Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification

Abstract

The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors.

Hunting Molecular Targets for Anticancer Reagents by Chemical Proteomics

“…Furthermore, the instability of the conventional stationary matrices (such as resin, agarose, polyglycidylmethacrylate, and magnetic particles [28][29][30][31][32][33][34] ) might lead to low efficiency in affinity-based target identification. In order to overcome such drawbacks, many researchers have sought to develop innovative solutions for eliminating the unfavorable nonspecifically bound proteins, termed background proteins.…”

Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography

“…Thus, specific binding proteins can be quantitatively found from among a huge amount of non-specific binders. system consisting of ampicillin-and penicillin-binding proteins to evaluate non-specific binders when the compound was immobilized with different amino-reactive beads [34]. They succeeded in identifying a primary target as well as its associated protein complex by using Dynal M-270 magnetic beads to pull down drug-binding proteins.…”

Mass spectrometry approaches to monitor protein–drug interactions

“…The laborious synthesis of inactive analogs might be avoided by adding a competition-step with the free compound into the experimental work-flow (Fig 3b). In this approach, target proteins are identified by their reduced [120] binding to the affinity matrix in the presence of excess free compound as compared to the vehicle control [81][82][83][84][85][86]. Probe matrices or reactive probes selectively binding to a wide range of proteins of a particular target class can be employed to establish target selectivity of free test compounds in competition-based experiments.…”