Promyelinating drugs ameliorate oligodendrocyte pathologies in a mouse model of Krabbe disease.

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.

Development of a neutralizing antibody specific for the active form of matrix metalloproteinase-13.

Matrix metalloproteinase-13 (MMP-13) is important in the pathology of osteoarthritis (OA). Although MMP-13 is considered a therapeutic target for OA, it is unclear how MMP-13 activity is regulated by the system that comprises various proteinases and their inhibitors. MMP-13 neutralizing antibodies could be a useful tool for investigating the involvement of MMP-13 in cartilage degeneration in OA-affected joints because antibodies possess high affinity and specificity compared with low-molecular weight chemical compounds. On the basis of three-dimensional structure and amino acid sequence information on MMP-13, we selected an appropriate antigen peptide and generated a neutralizing antibody by immunizing mice with the antigen. The most significant property of monoclonal antibody 14D10 was the specific binding to the active form of MMP-13, but not to the latent form, or other MMPs. With this property, active MMP-13 was measured selectively by an enzyme-linked immunosorbet assay. Furthermore, 14D10 suppressed the cleavage of type II collagen in human articular chondrocyte cultures, and 14D10 is thought to inhibit MMP-13 activity effectively. Thus, the highly selective MMP-13 neutralizing antibody (14D10) might be a useful tool for investigating the mechanism of type II collagen degradation in arthritic pathology.

Macrocyclic Peptides as a Novel Class of NNMT Inhibitors: A SAR Study Aimed at Inhibitory Activity in the Cell.

Nicotinamide N-methyltransferase (NNMT), which catalyzes the methylation of nicotinamide, is a cytosolic enzyme that has attracted much attention as a therapeutic target for a variety of diseases. However, despite the considerable interest in this target, reports of NNMT inhibitors have still been limited to date. In this work, utilizing in vitro translated macrocyclic peptide libraries, we identified peptide 1 as a novel class of NNMT inhibitors. Further exploration based on the X-ray cocrystal structures of the peptides with NNMT provided a dramatic improvement in inhibitory activity (peptide 23: IC50 = 0.15 nM). Furthermore, by balance of the peptides' lipophilicity and biological activity, inhibitory activity against NNMT in cell-based assay was successfully achieved (peptide 26: cell-based IC50 = 770 nM). These findings illuminate the potential of cyclic peptides as a relatively new drug discovery modality even for intracellular targets.

Artepillin C, a Typical Brazilian Propolis-Derived Component, Induces Brown-Like Adipocyte Formation in C3H10T1/2 Cells, Primary Inguinal White Adipose Tissue-Derived Adipocytes, and Mice.

Induction of brown-like adipocytes (beige/brite cells) in white adipose tissue (WAT) suggests a new approach for preventing and treating obesity via induction of thermogenesis associated with uncoupling protein 1 (UCP1). However, whether diet-derived factors can directly induce browning of white adipocytes has not been well established. In addition, the underlying mechanism of induction of brown-like adipocytes by diet-derived factors has been unclear. Here, we demonstrate that artepillin C (ArtC), which is a typical Brazilian propolis-derived component, significantly induces brown-like adipocytes in murine C3H10T1/2 cells and primary inguinal WAT (iWAT)-derived adipocytes. This significant induction is due to activation of peroxisome proliferator-activated receptor γ and stabilization of PRD1-BF-1-RIZ1 homologous domain-containing protein-16 (PRDM16). Furthermore, the oral administration of ArtC (10 mg/kg) for 4 weeks significantly induced brown-like adipocytes accompanied by significant expression of UCP1 and PRDM16 proteins in iWAT of mice, and was independent of the ß3-adrenergic signaling pathway via the sympathetic nervous system. These findings may provide insight into browning of white adipocytes including the molecular mechanism mediated by dietary factors and demonstrate that ArtC has a novel biological function with regard to increasing energy expenditure by browning of white adipocytes.

Development of a novel immunoassay for the measurement of type II collagen neoepitope generated by collagenase cleavage.

BACKGROUND: To evaluate cartilage degeneration in arthritis, we developed a novel enzyme-linked immunosorbent assay (ELISA) with the capacity to determine urinary concentrations of type II collagen neoepitope (CIINE) generated by collagenase cleavage. METHODS: Two monoclonal antibodies, 20A10 and 6G4, were generated. Of these antibodies, 20A10 recognized CIINE regardless of hydroxylation of Pro77¹, and 6G4 recognized the type II collagen-specific region adjacent to the neoepitope. A sandwich ELISA was constructed using these antibodies. RESULTS: The ELISA positively determined CIINE concentrations from human and dog urine samples, and from tissue culture supernatant of rat and bovine cartilage. Validation with human urine samples revealed that the ELISA had a detection limit of 100 pmol/l, with intra- and inter-assay coefficients of less than 15%. Recovery of extraneously added CIINE peptide to human urine samples was 83.1-104%. Measurement with the ELISA demonstrated that urine samples from OA patients contained CIINE at significantly higher concentrations compared with those from healthy controls (P<0.01). CONCLUSIONS: The ELISA can determine the CIINE concentration in human urine sensitively and accurately. This assay may also be useful to determine the concentration of CIINE of various animal samples.