Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24

Abstract

Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or nonspecific binding to unintended membrane protein targets; however, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids, and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs but also highlight new approaches for documenting off-target drug binding.

Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology

“…However, a peptide substrate lacking this farnesyl-cysteine (and four additional residues N-terminal to it) now exhibited multiple cleavage products in the vicinity of the distal site, including not only the canonical Prelamin A site but also two other adjacent positions. (A later followup study from the same group subsequently reported only a single cleavage of the same, truncated peptide substrate lacking the farnesylcysteine and four additional N-terminal residues, but did report "… slow product formation …" compared to the farnesylated version [38].) Both studies demonstrate that, for peptides derived from Prelamin A, deletion of residues at a location removed from the canonical cleavage site of Ste24 has a marked effect on HsSte24 proteolytic activity.…”

“…Improper prelamin A processing is also correlated with deleterious alterations to adipose tissue localization and accumulation, known as lipodystrophy, due to the toxic effects of prelamin A accumulation leading to altered expression of genes responsible for adipocyte proliferation and differentiation [33,34]. Additionally, the inability of some AIDS patients to produce and maintain adipose tissue, acquired from antiretroviral therapy, likely results from off-target interactions of HIV (aspartyl) protease inhibitor drugs with HsSte24 [35][36][37][38]. Because of HsSte24's roles in these syndromes, the Zmpste24 −/− mouse has been suggested as a model for senescent wound healing [39] and lipodystrophy [40].…”

Mass spectrometry-enabled structural biology of membrane proteins

“…However, the advent of Orbitrap instrumentation with higher sensitivity and resolving power has enabled applications to be developed that would be impossible with older generations of instruments. For example, the Orbitrap has enabled the study of multiple concomitant binding events to MPs for lipids that differ in mass by as little as 12 Da [133], allowing the relative binding affinities of different lipids/ligands to be quantified, the study of lipids co-purified with a MP, and drug binding in the presence of lipids [131,133]. Indeed, the resolving power of the Orbitrap instrument may also assist with the identification of proteoforms, glycoforms and post-translational modifications.…”

“…Studies of the ABC transporter P-gp [27] revealed that this protein preferentially binds to negatively charged lipids and cardiolipins (over zwitterionic lipids), and that binding to an inhibitor enhances cardiolipin binding, demonstrating that native MS could be used to probe synergistic binding events. Native MS has also been used to capture off-target drug interactions with MPs [131] and, excitingly, to monitor ligand binding to G-protein-coupled receptors (GPCRs), enabling native MS methods to be applied to GPCR drug discovery [132].…”

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

“…Yeast 30,33 and human 23,30,[34][35][36][37] Ste24 orthologs have been the most rigorously characterized Ste24 family members both functionally and structurally. Additionally, functional complementation between human and yeast Ste24 orthologs has been established in vivo where HsSte24 rescues defects in a-factor biogenesis associated with Ste24p knockout yeast (ste24Δ).…”