stage. Cytological analyses showed that 30 °C promoted autophagy and autolysosome formation in both anther wall cells and microspores in developing anthers of WT, but the atg5-1 mutant did not show completion of tapetum degeneration and microspore maturation. HT upregulated hydrogen peroxide and dehydroascorbate reductase 1 production in both WT and atg5-1 anthers, but the basal levels were already higher in the mutant. HT repressed expression of UNDEAD and its regulator MYB80, which are required for tapetal programmed cell death (PCD) for proper pollen development. Taken together, our results suggest that autophagy functions in tapetum degeneration and pollen development during HT-caused tapetal PCD abortion.

Discovery of novel PTHR1 antagonists: Design, synthesis, and structure activity relationships.

The discovery and optimization of a novel series of PTHR1 antagonists are described. Starting from known PTHR1 antagonists, we identified more potent 1,4-benzodiazepin-2-one derivatives by means of a scaffold-hopping approach. The representative compound 23 (DS08210767) exhibited nanomolar-level PTHR1 antagonist activity and potential oral bioavailability in a pharmacokinetic study.