Query-guided protein-protein interaction inhibitor discovery

Abstract

Protein-protein interactions (PPIs) are central to biological mechanisms, and can serve as compelling targets for drug discovery. Yet, the discovery of small molecule inhibitors of PPIs remains challenging given the large and typically shallow topography of the interacting protein surfaces. Here, we describe a general approach to the discovery of orthosteric PPI inhibitors that mimic specific secondary protein structures. Initially, hot residues at protein-protein interfaces are identified in silico or from experimental data, and incorporated into secondary structure-based queries. Virtual libraries of small molecules are then shape-matched against the queries, and promising ligands docked to target proteins. The approach is exemplified experimentally using two unrelated PPIs that are mediated by an α-helix (p53/hDM2) and a β-strand (GKAP/SHANK1-PDZ). In each case, selective PPI inhibitors are discovered with low μM activity as determined by a combination of fluorescence anisotropy and ¹H-¹⁵N HSQC experiments. In addition, hit expansion yields a series of PPI inhibitors with defined structure-activity relationships. It is envisaged that the generality of the approach will enable discovery of inhibitors of a wide range of unrelated secondary structure-mediated PPIs.

Fig. 1. Schematic illustrating query-guided PPI inhibitor discovery in which small-molecules can potentially mimic any secondary structure. The discovery workflow is implemented through several stages that combine established computational tools and experimental validation. Initially a query is built that incorporates the key secondary structural motif and hot residues from the PPI. A virtual library of small molecules is then shape-matched against the query, and promising compounds docked against the target protein. Candidate inhibitors are then subjected to experimental screening and characterisation, enabling selection of hits for: (i) clustering, near neighbour expansion and further screening by reiteration of step II and III of the workflow; or (ii) further development.

Fig. 2. Discovery of small-molecule inhibitors of the p53/hDM2 interaction: (a) close-up of the p53/hDM2 interaction structure (PDB ID: 1YCR), p53 (cyan), with key side chains Phe19, Trp23 and Leu26 highlighted, docks into the hDM2 cleft with the hDM2 surface defined by the Phe19 (violet), Trp23 (magenta) and Leu 26 (green) pockets, into which each corresponding hydrophobic amino acid projects; (b) ribbon representation of the p53_17–29 transactivation domain with key side chains Phe19, Trp23 and Leu 26 highlighted together with the primary sequence (below); (c) query that incorporates the hot residues and a “hydrocarbon” backbone; (d) query that incorporates the hot residues and a peptidic backbone. (e) Overlay of exemplar shape-matched hit compounds and queries; (f) structures of the exemplar hit compounds as representatives of the most populated clusters; (g) ligand efficiency plot for the 37 hits obtained from the computational workflow (LE = 1.4 × pIC₅₀/nHA, nHA: number of non-hydrogen atoms, IC₅₀ obtained by fluorescence anisotropy competition: 150 nM hDM2 and p53_15–31Flu, 40 mM phosphate, pH 7.4, 200 mM NaCl and 0.02 mg ml⁻¹ BSA); (h) mapping of the chemical shift perturbations in hDM2 for hit compounds from cluster A, cluster B and cluster C. Colour variation is associated with a chemical shift perturbation that goes from 0 ppm (white) to 0.2 ppm (red).

Fig. 3. Development of a GKAP query for inhibitor screening against the GKAP/SHANK1-PDZ interaction; (a) close-up of the GKAP/SHANK1 PDZ interaction structure (PDB ID: 1Q3P), GKAP (cyan), with key side chains Thr3 and Leu5 highlighted, interacts with SHANK1 PDZ domain mainly through polar contacts (orange area); however, hydrophobic effects play a significant role in binding (magenta area); (b) cartoon representation of the GKAP_1–5 peptide with key side chains Thr3 and Leu5 highlighted together with the primary sequence (below); (c) X-ray crystal structure of Ac-Glu-Ala-Gln-Thr-Arg-Phe peptide (L6F) bound to SHANK1-PDZ (PDB ID: 7A00) illustrating good correspondence with the position of key recognition groups observed for the wild-type sequence (PDB ID: 1Q3P); (d) close up of the interactions between the C-terminus of the L6F GKAP peptide and SHANK1 PDZ (H-bonds orange, other contacts yellow dashed lines); (e) close up of the interactions between the C-terminus of the wt GKAP peptide and SHANK1 PDZ; (f) binding of wild type (left) and L6F (right) GKAP peptide to SHANK1, monitored by ITC (25 °C in 20 mM Tris, 150 mM NaCl, pH 7.5, heats of peptide dilution were subtracted from each measurement raw data) with data analysed using Microcal Origin 8 and fitted to a one-binding site model; (g) GKAP query whereby the Thr3 side chain is retained together with a Phe in lieu of a Leu side chain alongside key backbone donor (NH) and acceptor (CO) groups; (h) structure of the hit compounds Z-1 and Z-2 identified from single point screening workflow using the query in panel (g); (i) overlay of the compound Z-1 and the query (grey); (j) fluorescence anisotropy competition assay for compound Z-1 (FITC-GKAP 50 nM, SHANK1-PDZ 1 μM, pH 7.4, 20 mM Tris, 150 mM NaCl, 0.01% Triton-X-100 buffer); (k) expansion of the ¹H–¹⁵N HSQC spectra of ¹⁵N labelled SHANK1 in the absence (red) and presence of compound Z-1 (compound : protein molar ratio 1 : 1 yellow, 2 : 1 green, 4 : 1 blue). Peaks indicated with asterisk undergo significant changes in intensity upon binding (SHANK1-PDZ 50 μM, pH 7.4, 5 mM Tris, 100 mM NaCl buffer).

Fig. 4. Development of p53/hDM2 inhibitors, (a) synthetic route to hit A1; (b) selected chemical structures and associated IC₅₀ values (fluorescence anisotropy competition: 150 nM hDM2 and p53_15–31Flu, 40 mM phosphate, pH 7.4, 200 mM NaCl and 0.02 mg ml⁻¹ BSA) of developed inhibitors (increasing colour intensity of the wedges denotes increased potency, *reduced anisotropy in the assay attributed to solubility †non-specific interactions observed); (c) ligand efficiency plot for selected inhibitors shows exploration of the chemical space starting from selected hit A1 (green sphere). Coloured shapes are assigned to describe inhibitors from the first (triangle), second (square, diamond and inverse triangle) and third series (star). (d) Mapping of chemical shift perturbations in hDM2 for selected inhibitors (750 MHz, 100 mM phosphate, pH 7.4, 2.5% glycerol, 1 mM DTT, increasing concentration of compound was titrated into 50 μM hDM2 and CSP wasdetermined at 1 : 2 protein : compound ratio). Colour variation is associated with a chemical shift perturbation from 0 ppm (white) to 0.2 ppm (red).

Fig. 5. Molecular dynamics simulation of hit compound A1 indicates coexistence of two conformations at the hDM2 binding site. (a) Root mean square deviations during a 10 nanoseconds molecular dynamics simulation of A1/hDM2. Red represents the core and green represents the tail. (b) Chemical structure of A1 highlighting core and tail. (c) MD snapshot at 0 ns shows the folded conformation, which occupies only two of the three hot-spots, (d) MD snapshot at 8 ns shows the extended conformation which occupies the three hot-spots. p53 hot residue side chains are shown in cyan, Phe19, Trp23 and Leu26 pockets of hDM2 are highlighted in violet, magenta and green, respectively.