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Novel Fluorescent Dye ZMB741 for Podocytopathy Drug Screening
In order to develop new fluorescent dye that can be used for in vivo phenotypic screening, which is currently in short supply in the global market, transfected zebrafish (MieKomachi001) were reared in a 96-well ZFplate, and one fluorescent pigment from a library of approximately 600 fluorescent pigments was added to the rearing water. A final concentration of 5 μM of each fluorophore from a library of approximately 600 fluorophores was added to the rearing water, and image screening was performed 24 hours later. The results showed that several fluorochromes selectively visualized blood vessels, with ZMB741 having the highest fluorescence intensity. Subsequently, ZMB741 was found to bind to a large number of plasma proteins rather than to the vascular wall after invasive administration, leading to large-scale in vivo phenotypic screening. For example, apolipoprotein A-I (Kd=0.84μM) and hemopexin (Kd=3.13μM) bind to most plasma proteins with slightly different affinities, which is the basis for their great usefulness in in vivo proteinuria quantitative analysis. Furthermore, existing tracers such as fluorescent dextran, EB, and ICG can only be administered invasively in zebrafish, such as via intravascular injection. In addition, free ZMB741 in aqueous solution shows almost no fluorescence, but when bound to plasma proteins, the fluorescence quantum yield increases at a higher level than existing tracers, enabling analysis of plasma protein dynamics at higher sensitivity than with existing tracers and allowing analysis of medically important blood brain barrier (BBB) breakdown (ACS Chem. Neurosci. 2013, 4, 1183-1193) and in vivo imaging of podocytopathy proteinuria.
Since the discovery in 1998 of nephrin, a protein that is a component of the slit membrane of podocytes, there has been a dramatic increase in podocyte findings, and it became clear that podocyte injury causes irreversible hemarthrosclerosis and, eventually, chronic kidney disease (CKD). Podocyte injury is a pathophysiological factor in the pathogenesis of CKD. The kidney diseases in which podocyte injury is the primary focus of the pathology are collectively referred to as podocytopathies, typically including the diseases of the primary nephrotic syndrome. Glucocorticoids and immunosuppressive drugs used in the treatment of these diseases have been reported to have direct podocytoprotective effects other than immunological effects. However, there are no globally proven therapies that are potently protective of podocytes and inhibit the progression of CKD. It is hoped that the mechanisms of podocytosis onset and progression will be elucidated, and specific treatment and prevention methods will be developed. Therefore, we created a zebrafish model of podocytosis by KO and KD of nephrin genes and others, and constructed an in vivo proteinuria individual quantification system using ZMB741. This in vivo phenotypic screening has resulted in at least two drug candidates with different mechanisms of action as a result of repositioning screening of existing drugs. We are currently analyzing the therapeutic mechanism of these new drug candidates for podocytosis and ultimately aiming for clinical application.
Novel Fluorescent Dye ZMB741 for Podocytopathy Drug Screening
⾎液脳関⾨ (BBB) 破綻(ACS Chem. Neurosci. 2013, 4, 1183?1193)