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Browsing by Author "Huang, Rong"
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Item Exploring Unconventional SAM Analogues To Build Cell-Potent Bisubstrate Inhibitors for Nicotinamide N-Methyltransferase(Wiley, 2022) Iyamu, Iredia D.; Vilseck, Jonah Z.; Yadav, Ravi; Noinaj, Nicholas; Huang, Rong; Biochemistry and Molecular Biology, School of MedicineNicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9 nM in a biochemical assay and a cellular IC50value of 1.9µM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399’s low nM binding affinity. Notably, II399 is 1,000-fold more selective for NNMT than closely related methyltransferases. We expect that II399would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.Item Exploring Unconventional SAM Analogues To Build Cell-Potent Bisubstrate Inhibitors for Nicotinamide N-Methyltransferase(Wiley, 2022) Iyamu, Iredia D.; Vilseck, Jonah Z.; Yadav, Ravi; Noinaj, Nicholas; Huang, Rong; Biochemistry and Molecular Biology, School of MedicineNicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9 nM in a biochemical assay and a cellular IC50 value of 1.9 μM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399′s low nM binding affinity. Notably, II399 is 1 000-fold more selective for NNMT than closely related methyltransferases. We expect that II399 would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.Item Improved Cell-Potent and Selective Peptidomimetic Inhibitors of Protein N-Terminal Methyltransferase 1(MDPI, 2022-02) Dong, Guangping; Iyamu, Iredia D.; Vilseck, Jonah Z.; Chen, Dongxing; Huang, Rong; Biochemistry and Molecular Biology, School of MedicineProtein N-terminal methyltransferase 1 (NTMT1) recognizes a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) and transfers 1–3 methyl groups to the N-terminal region of its substrates. Guided by the co-crystal structures of NTMT1 in complex with the previously reported peptidomimetic inhibitor DC113, we designed and synthesized a series of new peptidomimetic inhibitors. Through a focused optimization of DC113, we discovered a new cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM). GD562 exhibited improved inhibition of the cellular N-terminal methylation levels of both the regulator of chromosome condensation 1 and the oncoprotein SET with an IC50 value of ~50 µM in human colorectal cancer HCT116 cells. Notably, the inhibitory activity of GD562 for the SET protein increased over 6-fold compared with the previously reported cell-potent inhibitor DC541. Furthermore, GD562 also exhibited over 100-fold selectivity for NTMT1 against several other methyltransferases. Thus, this study provides a valuable probe to investigate the biological functions of NTMT1.Item The role of Meteorin-like in skeletal development and bone fracture healing(Wiley, 2022) Huang, Rong; Balu, Abhinav R.; Molitoris, Kristin H.; White, James P.; Robling, Alexander G.; Ayturk, Ugur M.; Baht, Gurpreet S.; Anatomy, Cell Biology and Physiology, School of MedicineMeteorin‐like protein (Metrnl), homologous to the initially identified neurotrophic factor Meteorin, is a secreted, multifunctional protein. Here we used mouse models to investigate Metrnl's role in skeletal development and bone fracture healing. During development Metrnl was expressed in the perichondrium and primary ossification center. In neonates, single cell RNA‐seq of diaphyseal bone demonstrated strongest expression of Metrnl transcript by osteoblasts. In vitro, Metrnl was osteoinductive, increasing osteoblast differentiation and mineralization in tissue culture models. In vivo, loss of Metrnl expression resulted in no change in skeletal metrics in utero, at birth, or during postnatal growth. Six‐week‐old Metrnl‐null mice displayed similar body length, body weight, tibial length, femoral length, BV/TV, trabecular number, trabecular thickness, and cortical thickness as littermate controls. In 4‐month‐old mice, lack of Metrnl expression did not change structural stiffness, ultimate force, or energy to fracture of femora under 3‐point‐bending. Last, we investigated the role of Metrnl in bone fracture healing. Metrnl expression increased in response to tibial injury, however, loss of Metrnl expression did not affect the amount of bone deposited within the healing tissue nor did it change the structural parameters of healing tissue. This work identifies Metrnl as a dispensable molecule for skeletal development. However, the osteoinductive capabilities of Metrnl may be utilized to modulate osteoblast differentiation in cell‐based orthopedic therapies.