qHTS Assay for Inhibitors of RanGTP induced Rango (Ran-regulated importin-beta cargo) - Importin beta complex dissociation
The small GTPase Ran regulates transport of macromolecules between the nucleus and the cytoplasm (nuclear transport) and has important functions in mitotic spindle assembly, nuclear envelope (NE) formation and nuclear pore complex (NPC) assembly . RanGTP gradient is thought to promote spindle assembly by providing a spatial clue to microtubule nucleation and Aurora A activation . Because more ..
BioActive Compounds: 242
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03 MH090808-01A1
Assay Submitter (PI): Petr Kalab
NCGC Assay Overview:
The small GTPase Ran regulates transport of macromolecules between the nucleus and the cytoplasm (nuclear transport) and has important functions in mitotic spindle assembly, nuclear envelope (NE) formation and nuclear pore complex (NPC) assembly . RanGTP gradient is thought to promote spindle assembly by providing a spatial clue to microtubule nucleation and Aurora A activation . Because the localized release and binding of importin alpha cargos relays this spatial clue to SAFs, inhibitors of the Rango - importin beta complex can then be used in further understanding their role in cell cycle specifically in mitosis progression. In addition RanGTP, importin beta, and importin alpha has been shown to regulate the activation of Aurora A though its binding to TPX2 [2-4]. Activation of Aurora A has been linked to different types of cancer and a number of Aurora kinase inhibitors are being developed as potential canter therapeutics [5-6]; therefore, identified inhibitors of this complex could be developed into potential anti-mitotic probes for cancer treatment.
Purified recombinant proteins Rango - importin beta and RanGTP-RCC1 stock premixes were provided by the assay submitter. The assay used a fluorescence resonance energy transfer (FRET) Rango biosensor that contains the IBB of human snurportin 1 flanked by yellow fluorescent protein (EYFP) at the amino terminus and cerulean CFP10 at the carboxy terminus. Rango assumes extended conformation when bound to importin beta and displays low FRET. RanGTP binding to the complex (in the presence of RCC1) releases the Rango from the complex. The flexibility of the IBB in the freed Rango allows the donor - acceptor fluorophores to interact, resulting in a high FRET signal . Inhibitors will therefore be identified based on the ability to interfere with the FRET signal increase. DMSO treated and no RanGTP-RCC1 (buffer only) wells were used as negative and positive controls respectively. FRET readings were obtained at two time points. FRET reading prior to importin beta addition (pre-read) were used flag potential compound interference; FRET reading after importin beta addition (post-read) were used to identify inhibitors.
NCGC Assay Protocol Summary:
Two and a half uL/well of Rango- importin beta premix solution (0.1 uM Rango, 0.25 uM importin beta, 1x PBS, 1mM DTT, 0.02% Tween-20, 0.1% BSA final concentrations) was dispensed into 1536-well assay plates (Greiner, solid black medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Compound solution (23 nL) was transferred to the assay plate using Kalypsys pin tool equipped with a 1536-pin tool and incubated at room temperature for 5 min followed by a FRET pre-read using the PerkinElmer Envision plate reader. Two and a half uL/well of RanGTP-RCC1 premix solution (0.6 uM Ran, 2mM GTP, 0.2 uM RCC1, 1x PBS, 1mM DTT, 0.02% Tween-20, 0.1% BSA final concentrations) were then added and incubated at room temperature giving a final reaction volume of 5uL. After 45min FRET post-read was obtained via the Envision plate reader using the same optics and settings as the pre-read (CFP/YFP optimized dual optical module D450/D505; excitation mirror CFP 430/24 nm; and emission filters CFP 486/10 nm and YFP 530/10 nm).
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)