Neuroprotection and axon regeneration by novel low-molecular-weight compounds through the modification of DOCK3 conformation.

Dedicator of cytokinesis 3 (DOCK3) is an atypical member of the guanine nucleotide exchange factors (GEFs) and plays important roles in neurite outgrowth. DOCK3 forms a complex with Engulfment and cell motility protein 1 (Elmo1) and effectively activates Rac1 and actin dynamics. In this study, we screened 462,169 low-molecular-weight compounds and identified the hit compounds that stimulate the interaction between DOCK3 and Elmo1, and neurite outgrowth in vitro. Some of the derivatives from the hit compound stimulated neuroprotection and axon regeneration in a mouse model of optic nerve injury. Our findings suggest that the low-molecular-weight DOCK3 activators could be a potential therapeutic candidate for treating axonal injury and neurodegenerative diseases including glaucoma.

Patritumab deruxtecan (HER3-DXd), a novel HER3 directed antibody drug conjugate, exhibits in vitro activity against breast cancer cells expressing HER3 mutations with and without HER2 overexpression.

ErbB3 (HER3), a member of the HER family, is overexpressed in various cancers and plays an important role in cell proliferation and survival. Certain HER3 mutations have also been identified as oncogenic drivers, making them potential therapeutic targets. In the current study, antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3 directed antibody drug conjugate, was evaluated in tumor models with clinically reported HER3 mutations. MDA-MB-231, a HER3-negative human triple-negative breast cancer cell line, was transduced with lentiviral vectors encoding HER3 wild type (HER3WT), one of 11 HER3 mutations, or HER3 empty vector (HER3EV), in the presence/absence of HER2 overexpression. Targeted delivery of HER3-DXd was assessed using cell-surface binding, lysosomal trafficking, and cell-growth inhibition assays. HER3-DXd bound to the surface of HER3WT and mutant cells in a similar, concentration-dependent manner but not to HER3EV. HER3-DXd was translocated to the lysosome, where time- and concentration-dependent signals were observed in the HER3 mutant and HER3WT cells. HER3-DXd inhibited the growth of HER3WT and HER3 mutant cells. HER3-DXd activity was observed in the presence and absence of HER2 overexpression. These data suggest that HER3-DXd may have activity against tumors expressing wild type HER3 or clinically observed HER3 mutations, supporting further clinical evaluation.

Direct and quantitative analysis of altered metabolic flux distributions and cellular ATP production pathway in fumarate hydratase-diminished cells.

Fumarate hydratase (FH) is an enzyme in the tricarboxylic acid (TCA) cycle, biallelic loss-of-function mutations of which are associated with hereditary leiomyomatosis and renal cell cancer. However, how FH defect modulates intracellular metabolic fluxes in human cells has remained unclear. This study aimed to reveal metabolic flux alterations induced by reduced FH activity. We applied 13C metabolic flux analysis (13C-MFA) to an established cell line with diminished FH activity (FHdim) and parental HEK293 cells. FHdim cells showed reduced pyruvate import flux into mitochondria and subsequent TCA cycle fluxes. Interestingly, the diminished FH activity decreased FH flux only by about 20%, suggesting a very low need for FH to maintain the oxidative TCA cycle. Cellular ATP production from the TCA cycle was dominantly suppressed compared with that from glycolysis in FHdim cells. Consistently, FHdim cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FHdim cells we demonstrated that FH defect led to suppressed pyruvate import into mitochondria, followed by downregulated TCA cycle activity and altered ATP production pathway balance from the TCA cycle to glycolysis. We confirmed that 13C-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect.

Pharmacological profile of CS-3150, a novel, highly potent and selective non-steroidal mineralocorticoid receptor antagonist.

The present study was designed to characterize the pharmacological profile of CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist. In the radioligand-binding assay, CS-3150 inhibited (3)H-aldosterone binding to mineralocorticoid receptor with an IC50 value of 9.4nM, and its potency was superior to that of spironolactone and eplerenone, whose IC50s were 36 and 713nM, respectively. CS-3150 also showed at least 1000-fold higher selectivity for mineralocorticoid receptor over other steroid hormone receptors, glucocorticoid receptor, androgen receptor and progesterone receptor. In the reporter gene assay, CS-3150 inhibited aldosterone-induced transcriptional activation of human mineralocorticoid receptor with an IC50 value of 3.7nM, and its potency was superior to that of spironolactone and eplerenone, whose IC50s were 66 and 970nM, respectively. CS-3150 had no agonistic effect on mineralocorticoid receptor and did not show any antagonistic or agonistic effect on glucocorticoid receptor, androgen receptor and progesterone receptor even at the high concentration of 5µM. In adrenalectomized rats, single oral administration of CS-3150 suppressed aldosterone-induced decrease in urinary Na(+)/K(+) ratio, an index of in vivo mineralocorticoid receptor activation, and this suppressive effect was more potent and longer-lasting than that of spironolactone and eplerenone. Chronic treatment with CS-3150 inhibited blood pressure elevation induced by deoxycorticosterone acetate (DOCA)/salt-loading to rats, and this antihypertensive effect was more potent than that of spironolactone and eplerenone. These findings indicate that CS-3150 is a selective and highly potent mineralocorticoid receptor antagonist with long-lasting oral activity. This agent could be useful for the treatment of hypertension, cardiovascular and renal disorders.

[Syndrome of inappropriate secretion of ADH following chemoradiation therapy].

