Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally ...compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.
We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.
We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.
Automated cell-based high-throughput screening (HTS) is a powerful tool in drug discovery, and it is increasingly being recognized that three-dimensional (3D) models, which more closely mimic ...in vivo-like conditions, are desirable screening platforms. One limitation hampering the development of 3D HTS is the lack of suitable 3D culture scaffolds that can readily be incorporated into existing HTS infrastructure. We now show that β-hairpin peptide hydrogels can serve as a 3D cell culture platform that is compatible with HTS. MAX8 β-hairpin peptides can physically assemble into a hydrogel with defined porosity, permeability and mechanical stability with encapsulated cells. Most importantly, the hydrogels can then be injected under shear-flow and immediately reheal into a hydrogel with the same properties exhibited prior to injection. The post-injection hydrogels are cell culture compatible at physiological conditions. Using standard HTS equipment and medulloblastoma pediatric brain tumor cells as a model system, we show that automatic distribution of cell-peptide mixtures into 384-well assay plates results in evenly dispensed, viable MAX8-cell constructs suitable for commercially available cell viability assays. Since MAX8 peptides can be functionalized to mimic the microenvironment of cells from a variety of origins, MAX8 peptide gels should have broad applicability for 3D HTS drug discovery.
RAD51 is a key protein of homologous recombination that plays a critical role in the repair of DNA double-strand breaks (DSB) and interstrand cross-links (ICL). To better understand the cellular ...function(s) of human RAD51, we propose to develop specific RAD51 inhibitors. RAD51 inhibitors may also help to increase the potency of anticancer drugs that act by inducing DSBs or ICLs, e.g., cisplatin or ionizing radiation. In vitro, RAD51 promotes DNA strand exchange between homologous ss- and dsDNA. Here, we developed a DNA strand exchange assay based on fluorescence resonance energy transfer and used this assay to identify RAD51 inhibitors by high-throughput screening of the NIH Small Molecule Repository (>200,000 compounds). Seventeen RAD51 inhibitors were identified and analyzed for selectivity using additional nonfluorescent DNA-based assays. As a result, we identified a compound (B02) that specifically inhibited human RAD51 (IC50 = 27.4 μM) but not its E. coli homologue RecA (IC50 > 250 μM). Two other compounds (A03 and A10) were identified that inhibited both RAD51 and RecA but not the structurally unrelated RAD54 protein. The structure−activity relationship (SAR) analysis allowed us to identify the structural components of B02 that are critical for RAD51 inhibition. The described approach can be used for identification of specific inhibitors of other human proteins that play an important role in DNA repair, e.g., RAD54 or Bloom’s syndrome helicase.
High-throughput screening against the human sirtuin SIRT1 led to the discovery of a series of indoles as potent inhibitors that are selective for SIRT1 over other deacetylases and NAD-processing ...enzymes. The most potent compounds described herein inhibit SIRT1 with IC50 values of 60−100 nM, representing a 500-fold improvement over previously reported SIRT inhibitors. Preparation of enantiomerically pure indole derivatives allowed for their characterization in vitro and in vivo. Kinetic analyses suggest that these inhibitors bind after the release of nicotinamide from the enzyme and prevent the release of deacetylated peptide and O-acetyl-ADP-ribose, the products of enzyme-catalyzed deacetylation. These SIRT1 inhibitors are low molecular weight, cell-permeable, orally bioavailable, and metabolically stable. These compounds provide chemical tools to study the biology of SIRT1 and to explore therapeutic uses for SIRT1 inhibitors.
Substrate-specific Activation of Sirtuins by Resveratrol Kaeberlein, Matt; McDonagh, Thomas; Heltweg, Birgit ...
Journal of biological chemistry/The Journal of biological chemistry,
04/2005, Letnik:
280, Številka:
17
Journal Article
Recenzirano
Odprti dostop
Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported ...to act as a sirtuin activator, and this property has been proposed to account for its anti-aging effects. We show here that resveratrol is a substrate-specific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined.
Here we offer perspectives on phenotypic screening based on a wide-ranging discussion entitled “Phenotypic screening, target ID, and multi-omics: enabling more disease relevance in early discovery?” ...at the Screen Design and Assay Technology Special Interest Group Meeting at the 2023 SLAS Conference. During the session, the authors shared their own experience from within their respective organizations, followed by an open discussion with the audience. It was recognized that while substantial progress has been made towards translating disease-relevant phenotypic early discovery into clinical success, there remain significant operational and scientific challenges to implementing phenotypic screening efforts, and improving translation of screening hits comes with substantial resource demands and organizational commitment. This Perspective assesses progress, highlights pitfalls, and offers possible solutions to help unlock the therapeutic potential of phenotypic drug discovery. Areas explored comprise screening and hit validation strategy, choice of cellular model, moving beyond 2D cell culture into three dimensions, and leveraging high-dimensional data sets downstream of phenotypic screens.
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