Hiroshi Ohmoto

Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: Metalloproteinase inhibitors as a new therapy

G-protein–coupled receptor (GPCR) agonists are well-known inducers of cardiac hypertrophy. We found that the shedding of heparin-binding epidermal growth factor (HB-EGF) resulting from metalloproteinase activation and subsequent transactivation of the epidermal growth factor receptor occurred when cardiomyocytes were stimulated by GPCR agonists, leading to cardiac hypertrophy. A new inhibitor of HB-EGF shedding, KB-R7785, blocked this signaling. We cloned a disintegrin and metalloprotease 12 (ADAM12) as a specific enzyme to shed HB-EGF in the heart and found that dominant-negative expression of ADAM12 abrogated this signaling. KB-R7785 bound directly to ADAM12, suggesting that inhibition of ADAM12 blocked the shedding of HB-EGF. In mice with cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF and attenuated hypertrophic changes. These data suggest that shedding of HB-EGF by ADAM12 plays an important role in cardiac hypertrophy, and that inhibition of HB-EGF shedding could be a potent therapeutic strategy for cardiac hypertrophy.

Activity-based kinase profiling of approved tyrosine kinase inhibitors.

The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity‐based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease‐relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the Km for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC50 values of the inhibitors against each kinase were compared with the estimated plasma‐free concentration (calculated from published pharmacokinetic parameters of plasma Ctrough and Cmax values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off‐target kinases in drug side effects. Thus, large‐scale kinase profiling at both Km and physiological ATP concentrations could be useful in characterizing the targets and off‐targets of kinase inhibitors.

Therapeutic potential of a novel synthetic selectin blocker, OJ-R9188, in allergic dermatitis

We investigated the ability of a newly synthesized sugar derivative, OJ‐R9188, {N‐(2‐tetradecylhexadecanoyl)‐O‐(L‐alpha‐fucofuranosyl)‐D‐seryl}‐L‐glutamic acid 1‐methylamide 5‐L‐arginine salt, to block binding of selectins to their ligands in vitro and inhibit the infiltration of leukocytes in vivo. OJ‐R9188 prevented the binding of human E‐, P‐ and L‐selectin‐IgG fusion proteins to immobilized sialyl Lewisx (sLex)‐pentasaccharide glycolipid, with IC50 values of 4.3, 1.3, and 1.2 μM, respectively. In a mouse model of thioglycollate‐induced peritonitis, OJ‐R9188 at 10 mg kg−1, i.v. inhibited neutrophil accumulation in the peritoneal cavity. In the IgE‐mediated skin reaction, OJ‐R9188 at 3 and 10 mg kg−1, i.v. significantly inhibited extravasation of neutrophils and eosinophils into the inflammatory sites and at 10 mg kg−1, i.v. also inhibited infiltration caused by picryl chloride‐induced delayed‐type hypersensitivity in mice. These results suggest that OJ‐R9188 may be a useful selectin blocker, with activity against human and mouse E‐, P‐ and L‐selectins in vitro and in vivo, and that blocking selectin‐sLex binding is a promising strategy for the treatment of allergic skin diseases.

Regenerative and therapeutic effects of heparin-binding epidermal growth factor-like growth factor on diabetes by gene transduction through retrograde pancreatic duct injection of adenovirus vector.

Objectives: In the adult pancreas, pre-existing β cells, stem cells, and endocrine progenitor cells residing in the duct lining are considered important sources for β-cell regeneration. A member of the epidermal growth factor (EGF) family, heparin binding (HB)-EGF, may promote this process. We examined whether HB-EGF gene transduction into duct cells could promote β-cell regeneration. Methods: We administered an HB-EGF adenovirus vector construct to male Institute of Cancer Research mice by retrograde injection through the pancreatic duct. We also performed HB-EGF gene transduction into cultured duct cells. Results: On immunohistochemical and histomorphometric analysis of the experimental group, insulin-positive cells differentiated from duct cells, and the 5-bromo-2-deoxyuridine labeling index of β cells was significantly increased. β-cell mass was also increased, and the glucose tolerance of diabetic mice was improved at 12 weeks after injection. Using cultured pancreatic duct cells, we confirmed that HB-EGF gene transduction induced both insulin gene expression and insulin production by these cells. Conclusions: These results indicate that HB-EGF gene transduction into adult pancreatic duct cells not only promotes the proliferation of pre-existing β cells but also leads to β-cell differentiation from duct cells, and the resulting increase in β-cell mass improves glucose tolerance.

Synthesis of sialyl lewis x pentasaccharide analogue for high-throughput screening of selectin blockers

We have developed an effective synthesis of sLe(x) pentasaccharide glycolipid analogue 2. As a part of application of sLe(x) pentasaccharide glycolipid 2 synthesized here, we have investigated the construction of a high-through-put screening system for discovery of selectin blockers. As a result, it was found that compound 2 was a useful ligand for in vitro ELISA assay and could be an important material for high-throughput screening of selectin blockers.