Identification of molecular glues of the SLP76/14-3-3 protein-protein interaction

Abstract

The stabilisation of protein-protein interactions (PPIs) through molecular glues is a novel and promising approach in drug discovery. In stark contrast to research in protein-protein inhibition the field of stabilisation remains underdeveloped with comparatively few examples of small-molecule stabilisers of PPIs reported to date. At the same time identifying molecular glues has received recent sustained interest, especially in the fields of targeted protein degradation and 14-3-3 PPIs. The hub-protein 14-3-3 has a broad interactome with more than 500 known protein partners which presents a great opportunity for therapeutic intervention. In this study we have developed an HTRF assay suitable for HTS of the 14-3-3/SLP76 PPI and have completed a proof of concept screen against a chemically diverse library of 20 K molecules. The adaptor protein SLP76 has been reported to interact with 14-3-3 proteins downstream of the TCR playing an important role in mediating its own proteasomal degradation. We believe that stabilisation of this PPI could be exploited to potentiate degradation of SLP76 and therefore inhibit TCR signalling. This would represent an interesting alternative to other approaches in the field of targeted protein degradation. Here we disclose 16 novel stabilisers of the 14-3-3/SLP76 PPI across multiple different chemotypes. Based on the early results presented here we would recommend this approach to find molecular glues with broad applicability in the field of 14-3-3 PPIs.

Fig. 1. Assay set-up for the 14-3-3γ/SLP76 PPI. a) SPR binding assay of the SLP76-SH2 construct binding to 14-3-3γ in the presence of 5% DMSO. The K_D value has been extrapolated from the binding curve reported on the right of the sensorgrams. b) HTRF assay set-up. The two proteins of interest, SLP76 and 14-3-3γ have been labelled with Tb and AF647, respectively, to generate a suitable matched pair of donor molecule (Tb) and acceptor molecule (AF647). The donor molecule, Tb labelled 14-3-3, is excited at the proper wavelength of 337 nm. When the acceptor is not in the proximity (no binding) no signal is detected. When SLP76 bound to 14-3-3 brings the acceptor molecule AF647 close to the donor, energy transfer occurs between the fluorophores pair. A radiation with the wavelength of 665 nm is consequently emitted to generate a read-out (665/620 nm). All HTRF protein systems used in this work are reported in the legend.

Fig. 2. Graphical representation of the high throughput screening performance. a) Dots representation of the HTS in the plate format. Potential inhibitors are represented as grey dots, potential stabilisers are represented as green dots. DMSO control and negative control are grouped in plate columns 1, 2 and 23, 24 respectively. The calculated overall Z′ factor for the assay was 0.72. b) An initial cut-off of 20% stabilisation was used to select hits to follow-up.

Fig. 3. Graphical representation of the repeated 1136 hits tested on 14-3-3/SLP76 and PPI X. a) Representation of the hits expressed as percent inhibition against 14-3-3γ/SLP76 and IL17/IL17R. Potential 14-3-3/SLP76 stabilisers are represented as green dots. b) Selection conditions applied to the hits were to retain compounds that showed an effect on SLP76 ≥ −30% and simultaneously at least a two-fold greater stabilisation than IL17 PPI, SPL76 ≥ 2*IL17.

Fig. 4. Representation of the 64 small molecules selected on the basis of SLP76 stabilisation and IL17/IL17R counter-screen and 14-3-3γ/SLP76 dose response curves for the best 16 compounds. a) Selection of 64 small molecules represented as green dots within dotted lines. b) Dose response curves of the best 16 out of the 64 compounds tested. Estimated EC50 values for every compound are reported in (Table 1).

Fig. 5. Comparison of the dose response assay of the 16 compounds on the systems IL17/IL17R, 14-3-3ζ/p27 and 14-3-3γ/SLP76. a) Dose response of the 16 compounds on IL17/IL17R. None of the compounds showed an effect on IL17/IL17R. b) Dose response of the 16 compounds on 14-3-3ζ/p27. 8 out 16 compounds showed an effect greater than 1.5 normalised response. c) All 16 compounds repeated on 14-3-3γ/SLP76.

Fig. 6. Combined set of assays example for the compound 2 and 4. a) HTRF dose response comparison of the three systems used. HTRF dose ratio assay on the 14-3-3γ/SLP76-SH2 system. The arrow represents the increment in potency upon increasing compound concentration. Sensorgrams generated by SLP76-SH2 flowing over 14-3-3γ in absence (as reference) and in the presence of different concentration of compounds. The plot of the reference points taken from the sensogram used to extrapolate the SPR apparent EC50 is reported to the right. The reference points before and after the dissociation phase are highlighted in the sensograms (bottom left). b) The same set of assays are repeated on compound 4.