Novel TAZ modulators enhance myogenic differentiation and muscle regeneration.

BACKGROUND AND PURPOSE: The transcriptional co-activator with PDZ-binding motif (TAZ) is a key controller of mesenchymal stem cell differentiation through its nuclear localization and subsequent interaction with master transcription factors. In particular, TAZ directly associates with myoblast determining protein D (MyoD) and activates MyoD-induced myogenic gene expression, thereby enhancing myogenic differentiation. Here, we have synthesized and characterized low MW compounds modulating myogenic differentiation via induction of TAZ nuclear localization. EXPERIMENTAL APPROACH: COS7 cells stably transfected with GFP-TAZ were used in a high content imaging screen for compounds specifically enhancing nuclear localization of TAZ. We then studied the effects of such TAZ modulators on myocyte differentiation of C2C12 cells and myogenic transdifferentiation of mouse embryonic fibroblast cells in vitro and muscle regeneration in vivo. KEY RESULTS: We identified two TAZ modulators, TM-53, and its structural isomer, TM-54. Each compound strongly enhanced nuclear localization of TAZ by reducing S89-phosphorylation and dose-dependently augmented myogenic differentiation and MyoD-mediated myogenic transdifferentiation through an activation of MyoD-TAZ interaction. The myogenic stimulatory effects of TM-53 and TM-54 were impaired in the absence of TAZ, but retrieved by the restoration of TAZ. In addition, administration of TM-53 and TM-54 enhanced injury-induced muscle regeneration in vivo and attenuated myofiber injury in vitro. CONCLUSIONS AND IMPLICATIONS: The novel TAZ modulators TM-53 and TM-54 accelerated myogenic differentiation and improved muscle regeneration and function after injury, demonstrating that low MW compounds targeting the nuclear localization of TAZ have beneficial effects in skeletal muscle regeneration and in recovery from muscle degenerative diseases.

Transport characteristics and transporter-based drug-drug interactions of TM-25659, a novel TAZ modulator.

The in vitro metabolic stability and transport mechanism of TM-25659, a novel TAZ modulator, was investigated in human hepatocytes and human liver microsomes (HLMs) based on the preferred hepatobiliary elimination in rats. In addition, the in vitro transport mechanism and transporter-mediated drug-drug interactions were evaluated using oocytes and MDCKII cells overexpressing clinically important drug transporters. After a 1 h incubation in HLMs, 92.9 ± 9.5% and 95.5 ± 11.6% of the initial TM-25659 remained in the presence of NADPH and UDPGA, respectively. Uptake of TM-25659 readily accumulated in human hepatocytes at 37 ºC (i.e. 6.7-fold greater than that at 4 ºC), in which drug transporters such as OATP1B1 and OATP1B3 were involved. TM-25659 had a significantly greater basal to apical transport rate (5.9-fold) than apical to basal transport rate in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and overexpressing cells revealed that MRP2 was involved in the transport of TM-25659. These results, taken together, suggested that TM-25659 can be actively influxed into hepatocytes and undergo biliary excretion without substantial metabolism. Additionally, TM-25659 inhibited the transport activities of OATP1B1 and OATP1B3 with IC50 values of 36.3 and 25.9 µm, respectively. TM-25659 (100 µm) increased the accumulation of the probe substrate by 160% and 213%, respectively, through the inhibition of efflux function of P-gp and MRP2. In conclusion, OATP1B1, OATP1B3, P-gp and MRP2 might be major transporters responsible for the pharmacokinetics and drug-drug interaction of TM-25659, although their contribution to in vivo pharmacokinetics needs to be further investigated.

A novel anti-neuroinflammatory pyridylimidazole compound KR-31360.

Excessive microglial activation with overexpression of proinflammatory cytokines and oxidative stress products is linked to the progression of several neurodegenerative diseases; therefore, suppression of microglial activation is a potential therapeutic approach against these diseases. Since nitric oxide (NO) is one of the major inflammatory mediators that are produced by activated microglia, inhibitory effects of novel synthetic compounds on microglial NO production were investigated. From the mouse microglia cell-based assays, an imidazo [4,5-b] pyridine compound KR-31360 was identified as an inhibitor of microglial NO production with an IC(50) value of 2 microM. Structure-activity relationship study indicated that 5-position of imidazo [4,5-b] pyridine ring is critical for the activity. KR-31360 also inhibited lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor alpha (TNF-alpha) and transcription of TNF-alpha, interleukin-1 beta, and inducible nitric oxide synthase as well as activation of nuclear factor kappa B and mitogen-activated protein kinases. KR-31360 was neuroprotective by suppressing microglial neurotoxicity in a microglia-neuron coculture. The neuroprotective activity of the compound was most effective when microglia were pretreated with the compound prior to LPS challenge. The inhibitory effect of KR-31360 on microglial activation was further demonstrated in a mouse neuroinflammation model in vivo: compared to vehicle-injected animals, KR-31360 injection attenuated LPS-induced microglial activation as evidenced by isolectin B4 staining and proinflammatory gene expression of brain sections. DNA microarray analysis supported that KR-31360 targeted Toll-like receptor 4 pathways. In addition to being a new drug candidate against neuroinflammatory diseases, the compound may be a powerful tool for the better understanding of microglia biology and neuroinflammation.