Screening cascade targeting PPIs


Screening cascades need to be designed specifically for the target chosen and for the biological activity for the target. Projects targeting the interactions between two proteins (protein-protein interaction [PPI]) targets frequently have no enzyme activity that can be measured and therefore the screening cascade needs to employ different non-enzymatic methods to measure compound efficacy. Whilst there are many methods around a recent paper published exemplifies an excellent screening cascade for targeting a PPI (1).

In this paper the group were targeting a DNA-damage repair complex involved in the Fanconi anaemia pathway, a mechanism involved in tumour resistance. Specifically the interaction of FA complementation M group (FANCM) and the RecQ-mediated genome instability protein (RMI) complex.

The screening cascade used a fluorescence polarisation (FP) assay as the primary screen, followed by hit confirmation in an alpha screen-based proximity assay to demonstrate inhibition of the PPI. Surface Plasmon Resonance (SPR) and Isothermal titration calorimetry (ITC) were then used to demonstrate compound binding to the RMI core complex.

The group was specifically targeting the interaction within the FANCM complex, between RMI and MM2 and the FP assay was designed to measure this interaction. Therefore a standard format (figure 1) with the labelled MM2 protein having a Kd <5 nM. The assay was miniaturised to 384 format and the screen produced Z` of 0.53. The alpha screen assay (figure 2) was a proximity-based assay with MM2 bound to the streptavidin donor bead and the RMI complex bound to the acceptor bead. The Z` for the alpha screen assay (0.75) was better than the FP assay. Despite this the FP assay was chosen for the initial single point screen rather than the alpha screen, presumably on cost grounds.

fig1Trevor

Figure 1 Schematic of the fluorescence polarisation (FP)

fig2Trevor

Figure 2 Schematic of the alpha screen proximity assay

The alpha screen was therefore used as the counter screen and interestingly on the hit published there was a 10 fold discrepancy in the IC50 from the FP assay (450 uM) vs. alpha screen (36 uM). Direct interaction of this hit to the RMI complex was confirmed by both SPR and ITC that produced a consistent Kd of 7.8 and 3.4 uM respectively.

What is particularly nice about this cascade is that three of the screens, the FP assay, alpha screen and SPR can all be run in high throughput therefore although the publication only talks about a pilot screen of 74,000 compounds it could easily be scaled up to many more compounds. The only lower-throughput assay is the ITC, which is at the end of the cascade.

 

Blog writted by Trevor Askwith

References

  1. Voter AF, Manthei K a, Keck JL. A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway. J. Biomol. Screen. (March 8, 2016). doi: 10.1177/1087057116635503.
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