Linking Ubiquitin Research to Drug Discovery

Case studies that may be addressed by Ubiquigent Drug Discovery Services employing Horizon’s cell line technology

Abbreviations:

KO = Knock Out                 WT = Wild Type

Target discovery and validation:

Protein target modifications

Select the question below to see the answers

Is the abundancy of my protein of interest regulated by the ubiquitin system? What happens to the abundancy of my protein of interest if a component of the ubiquitin system is knocked out or mutated in a site directed manner such as a; 1) DUB catalytic site mutation, 2) full DUB KO, 3) KO of an E3 ligase or a sub-component of an E3 ligase such as a substrate binding adaptor, 4) KO of an E2 conjugating enzyme?

Monitor up or down regulation of your protein of interest in the ubiquitin system modified relative to the control WT HAP1 cell line.

Readout methods may include for example; 1) Western blotting if an antibody is available, 2) tagging your protein of interest with a fluorescent protein and monitoring changes in fluorescence.

Is the localisation of my protein of interest regulated by the ubiquitin system? What happens to the localisation of my protein of interest if a component of the ubiquitin system is knocked out or mutated in a site directed manner as above?

Monitor the sub-cellular localisation and/or protein/protein interactions of your protein of interest tagged with a fluorescent protein.

Is my cell phenotype of interest regulated by the ubiquitin system? What happens to a phenotype of the HAP1 cell line (in the presence and/or absence of other ligands such as growth factors) if a component of the ubiquitin system is knocked out or mutated in a site directed manner as above?

We can transfer your phenotypic assay of choice to Ubiquigent and monitor the outcome of addressing this question using modified and control   HAP1 cell lines.

Do specific mutations in my ubiquitin system protein of interest modify the proteins with which it interacts?

We can perform pull down experiments and mass spectrometry identification of the proteins associated with your WT and mutant ubiquitin system protein of choice.

Is there redundancy in my cell based model to tolerate KO of a specific ubiquitin system protein? For example for identifying synthetic lethal pathways.

We can transfer your phenotypic assay of choice to Ubiquigent and monitor pathway redundancies.

Address any of the above questions but where you wish to KO or mutate a non-ubiquitin system protein and determine its effect on an ubiquitin system protein.

Methodologies as above.

I'd like to find the gene(s) important in my ubiquitin system or ubiquitin system regulated pathway of interest.

Utilising the ‘gene trapping’ platform we can expose a library of cells – in which 99% of the expressed genes have been inactivated – to an assay [provided by the client or that is developed with the client] that is capable of selecting cells with a specific phenotype.  Using this approach we can identify genes that are critical to your ubiquitin system pathway of interest.

Gene promoters

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What is the activity of my ubiquitin system gene promoter under control and other experimental conditions?

We can fuse a reporter gene downstream of your ubiquitin system promoter of interest and measure reporter activity under control and experimental conditions.

Compound mechanism of action studies

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Does my compound effect it's mechanism of action via the predicted target? For example your Ubiquigent generated DUBprofiler™ data may suggest that the mechanism of action of your compound may be via the inhibition of DUB A, B or C and you wish to determine which DUB inhibition is relevant to the mechanism of action of the compound (NB: A custom cell line carrying multiple DUB KOs or site directed mutations can be generated for you).

Do we see a similar phenotype in WT cells treated with your compound compared to the mutant cell line where the predicted target of your inhibitor has been knocked out? If so then the inhibition of the predicted target of your compound may indeed explain the compound’s mechanism of action.

Furthermore does my compound have no further effect on the cell line carrying a mutation or KO in the predicted target protein of the compound?

Biomarkers for patient stratification/personalised medicine

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Does a specific defined mutation(s) define responsive or resistant patient populations?

Generate a cell line carrying the mutation(s) in order to test or confirm the patient stratification hypothesis using a tool compound and/or an siRNA or similar approach.

Applying a clinically relevant genetic background

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Can I perform any of the above experiments against a clinically relevant genetic background?

Yes.  We can have cell lines generated carrying any one or multiple genetic modifications in order to model a clinically relevant genetic background of choice.