title: 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-(2)H] (13)CH3-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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA Co-first authors.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Chimenti
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA Co-first authors.
authors.forename: Stacie L
authors.initials: SL
authors.lastname: Bulfer
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.
authors.forename: R Jeffrey
authors.initials: RJ
authors.lastname: Neitz
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Renslo
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: James
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Arkin
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA michelle.arkin@ucsf.edu Mark.Kelly@ucsf.edu.
authors.forename: Mark J S
authors.initials: MJ
authors.lastname: Kelly
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021281:Quantitative Structure-Activity Relationship; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D008024:Ligands; D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; C000614178:VCP protein, human; D000074405:Valosin Containing Protein
keywords: AAA+ ATPase p97; fragment-based drug design; valosin-containing protein (VCP)