Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90

• Published in: European journal of pharmaceutical sciences Vol. 76; pp. 83 - 94
• Main Authors: Riccardi Sirtori, Federico, Caronni, Dannica, Colombo, Maristella, Dalvit, Claudio, Paolucci, Mauro, Regazzoni, Luca, Visco, Carlo, Fogliatto, Gianpaolo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30-08-2015
• Subjects: Antitumor agents; Automation, Laboratory; Binding Sites; Binding, Competitive; Crystallography, X-Ray; Drug Discovery - methods; Flow Injection Analysis; Fluorescence Polarization; Fragment based approach; High-Throughput Screening Assays; Hsp90; HSP90 Heat-Shock Proteins - antagonists & inhibitors; HSP90 Heat-Shock Proteins - chemistry; HSP90 Heat-Shock Proteins - metabolism; Ligands; Magnetic Resonance Spectroscopy; Mass spectrometry; Medicinal chemistry; Protein Binding; Reproducibility of Results; Small Molecule Libraries; Spectrometry, Mass, Electrospray Ionization; Hsp90; Medicinal chemistry; Antitumor agents; Mass spectrometry; Fragment based approach
• ISSN: 0928-0987; 1879-0720
• DOI: 10.1016/j.ejps.2015.05.001

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ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand–target complexes in the gas phase and allows inference of ligand–target binding in solution.
In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer.
In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100μM, were tested to assess the reliability of ESI-MS for the study of protein–ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry.
After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90.
In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments.
These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with unpredicted or allosteric sites, without the need of any binding probes.