A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP

Abstract

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U- H]13 CH 3 -methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding.Keywords fragment-based drug design, AAA+ ATPase p97, valosin-containing protein (VCP)

Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97

“…The affinity (Kd) of ADP to p97-ND1 was measured to be 200 ± 14 nM using a Langmuir model based on steady state measurements of a dilution series. This value is in agreement with reported affinities for ND1 constructs of similar lengths; e.g., a value of 200 nM was reported for an ND1 fragment spanning amino acids 1-458, and a value of 98 nM for an ND1 fragment spanning amino acids 1-480 [26,29]. The kinetic analysis of the sensorgrams indicated a clear 1:1 binding model for ADP.…”

“…The remaining fragments showed maximum STD-effects in a range from 10.5% (TROLL9) up to 72.5% (TROLL6). Similar results were obtained in the previously reported fragment screen with p97 [26]. An overview of the STD-effects is shown in Fig.…”

2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition

“…Several classes of inhibitors, including those that act in an allosteric or competitive manner, have been identified that impair p97 ATPase activity (10–14, 21–27). Determination of the cryo-EM structure at 2.3 Å resolution of p97 in complex with UPCDC30245, a phenyl indole derivative (fig.…”

Small‐Molecule Inhibitors of the Proteasome's Regulatory Particle

“…To explore the multiple binding site on p97 and its dynamics further, a 2485‐member fragment library was screened by surface plasmon resonance (SPR), followed by NMR spectroscopy, to identify fragments that bound to the D1 or N domain of p97 . Even though this strategy is less high‐throughput than previously mentioned assays, it is useful in identifying fragments for studying p97’s dynamic nature, and the results could be usable for further lead optimization of future inhibitors.…”