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Detection and structural characterization of ras oncoprotein-inhibitors complexes by electrospray mass spectrometry
A K Ganguly1, B N Pramanik, E C Huang
1Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Abstract:
MS based methodology employing electrospray ionization (ESI) is described for the detection of ternary complexes in which SCH 54292 or SCH 54341 and GDP are noncovalently bound to oncogenic ras protein. The observed molecular weights of 19,816 and 19,570 Da confirmed the presence of noncovalent complexes of ras-GDP-SCH 54292 and ras-GDP-SCH 54341, respectively. We have also performed selective chemical modification of lysine residues of the ras protein complex followed by enzymatic digestion and on-line LC-ESI MS peptide mapping to determine protein-drug binding topography. There was a good correlation between nucleotide exchange inhibition as determined by the enzyme assay and evidence of complex formation as determined by MS.
Insights
Mass spectrometry detects ternary complexes of oncogenic ras protein with GDP and inhibitors SCH 54292 or SCH 54341. This method confirms drug binding and aids in understanding protein-drug interactions for potential therapeutic development.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Pharmacology
Background:
- Oncogenic ras proteins are key targets in cancer therapy.
- Understanding the noncovalent binding of small molecules to ras is crucial for drug development.
Purpose of the Study:
- To develop and validate a mass spectrometry (MS) based methodology for detecting ternary complexes of ras protein.
- To investigate the binding of inhibitors SCH 54292 and SCH 54341 to ras-GDP complexes.
- To map the protein-drug binding topography using chemical modification and MS.
Main Methods:
- Electrospray ionization mass spectrometry (ESI-MS) for complex detection.
- Selective chemical modification of lysine residues.
- Enzymatic digestion and on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) peptide mapping.
Main Results:
- Detection of noncovalent ras-GDP-SCH 54292 (19,816 Da) and ras-GDP-SCH 54341 (19,570 Da) complexes.
- Identification of drug binding sites on the ras protein through peptide mapping.
- Correlation between MS-detected complex formation and enzyme assay results for nucleotide exchange inhibition.
Conclusions:
- ESI-MS is a robust method for characterizing noncovalent ternary complexes involving oncogenic ras.
- The study provides insights into the binding interactions of SCH 54292 and SCH 54341 with ras protein.
- This approach facilitates the structural elucidation of protein-drug interactions relevant to cancer therapy.