10 research outputs found
An engineered gastrointestinally stable microbial leucine decarboxylase for potential treatment of maple syrup urine disease
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The GluN2B subunit represents a major functional determinant of NMDA receptors in human induced pluripotent stem cell-derived cortical neurons
Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However, few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study, we assessed expression, functionality, and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC)-derived cortical neurons (iCell Neurons and iCell GlutaNeurons). We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents. Keywords: Human induced pluripotent stem cell-derived neurons, iCell Neurons, iCell GlutaNeurons, NMDA receptors, GluN2B, Positive allosteric modulator
Identification of a Key Residue Mediating Bone Morphogenetic Protein (BMP)-6 Resistance to Noggin Inhibition Allows for Engineered BMPs with Superior Agonist Activity*
Bone morphogenetic proteins (BMPs) are used clinically to induce new bone formation in spinal fusions and long bone non-union fractures. However, large amounts of BMPs are needed to achieve these effects. BMPs were found to increase the expression of antagonists, which potentially limit their therapeutic efficacy. However, the relative susceptibility of osteoinductive BMPs to different antagonists is not well characterized. Here we show that BMP-6 is more resistant to noggin inhibition and more potent in promoting osteoblast differentiation in vitro and inducing bone regeneration in vivo when compared with its closely related BMP-7 paralog. Noggin was found to play a critical role as a negative feedback regulator of BMP-7 but not BMP-6-induced biological responses. Using BMP-6/7 chimeras, we identified lysine 60 as a key residue conferring noggin resistance within the BMP-6 protein. A remarkable correlation was found between the presence of a lysine at this position and noggin resistance among a panel of osteoinductive BMPs. Introduction of a lysine residue at the corresponding positions of BMP-2 and BMP-7 allowed for molecular engineering of recombinant BMPs with increased resistance to noggin antagonism
BMP-2/4 and BMP-6/7 Differentially Utilize Cell Surface Receptors to Induce Osteoblastic Differentiation of Human Bone Marrow-derived Mesenchymal Stem Cells*S⃞
Bone morphogenetic proteins (BMPs) are members of the transforming growth
factor-β superfamily of growth factors and are used clinically to induce
new bone formation. The purpose of this study was to evaluate receptor
utilization by BMP-2, BMP-4, BMP-6, and BMP-7 in primary human mesenchymal
stem cells (hMSC), a physiologically relevant cell type that probably mediates
the in vivo effects of BMPs. RNA interference-mediated gene knockdown
revealed that osteoinductive BMP activities in hMSC are elicited through the
type I receptors ACVR1A and BMPR1A and the type II receptors ACVR2A and BMPR2.
BMPR1B and ACVR2B were expressed at low levels and were not found to play a
significant role in signaling by any of the BMPs evaluated in this study. Type
II receptor utilization differed significantly between BMP-2/4 and BMP-6/7. A
greater reliance on BMPR2 was observed for BMP-2/4 relative to BMP-6/7,
whereas ACVR2A was more critical to signaling by BMP-6/7 than BMP-2/4.
Significant differences were also observed for the type I receptors. Although
BMP-2/4 used predominantly BMPR1A for signaling, ACVR1A was the preferred type
I receptor for BMP-6/7. Signaling by both BMP-2/4 and BMP-6/7 was mediated by
homodimers of ACVR1A or BMPR1A. A portion of BMP-2/4 signaling also required
concurrent BMPR1A and ACVR1A expression, suggesting that BMP-2/4 signal in
part through ACVR1A/BMPR1A heterodimers. The capacity of ACVR1A and BMPR1A to
form homodimers and heterodimers was confirmed by bioluminescence resonance
energy transfer analyses. These results suggest different mechanisms for
BMP-2/4- and BMP-6/7-induced osteoblastic differentiation in primary hMSC
The GluN2B subunit represents a major functional determinant of NMDA receptors in human induced pluripotent stem cell-derived cortical neurons
Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase
Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase
NF-κB-inducing
kinase (NIK) is a protein kinase central to
the noncanonical NF-κB pathway downstream from multiple TNF
receptor family members, including BAFF, which has been associated
with B cell survival and maturation, dendritic cell activation, secondary
lymphoid organ development, and bone metabolism. We report herein
the discovery of lead chemical series of NIK inhibitors that were
identified through a scaffold-hopping strategy using structure-based
design. Electronic and steric properties of lead compounds were modified
to address glutathione conjugation and amide hydrolysis. These highly
potent compounds exhibited selective inhibition of LTβR-dependent
p52 translocation and transcription of NF-κB2 related genes.
Compound <b>4f</b> is shown to have a favorable pharmacokinetic
profile across species and to inhibit BAFF-induced B cell survival
in vitro and reduce splenic marginal zone B cells in vivo
Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase
NF-κB-inducing
kinase (NIK) is a protein kinase central to
the noncanonical NF-κB pathway downstream from multiple TNF
receptor family members, including BAFF, which has been associated
with B cell survival and maturation, dendritic cell activation, secondary
lymphoid organ development, and bone metabolism. We report herein
the discovery of lead chemical series of NIK inhibitors that were
identified through a scaffold-hopping strategy using structure-based
design. Electronic and steric properties of lead compounds were modified
to address glutathione conjugation and amide hydrolysis. These highly
potent compounds exhibited selective inhibition of LTβR-dependent
p52 translocation and transcription of NF-κB2 related genes.
Compound <b>4f</b> is shown to have a favorable pharmacokinetic
profile across species and to inhibit BAFF-induced B cell survival
in vitro and reduce splenic marginal zone B cells in vivo