Detection of FIP1L1-PDGFRA fusion gene-positive cells in the skin lesion of a patient with hypereosinophilic syndrome.

Hypereosinophilic syndrome (HES) is a heterogeneous group of diseases, characterized by persistent hypereosinophilia and end-organ damage. The FIP1L1-PDGFRA (F/P) fusion gene is found in 3-25% of patients with HES and is an oncogenic driver of myeloid neoplasms with clonal eosinophilia. Although cutaneous symptoms are the most common type of symptom in patients who have F/P fusion gene-positive HES (F/P HES), histological reports are limited. We herein present the case of a 78-year-old man with erythematous macules and severe pruritus on his trunk and extremities. Laboratory investigations revealed marked eosinophilia and elevated serum vitamin B12. A histological examination showed massive infiltration of eosinophils and mast cells around the vessels in the upper dermis. Fluorescence in situ hybridization revealed F/P fusion genes in nuclei in the peripheral blood and the skin lesion. The patient was diagnosed with F/P HES, and showed an excellent clinical and haematological response to imatinib.

A reconstituted depolarization-induced Ca2+ release platform for validation of skeletal muscle disease mutations and drug discovery.

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs through the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including ß1a, Stac3 adaptor protein, and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for the validation of mutations and drug discovery for skeletal muscle diseases.

Development of a water-soluble ryanodine receptor 1 inhibitor.

Ryanodine receptor 1 (RyR1) is a Ca2+-release channel expressed on the sarcoplasmic reticulum (SR) membrane. RyR1 mediates release of Ca2+ from the SR to the cytoplasm to induce muscle contraction, and mutations associated with overactivation of RyR1 cause lethal muscle diseases. Dantrolene sodium salt (dantrolene Na) is the only approved RyR inhibitor to treat malignant hyperthermia patients with RyR1 mutations, but is poorly water-soluble. Our group recently developed a bioassay system and used it to identify quinoline derivatives such as 1 as potent RyR1 inhibitors. In the present study, we focused on modification of these inhibitors with the aim of increasing their water-solubility. First, we tried reducing the hydrophobicity by shortening the N-octyl chain at the quinolone ring of 1; the N-heptyl compound retained RyR1-inhibitory activity, but the N-hexyl compound showed decreased activity. Next, we introduced a more hydrophilic azaquinolone ring in place of quinolone; in this case, only the N-octyl compound retained activity. The sodium salt of N-octyl azaquinolone 7 showed similar inhibitory activity to dantrolene Na with approximately 1,000-fold greater solubility in saline.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

LRBA is essential for urinary concentration and body water homeostasis.

Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.

A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Development of Androgen-Antagonistic Coumarinamides with a Unique Aromatic Folded Pharmacophore.

First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide (2a) and bicalutamide (3), are effective for most prostate cancer patients, but resistance often appears after several years due to the mutation of AR. Second-generation AR antagonists are effective against some of these castration-resistant prostate cancers, but their structural variety is still limited. In this study, we designed and synthesized 4-methyl-7-(N-alkyl-arylcarboxamido)coumarins as AR antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent SC-3 cells. Coumarinamides with a secondary amide bond did not show inhibitory activity, but their N-methylated derivatives exhibited AR-antagonistic activity. Especially, 19b and 31b were more potent than the lead compound 7b, which was comparable to hydroxyflutamide (2b). Conformational analysis showed that the inactive coumarinamides with a secondary amide bond have an extended structure with a trans-amide bond, while the active N-methylated coumarinamides have a folded structure with a cis-amide bond, in which the two aromatic rings are placed face-to-face. Docking study suggested that this folded structure is important for binding to AR. Selected coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR, and they had weak progesterone receptor (PR)-antagonistic activity. The folded coumarinamide structure appears to be a unique pharmacophore, different from those of conventional AR antagonists.

Development of Boron-Cluster-Based Progesterone Receptor Antagonists Bearing a Pentafluorosulfanyl (SF5) Group.

