Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface

Abstract

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed.

Figure 1. Structural Characterization of Small Molecules Targeting the pVHL:HIF-1α Interactions

(A) General binding mode of small molecule inhibitors of pVHL:HIF-1α, based on the crystal structure of the VCB ternary complex with 1 bound, as shown in (D). The small molecule is shown in stick representation, the left-hand side (LHS) and right-hand side (RHS) groups are shown as green and red spheres, respectively, as represented in the model structure below. VCB is shown in surface representation (pVHL in green, Elongin C in gold, and Elongin B in purple). (B–E) Crystal structures of VCB in complex with a 19-mer HIF-1α peptide (DEALAHypYIPMDDDFQLRSF) (B), 3 (C), 1 (D), and 2 (E) are shown with the ligands as stick representation bound on the surface of pVHL (green). Residues in the HIF-1α binding site of pVHL are shown in magenta in stick representation. Residues are labeled in (C). See also Figure S1 and Table S1.

Figure 2. Biophysical Characterization of Compound 1 Binding to VCB

(A) WaterLogsy, STD, and CPMG NMR spectroscopy. (B) Isothermal titration calorimetry. (C) Fluorescence polarization. (D) Differential scanning fluorimetry. See also Figure S3.

Figure 3. Small Molecule Library Used to Probe the pVHL:HIF-1α Interaction

(A) Micromolar small molecule inhibitors 1–3 used for the biophysical and structural characterization of the pVHL:ligand interactions and fragments used to probe individual subsites. (B) Fragments designed to probe more than one subsite, including reference molecules used to calculate group efficiencies and group lipophilicity efficiencies. (C) Library of compounds placing a t-butyl, phenyl, pyridyl, or Me-(is)oxazole group at the left-hand site (LHS), right-hand site 1 (RHS1), or 2 (RHS2). ClogP values are not defined for groups and depend on the context of the whole molecule. The values listed were calculated for parent molecules containing a methyl attached to each group. See also Figure S2.

Figure 4. X-Ray Crystal Structure of Fragment 13 Bound to VCB, Solved at 2.50Å of Resolution

Fragment 13 is shown in stick representation (magenta carbons), whereas pVHL is shown in light yellow cartoon representation. Key amino acids interacting with the fragment are shown in sticks, with carbon atoms in the same color. The F_o-;F_c omit electron density map associated with the fragment is shown as a green mesh contoured at 3σ. See also Figure S1.

Figure 5. Group Efficiencies and Group Lipophilic Efficiencies of Small Molecules Binding to VCB

Values for GE (black) and GLE_AT (red) for each group at the different positions (LHS, RHS1, and RHS2) are listed in (A), and their distributions, according to position or group, respectively, are shown in (B).