A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2.
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ABSTRACT:
SUBMITTER: Bery N
PROVIDER: S-EPMC8034850 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2. We selected four compounds that bind to LMO2 but not when the anti-LMO2 intracellular antibody fragment is bound to it. These findings further illustrate the value of intracellular antibodies in the initial stages of drug discovery campaigns and more generally antibodies, or antibody fragments, can be the starting point for chemical compound development as surrogates of the antibody combining site.
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Project description:We have developed a robust in vivo small-molecule screen that modulates heart size and cardiomyocyte generation in zebrafish. Three structurally related compounds (Cardionogen-1 to Cardionogen-3) identified from our screen enlarge the size of the developing heart via myocardial hyperplasia. Increased cardiomyocyte number in Cardionogen-treated embryos is due to expansion of cardiac progenitor cells. In zebrafish embryos and murine embryonic stem (ES) cells, Cardionogen treatment promotes cardiogenesis during and after gastrulation, whereas it inhibits heart formation before gastrulation. Cardionogen-induced effects can be antagonized by increasing Wnt/?-catenin signaling activity. We demonstrate that Cardionogen inhibits Wnt/?-catenin-dependent transcription in murine ES cells and zebrafish embryos. Cardionogen can rescue Wnt8-induced cardiomyocyte deficiency and heart-specific phenotypes during development. These findings demonstrate that in vivo small-molecule screens targeting heart size can reveal compounds with cardiomyogenic effects and identify underlying target pathways.
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Project description:Highly conserved noncoding RNA (ncRNA) elements in viral genomes and transcripts offer new opportunities to expand the repertoire of drug targets for the development of antiinfective therapy. Ligands binding to ncRNA architectures are able to affect interactions, structural stability or conformational changes and thereby block processes essential for viral replication. Proof of concept for targeting functional RNA by small molecule inhibitors has been demonstrated for multiple viruses with RNA genomes. Strategies to identify antiviral compounds as inhibitors of ncRNA are increasingly emphasizing consideration of drug-like properties of candidate molecules emerging from screening and ligand design. Recent efforts of antiviral lead discovery for RNA targets have provided drug-like small molecules that inhibit viral replication and include inhibitors of human immunodeficiency virus (HIV), hepatitis C virus (HCV), severe respiratory syndrome coronavirus (SARS CoV), and influenza A virus. While target selectivity remains a challenge for the discovery of useful RNA-binding compounds, a better understanding is emerging of properties that define RNA targets amenable for inhibition by small molecule ligands. Insight from successful approaches of targeting viral ncRNA in HIV, HCV, SARS CoV, and influenza A will provide a basis for the future exploration of RNA targets for therapeutic intervention in other viral pathogens which create urgent, unmet medical needs. Viruses for which targeting ncRNA components in the genome or transcripts may be promising include insect-borne flaviviruses (Dengue, Zika, and West Nile) and filoviruses (Ebola and Marburg). WIREs RNA 2016, 7:726-743. doi: 10.1002/wrna.1373 For further resources related to this article, please visit the WIREs website.
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Project description:The outer membrane (OM) of gram-negative bacteria forms a protective layer around the cell that serves as a permeability barrier to prevent unrestricted access of noxious substances. The permeability barrier of the OM results partly from the limited pore diameters of OM diffusion channels. As a consequence, there is an "OM size-exclusion limit," and the uptake of bulky molecules with molecular masses of more than ? 600 Da is thought to be mediated by TonB-dependent, active transporters. Intriguingly, the OM protein CymA from Klebsiella oxytoca does not depend on TonB but nevertheless mediates efficient OM passage of cyclodextrins with diameters of up to ? 15 Å. Here we show, by using X-ray crystallography, molecular dynamics simulations, and single-channel electrophysiology, that CymA forms a monomeric 14-stranded ?-barrel with a large pore that is occluded on the periplasmic side by the N-terminal 15 residues of the protein. Representing a previously unidentified paradigm in OM transport, CymA mediates the passive diffusion of bulky molecules via an elegant transport mechanism in which a mobile element formed by the N terminus acts as a ligand-expelled gate to preserve the permeability barrier of the OM.
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Project description:LMO2 was first discovered through proximity to frequently occurring chromosomal translocations in T cell acute lymphoblastic leukaemia (T-ALL). Subsequent studies on its role in tumours and in normal settings have highlighted LMO2 as an archetypical chromosomal translocation oncogene, activated by association with antigen receptor gene loci and a paradigm for translocation gene activation in T-ALL. The normal function of LMO2 in haematopoietic cell fate and angiogenesis suggests it is a master gene regulator exerting a dysfunctional control on differentiation following chromosomal translocations. Its importance in T cell neoplasia has been further emphasized by the recurrent findings of interstitial deletions of chromosome 11 near LMO2 and of LMO2 as a target of retroviral insertion gene activation during gene therapy trials for X chromosome-linked severe combined immuno-deficiency syndrome, both types of event leading to similar T cell leukaemia. The discovery of LMO2 in some B cell neoplasias and in some epithelial cancers suggests a more ubiquitous function as an oncogenic protein, and that the current development of novel inhibitors will be of great value in future cancer treatment. Further, the role of LMO2 in angiogenesis and in haematopoietic stem cells (HSCs) bodes well for targeting LMO2 in angiogenic disorders and in generating autologous induced HSCs for application in various clinical indications.
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Project description:There are intense interests in discovering proregenerative medicine leads that can promote cardiac differentiation and regeneration, as well as repair damaged heart tissues. We have combined zebrafish embryo-based screens with cardiomyogenesis assays to discover selective small molecules that modulate heart development and regeneration with minimal adverse effects. Two related compounds with novel structures, named as Cardiomogen 1 and 2 (CDMG1 and CDMG2), were identified for their capacity to promote myocardial hyperplasia through expansion of the cardiac progenitor cell population. We find that Cardiomogen acts as a Wnt inhibitor by targeting ?-catenin and reducing Tcf/Lef-mediated transcription in cultured cells. CDMG treatment of amputated zebrafish hearts reduces nuclear ?-catenin in injured heart tissue, increases cardiomyocyte (CM) proliferation, and expedites wound healing, thus accelerating cardiac muscle regeneration. Importantly, Cardiomogen can alleviate the functional deterioration of mammalian hearts after myocardial infarction. Injured hearts exposed to CDMG1 display increased newly formed CMs and reduced fibrotic scar tissue, which are in part attributable to the ?-catenin reduction. Our findings indicate Cardiomogen as a Wnt inhibitor in enhancing injury-induced CM proliferation and heart regeneration, highlighting the values of embryo-based small molecule screens in discovery of effective and safe medicine leads.
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