12 research outputs found
In vivo tracking of human neural progenitor cells in the rat brain using bioluminescence imaging
AbstractBackgroundStem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity.New methodIn this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPCLuc2) in the rat striatum long-term.ResultsWe show that hNPCLuc2 are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPCLuc2 contralateral migration.Comparison with existing methodsSome of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood–brain barrier remains intact.ConclusionsThese new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders
Synergistic Effects of GDNF and VEGF on Lifespan and Disease Progression in a Familial ALS Rat Model
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle
Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors
Novel
engineered NanoLuc (Nluc) luciferase being smaller, brighter,
and superior to traditional firefly (Fluc) or <i>Renilla</i> (Rluc) provides a great opportunity for the development of numerous
biological, biomedical, clinical, and food and environmental safety
applications. This new platform created an urgent need for Nluc inhibitors
that could allow selective bioluminescent suppression and multiplexing
compatibility with existing luminescence or fluorescence assays. Starting
from thienopyrrole carboxylate <b>1</b>, a hit from a 42 000
PubChem compound library with a low micromolar IC<sub>50</sub> against
Nluc, we derivatized four different structural fragments to discover
a family of potent, single digit nanomolar, cell permeable inhibitors.
Further elaboration revealed a channel that allowed access to the
external Nluc surface, resulting in a series of highly potent cell
impermeable Nluc inhibitors with negatively charged groups likely
extending to the protein surface. The permeability was evaluated by
comparing EC<sub>50</sub> shifts calculated from both live and lysed
cells expressing Nluc cytosolically. Luminescence imaging further
confirmed that cell permeable compounds inhibit both intracellular
and extracellular Nluc, whereas less permeable compounds differentially
inhibit extracellular Nluc and Nluc on the cell surface. The compounds
displayed little to no toxicity to cells and high luciferase specificity,
showing no activity against firefly luciferase or even the closely
related NanoBit system. Looking forward, the structural motifs used
to gain access to the Nluc surface can also be appended with other
functional groups, and therefore interesting opportunities for developing
assays based on relief-of-inhibition can be envisioned
KRAS is vulnerable to reversible switch-II pocket engagement in cells.
Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer
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Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome
Disease-modifying therapies are needed for Fragile X Syndrome (FXS), as at present there are no effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological and ADMET properties, and its ability to improve upon memory performance in a mouse model of FXS, Fmr1
mice. This small molecule demonstrates good brain penetrance, low-nanomolar potency for the inhibition of HDAC6 (IC
= 2.3 nM), with at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms. Moreover, through its inhibition of the α-tubulin deacetylase domain of HDAC6 (CD2), in cells SW-100 upregulates α-tubulin acetylation with no effect on histone acetylation and selectively restores the impaired acetylated α-tubulin levels in the hippocampus of Fmr1
mice. Lastly, SW-100 ameliorates several memory and learning impairments in Fmr1
mice, thus modeling the intellectual deficiencies associated with FXS, and hence providing a strong rationale for pursuing HDAC6-based therapies for the treatment of this rare disease
Comprehensive Survey of CDK Inhibitor Selectivity in Live Cells with Energy Transfer Probes
A panel of cell-permeable energy transfer probes has been developed to quantify target occupancy for all 21 CDKs in live, intact cells. Here we present the first comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi’s and tool molecules. Here we provide a broadly applicable method for evaluating the selectivity of chemical matter for CDKs in living cells, and present a refined set of tool molecules to study CDK function
Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome
Disease-modifying therapies are needed for Fragile X Syndrome (FXS), as at present there are no effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological and ADMET properties, and its ability to improve upon memory performance in a mouse model of FXS, Fmr1-/- mice. This small molecule demonstrates good brain penetrance, low-nanomolar potency for the inhibition of HDAC6 (IC50 = 2.3 nM), with at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms. Moreover, through its inhibition of the α-tubulin deacetylase domain of HDAC6 (CD2), in cells SW-100 upregulates α-tubulin acetylation with no effect on histone acetylation and selectively restores the impaired acetylated α-tubulin levels in the hippocampus of Fmr1-/- mice. Lastly, SW-100 ameliorates several memory and learning impairments in Fmr1-/- mice, thus modeling the intellectual deficiencies associated with FXS, and hence providing a strong rationale for pursuing HDAC6-based therapies for the treatment of this rare disease.status: publishe