The progesterone receptor (PR) plays an important role in various physiological processes, especially in the female reproductive system, and abnormalities of PR function are associated with several diseases, including some types of cancer. Non-steroidal PR ligands are of interest as candidate drugs for treatment of PR-related diseases without the serious adverse effects that may be caused by steroidal ligands. For the development of non-steroidal PR ligands, both a hydrophobic backbone and a polar functional group corresponding to the 3-carbonyl group of progesterone, which interacts with Gln725 and Arg766 of the PR-ligand binding domain, are critically important. We previously showed that carborane is a useful hydrophobic pharmacophore for PR antagonists, and in this work, we introduced the pentafluorosulfanyl (SF5) group as a novel polar functional group of carborane-based non-steroidal PR antagonists. All the synthesized SF5-containing carborane derivatives exhibited PR-antagonistic activity at micromolar or submicromolar concentration. Among them, compounds 11 are potent progesterone antagonists with submicromolar IC50 values.

Structural development of a type-1 ryanodine receptor (RyR1) Ca2+-release channel inhibitor guided by endoplasmic reticulum Ca2+ assay.

Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum (SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca2+ from the SR. Genetic mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and central core diseases, in which over-activation of RyR1 causes leakage of Ca2+ from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca2+ in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor. Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound. Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity. Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may be promising leads for clinically applicable RyR1 channel inhibitors.

Design, Synthesis and Biological Evaluation of Novel Nonsteroidal Progesterone Receptor Antagonists Based on Phenylamino-1,3,5-triazine Scaffold.

We report here the development of phenylamino-1,3,5-triazine derivatives as novel nonsteroidal progesterone receptor (PR) antagonists. PR plays key roles in various physiological systems, including the female reproductive system, and PR antagonists are promising candidates for clinical treatment of multiple diseases. By using the phenylamino-1,3,5-triazine scaffold as a template structure, we designed and synthesized a series of 4-cyanophenylamino-1,3,5-triazine derivatives. The synthesized compounds exhibited PR antagonistic activity, and among them, compound 12n was the most potent (IC50 = 0.30 µM); it also showed significant binding affinity to the PR ligand-binding domain. Docking simulation supported the design rationale of the compounds. Our results suggest that the phenylamino-1,3,5-triazine scaffold is a versatile template for development of nonsteroidal PR antagonists and that the developed compounds are promising lead compounds for further structural development of nonsteroidal PR antagonists.

Recent Advances in Chemical Tools for the Regulation and Study of Protein Lysine Methyltransferases.

Protein lysine methyltransferases (PKMTs) are epigenetic regulators that modulate gene transcription and physiological functions by catalyzing the post-translational methylation of specific lysine residues of substrate proteins, such as histones. They are considered to be candidate drugs for the treatment of various diseases, including acute myeloid leukemia, and in the past decade, potent and selective inhibitors of individual PKMTs have been developed. Some are currently under clinical trial. In this review, we will focus on some breakthrough PKMT inhibitors, and discuss chemistry-based methods available for elucidation of the physiological functions of PKMTs and methylated proteins.

Novel Nonsteroidal Progesterone Receptor (PR) Antagonists with a Phenanthridinone Skeleton.

The progesterone receptor (PR) plays an important role in various physiological systems, including female reproduction and the central nervous system, and PR antagonists are thought to be effective not only as contraceptive agents and abortifacients but also in the treatment of various diseases, including hormone-dependent cancers and endometriosis. Here, we identified phenanthridin-6(5H)-one derivatives as a new class of PR antagonists and investigated their structure-activity relationships. Among the synthesized compounds, 37, 40, and 46 exhibited very potent PR antagonistic activity with high selectivity for PR over other nuclear receptors. These compounds are structurally distinct from other nonsteroidal PR antagonists, including cyanoaryl derivatives, and should be useful for further studies of the clinical utility of PR antagonists.

Primary meningeal myxoid liposarcoma with aggressive behavior after recurrence: case report.

Although liposarcomas are the most common soft tissue sarcomas, their intracranial variants are extremely rare. Here, we present a case of a primary intracranial myxoid liposarcoma in a 23-year-old Japanese man who presented with generalized seizures and a mass in the left frontal lobe. The tumor was totally removed, and histological analyses pointed to liposarcoma. Thirteen years after his initial treatment, the patient presented with right-side weakness and local recurrence of tumor was discovered. Histology from the second resection confirmed the diagnosis of myxoid liposarcoma. Shortly after the second resection, progressive, new intracranial lesions were observed and despite a third resection, extensive intracerebral invasion by the tumor proved fatal. The histological features of myxoid liposarcoma were essentially similar with each recurrence, but the aggressive tumor behavior after the second operation did not align with expectations based on histological classification.

