STUDY OF COVALENT AND NON COVALENT INTERACTIONS OF BIOPOLYMER BY MASS SPECTROMETRY

ESI-MS screening methods directly detect ligand-target non covalent complexes in the gas phase and allow inference of affinity (and specificity) of the ligand-target interaction in solution [1, 2]. The identity of different complexes can be directly assessed as the mass of each molecule works as intrinsic label. Biopolymers can be screened either as a single component or a mixture of different targets; in this way it is possible to determine the selectivity of a new chemical entity for different targets. On the other hand, using ESI-MS it is also possible to identify, within a mixture, components that selectively bind the active site of a certain biopolymer and could be profitably used to screen libraries of known compounds. The aim of this PhD project was the study by ESI mass spectrometry of different noncovalent complexes formed with biopolymers identified as possible therapeutic targets (Mismatch Repair Mechanism and Hsp90). The noncovalent interaction between biological targets and possible ligands were studied in order to identify potential inhibitors. The binding affinities determined by ESI-MS were compared to existing data obtained by solution phase methods. A novel MS-based method was implemented for testing different biopolymer, of therapeutic interest, against a library of fragments (molecular weight 100-300 Da) constituted of about 2,000 compounds. Particularly this approach was used for the identification of new molecules able to recognize TG mismatched base pairs in DNA that are responsible for most of the common mutations leading to formation of tumors in humans. TG mismatches are particularly abundant in cells lacking mismatch repair mechanism (MMR). MMR is involved in the correction of DNA polymerase errors that escape proofreading activity. MMR deficiency increases 50-1000-fold spontaneous mutation rates (microsatellite instability MSI) and in addition, MMR deficiency can lead to resistance to several chemotherapeutic agents (DNA damaging agent) [3]. An ESI-MS method was used to study the complexes formed between different DNA duplexes and minor groove binders and intercalator compounds. A hairpin DNA sequence (CTGGsm) bearing a single T:G mismatch and a matched hairpin DNA sequence (CCGG) were prepared and used to set up the method. Two DNA sequences were also synthesized: a self complementary G:C rich DNA sequence (HFM) and a polyAT DNA duplex (A5TG). The association constants (KAs) were directly determined from the MS spectrum and the amount of bound ligand was used to determine the selectivity of a binder among the different DNA sequences. Minor groove binders confirmed their selectivity toward AT rich DNA duplex (A5TG) whereas a tris imidazole lexitropsin derivative proved to be selective for the TG mismatched DNA hairpin (CTGGsm) in agreement with NMR, SPR, and ITC studies. A medium throughput screening (MTS) method was setup for the screening of the fragment library (about 1,000 cps) and the procedure was validated studying the interactions between the two different DNA sequences (HFM and CTGGsm) and minor groove binders. The screening was performed on the fragment library and hit compounds were afterwards tested against HFM and CCGG in order to identify selective ligands. Among the different hits identified, a slight selectivity (1.3) for the single mismatched sequence was highlighted for the fragment FBA-05-094. Twenty close analogues of FBA-05-094 were subsequently tested against CTGGsm and CCGG DNA with the aim to find more selective and higher affinity ligands. The screening of the fragment library on CTGGsm was repeated using ligand mixtures (5 and 10 components) in order to improve the assay throughput. A good agreement between the results obtained by screening the compounds as single component and in mixture was found. We investigated also the binding of small molecules towards two enzymes (PKA and Hsp90) in order to verify the possibility to apply this procedure to protein targets. Hsp90 (heat shock protein 90) is a molecular chaperone and is one of the most abundant proteins expressed in cell [4]. Targeting Hsp90 with drugs has shown promising effects in clinical trials. Inhibition of the Hsp90 ATPase machinery by natural, semi- and totally synthetic inhibitors has shown promising results in clinic. The interactions between Hsp90 and different known ligands that bind either at N or C terminal binding domains were studied and the dissociation constants for this set of ligands (KD from 0.0007 uM to 103 uM) were previously determined by fluorescence polarization (FP). We found a good agreement between these values and the percentage of bound protein determined by mass spectrometry. A screening of the fragment library against Hsp90 was performed and the resulted hit compounds were compared to those obtained by using NMR FAXS technique[5]. A good correlation was found between the data obtained by MS and NMR screenings. Competition experiments using a known ATP competitor were performed and these studies highlighted the presence of a few ATP competitor ligands in agreement with X-ray experiments. [1] Ganem B, Li Y.-T., Henion JD. Detection of noncovalent Receptor-Ligand complexes by Mass Spectrometry. Journal of American Society for Mass Spectrometry, 113, 6294-6296 (1991) [2] Hofstadler SA, Sannes-Lowery KA. Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes. Nature Reviews Drug Discovery, 5(7), 585-595 (2006) [3] Stojic L., Brun R., Jiricny J., Mismatch repair and DNA damage signalling. DNA Repair, 3, 1091-1101 (2004). [4] Chaudhury S., Welch T.R., Blagg B.S., Hsp90 as a Target for Drug Development. ChemMedChem, 1331-1340 (2006). [5] Dalvit C., Fagerness P.E., Hadden D.T.A., Sarver R.W., Stockman B.J. Fluorine-NMR Experiments for High-Throughput Screening: Theoretical Aspects, Practical Considerations, and Range of Applicability. Journal of American Society for Mass Spectrometry, 125, 7696-7703 (2003)