We report a 69-year-old female patient with pulmonary adenocarcinoma complicated by the syndrome of inappropriate secretion of antidiuretic hormone(SIADH)following systemic chemotherapy with cisplatin(CDDP)and vinorelbine(VNR). She was admitted to our hospital for chemo-radiotherapy for advanced lung cancer, and became restless 4 hours after the administration of CDDP and VNR. Symptoms such as restlessness and incontinence were worsening despite the massive infusion that was completed. Laboratory examinations on day 6 after chemotherapy showed severe hyponatremia(107mEq/L)with decreased serum osmolarity(227mOsm/L)and increased urine osmolarity(452mOsm/L). The serum anti-diuretic hormone(ADH)level was elevated to 16. 7 pg/mL despite severe hyponatremia. She was diagnosed with SIADH and was treated with hypertonic saline infusion and fluid restriction. Her restlessness and other psychiatric symptoms were improved. The use of carboplatin and VNR in the subsequent course did not develop SIADH, indicating that the SIADH was induced by CDDP. Although SIADH following CDDP administration is rare, the electrolyte balance should be carefully monitored throughout the clinical course of chemo-radiation therapy, when psychiatric symptoms are found in patients with lung cancer.

The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3' poly(U) tail.

Hfq-dependent sRNAs contain, at least, an mRNA base-pairing region, an Hfq-binding site, and a Rho-independent terminator. Recently, we found that the terminator poly(U) of Escherichia coli sRNAs is essential for Hfq binding and therefore for riboregulation. In this study, we tried to identify additional components within Hfq-binding sRNAs required for efficient Hfq binding by using SgrS as a model. We demonstrate by mutational and biochemical studies that an internal hairpin and an immediately upstream U-rich sequence also are required for efficient Hfq binding. We propose that the functional Hfq-binding module of SgrS consists of an internal hairpin preceded by a U-rich sequence and a Rho-independent terminator with a long poly(U) tail. We also show that the Rho-independent terminator alone can act as a functional Hfq-binding module when it is preceded by an internal U-rich sequence. The 3' region of most known sRNAs share the features corresponding to either a double- or single-hairpin-type Hfq-binding module. We also demonstrate that increasing the spacing between the base-pairing region and the Hfq-binding module reduces or impairs the silencing ability. These findings allowed us to design synthetic Hfq-binding sRNAs to target desired mRNAs.

PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action.

Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.

Bovine 5-oxo-L-prolinase: simple assay method, purification, cDNA cloning, and detection of mRNA in the coronary artery.

RS-7897 is a novel antianginal nitrate containing an L-2-oxothiazolidine-4-carboxylic acid (OTCA) and unlike other organic nitrates does not induce nitrate tolerance. OTCA is known to be converted to L-cysteine (L-Cys) by rat 5-oxo-prolinase (5-OPase) and was detected in the plasma of RS-7897-treated dogs. Nitrate tolerance is considered to develop mainly through sulfhydryl depletion. We hypothesized that RS-7897-derived OTCA was converted into L-Cys by 5-OPase and supplied sulfhydryl groups in vascular smooth muscle cells, the targets of the nitrate. As the initial step for clarifying the presumed role of 5-OPase in RS-7897 metabolism, we established a non-radiochemical assay method highly specific for 5-OPase. Using this assay method, we purified 5-OPase from bovine kidney cytosol until homogeneous results were obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The catalytic activity of bovine 5-OPase to convert OTCA to L-Cys was confirmed, as was the case for the rat enzyme. Then the cDNA encoding bovine 5-OPase was cloned. The deduced amino acid sequence revealed that bovine 5-OPase was highly homologous to rat 5-OPase. Based on the bovine cDNA sequence, oligonucleotide primers were synthesized and used for RT-PCR. 5-OPase mRNA was significantly detected in the RT-PCR product of the bovine coronary artery, a major target organ of RS-7897. These results suggest that OTCA may be converted to L-Cys by 5-OPase in the mammalian coronary artery when treated with RS-7897, and thus generated L-Cys may reduce sulfhydryl depletion and contribute to the prevention of the development of nitrate tolerance.

An angiotensin II type 1 receptor antagonist, olmesartan medoxomil, improves experimental liver fibrosis by suppression of proliferation and collagen synthesis in activated hepatic stellate cells.

1. We studied the effect of a new angiotensin II type 1 (AT(1)) receptor antagonist, olmesartan medoxomil (olmesartan), on the fibrogenic responses in rat hepatic stellate cells (HSCs) and liver fibrogenesis. 2. Olmesartan (1 mg kg(-1) per day) was orally administered to fibrotic rats, induced by bile duct ligation. Liver hydroxyproline content, the mRNA expression of collagen alpha1(I) and alpha-smooth muscle actin (alpha-SMA), and plasma levels of transforming growth factor-beta1 (TGF-beta1) were significantly reduced by olmesartan treatment, suggesting that olmesartan improved liver fibrosis. Interestingly, AT(1) receptors were found to be expressed in alpha-SMA-positive cells in the fibrotic area of livers in bile duct-ligated rats by immunohistochemical analysis. Olmesartan treatment reduced the number of these cells. 3. In vitro experiments showed that angiotensin II (Ang II) treatment induced proliferation and collagen synthesis, and upregulated the profibrogenic cytokines, TGF-beta1 and connective tissue growth factor (CTGF), in rat primary HSCs. Olmesartan blocked all these effects of Ang II. 4. Based on these results, since activated HSCs were found to express AT(1) receptors and Ang II is thought to play an important role in the pathogenesis of liver fibrosis by binding to these receptors, olmesartan may act as a potent antifibrotic drug to suppress the proliferation, collagen synthesis and the expression of profibrogenic cytokines in activated HSCs by blocking these receptors.