Development of biotin-retinoid conjugates as chemical probes for analysis of retinoid function.

Herein, we report the rational design, synthesis and biological evaluation of conjugates consisting of the synthetic retinoid Am580 and biotin connected via a linker moiety. We found that the linking substructure between the retinoid part and the biotin part is critical for retaining the biological activity. Conjugate 4 with a shorter linker showed similar potency to endogenous retinoid ATRA (1) and the parent compound Am580 (2) for neural differentiation of mouse embryotic carcinoma P19 cells, and showed the same pattern of induction of gene expression. It is expected to be useful as a probe for investigations of retinoid function. The design rationale and structure-activity relationship of the linker moiety are expected to be helpful for developing biotin conjugates of other nuclear receptor ligands.

AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations.

Novel Non-steroidal Progesterone Receptor Ligands Based on m-Carborane Containing a Secondary Alcohol: Effect of Chirality on Ligand Activity.

The progesterone receptor (PR) controls various physiological processes, including the female reproductive system, and nonsteroidal PR ligands are considered to be drug candidates for treatment of various diseases without significant adverse effects. Here, we designed and synthesized m-carborane-based secondary alcohols and investigated their PR-ligand activity. All the synthesized alcohols exhibited PR-antagonistic activity at subnanomolar concentration. Among them, alcohols having a small alkyl side chain and a 4-cyanophenyl group also exhibited PR-agonistic activity in a relatively high concentration range. Optical resolution of secondary alcohols having a methyl side chain was performed, and the PR-ligand activity and PR-binding affinity of the purified enantiomers were examined. The chirality of the secondary alcohol appears to have a more significant influence on PR-agonistic activity than on antagonistic activity.

Development of N-(4-Phenoxyphenyl)benzenesulfonamide Derivatives as Novel Nonsteroidal Progesterone Receptor Antagonists.

We report here development of N-(4-phenoxyphenyl)benzenesulfonamide derivatives as a novel class of nonsteroidal progesterone receptor (PR) antagonists. PR plays key roles in various physiological systems, including the female reproductive system, and PR antagonists are candidates for clinical treatment of multiple diseases, including uterine leiomyoma, endometriosis, breast cancer, and some psychiatric disorders. We found that the benzenesulfonanilide skeleton functions as a novel scaffold for PR antagonists, and we adopted 3-chlorobenzenesulfonyl derivative 20a as a lead compound for structural development. Among the synthesized compounds, 3-trifluoromethyl derivative 32 exhibited the most potent PR-antagonistic activity, with high binding affinity for PR and selectivity over androgen receptor (AR). It is structurally distinct from other nonsteroidal PR antagonists, including cyanopyrrole derivatives, and further modification is expected to afford novel selective PR modulators.

Development of 6-arylcoumarins as nonsteroidal progesterone antagonists. Structure-activity relationships and fluorescence properties.

Progesterone is involved in multiple physiological processes, including female reproduction, via binding to the progesterone receptor (PR). We have developed 6-arylcoumarins such as 5 and 6 as non-steroidal PR antagonists with receptor-binding-dependent fluorescence. In this study, we investigated the structure-activity relationships and fluorescence properties of coumarin derivatives bearing a heterocyclic aromatic moiety. Among these derivatives, 7c (IC50: 34nM) and 10b (IC50: 24nM) showed more potent PR-antagonistic activity than lead compounds 5 (IC50: 500nM) and 6 (IC50: 65nM) in alkaline phosphatase (AP) assay. Compound 9b showed solvent-dependent fluorescence intensity, exhibiting strong fluorescence in the presence of PR LBD only in buffer solution. On the other hand, 10b showed a solvent-dependent shift of the fluorescence maximum wavelength in the presence of PR LBD. These results indicate that 6-arylcoumarin will be a useful scaffold for PR antagonists and fluorescent probes targeting PR.