Screening Derivatized Peptide Libraries for Tight Binding Inhibitors to Carbonic Anhydrase II by Electrospray Ionization-Mass Spectrometry

Abstract

This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development.

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

“…The ability to measure multiple binding equilibria simultaneously makes the ESI-MS assay well suited for screening libraries of compounds against target proteins [38,39]. Where direct detection of the protein-ligand complexes by ESI-MS is not possible due to factors such as high molecular weight (MW) or protein heterogeneity, the catch-and-release (CaR) ESI-MS assay can be employed [40][41][42][43][44][45][46][47]. In this assay, ligand binding is established by releasing (as ions) ligands bound to the protein in the gas phase using activation methods such as collisioninduced dissociation (CID).…”

Fourier transform-ion cyclotron resonance mass spectrometric resolution, identification, and screening of non-covalent complexes of Hck Src homology 2 domain receptor and ligands from a 324-member peptide combinatorial library

“…The gentle ionization provided by ESI has the potential to allow direct observation of this non-covalent complex in the gas phase in a mass spectrometer, and ultimately allow the identification of a few preferred ligands from a highly degenerate combinatorial library. For example, Gao et al used ESI FT-ICR MS to determine the components of a library of 289 sulfonamide-derivatized peptides that bound most strongly to carbonic anhydrase [12]. Here, we report the detection and characterization of the binding specificities of Hck SH2 domain protein to a compound library of 324 phosphorylated peptides by ESI FT-ICR mass spectrometry, and compare it to the results of solution screening.…”

“…A pilot demonstration of the screening of a highly degenerate combinatorial library by isolation and identification of non-covalent complexes in the gas phase has been published [12]. Nevertheless, the limitations of the mass spectrometric technique are not yet well defined, and comparison of solution phase and gasphase protein structure and reactivity has just begun [13].…”

Analysis and screening of combinatorial libraries using mass spectrometry

“…In this application, a combinatorial library is pre-incubated with a receptor in solution and then analyzed directly using electrospray in order to identify receptor-ligand complexes in the gas phase [52][53][54][55][56]. Once a receptor-ligand complex is ionized and trapped in the FT-ICR mass spectrometer, the mass difference between the complex and the receptor alone might be measured with sufficient resolution and accuracy to determine the mass(es) and perhaps elemental composition(s) of the ligand(s).…”

“…As an example, the FT-ICR mass spectrometric screening of a 289-peptide library for ligands to carbonic anhydrase is shown in Figure 8 [53]. After electrospray ionization of a carbonic anhydrase-ligand mixture, the receptor ligand complexes were isolated and trapped in the FT-ICR mass spectrometer ( Figure 8A).…”