Multitarget Affinity Specificity Screening

We have integrated high throughput sampling robotics and a custom fluidics module to rapidly characterize noncovalent biological complexes in order to identify small molecules that bind RNA targets using ESI-FTICR [30]. The multitarget affinity/specificity screening (MASS) assay takes advantage of the ''intrinsic mass'' label of each compound and target RNA by obtaining high resolution, high precision mass measurements of intact RNA-ligand complexes [13, 3133]. The identity of the small molecule(s) which bind, the RNA target to which it binds, and the compound-specific binding affinity can be determined in one set of rapid experiments. We have demonstrated the applicability of the MASS assay to screening natural product fractions for modified aminoglycosides that bind preferentially to the 16S A-site [34, 35].

At the core of the MASS approach is the premise that, in a solution containing multiple targets and multiple ligands, the molecular interaction between any given target-ligand pair is independent of the presence (or absence) of other li-gands and targets in solution. While FTICR is by no means the first or the only platform on which noncovalent complexes have been detected, the performance attributes of the platform make it well suited to analyze complex mixtures. Thus multiple targets can be screened against multiple ligands simultaneously. This concept is illustrated in Fig. 10.6. In a nondenaturing buffer solution a macromo-lecular target (RNA construct, protein, or mixed complex) is allowed to interact with a molecule, or a collection of molecules, of interest. The compound collection might represent a ''diversity'' collection intended to explore a range of hydrophilic/hydrophobic moieties, charge-carrying groups, and electron donor/ acceptor groups; it might represent a fraction from a natural product isolate for

10.3 Multitarget Affinity/Specificity Screening in a High Throughput Format 329

Fig. 10.6 Concept of multitarget affinity specificity screening (MASS). Macromolecular targets (typically structured RNA constructs or proteins) in nondenaturing buffers are mixed in solution with a collection of potential ligands derived from natural product fractions, combinatorial libraries, or other diverse compound collections. The mixture is analyzed by ESI-MS under gentle desolvation conditions that preserve noncovalent complexes; the exact molecular weight of compounds binding to targets (which can be used to derive an elemental composition), their relative (or absolute) affinity, and binding specificity of compounds are derived from the mass spectra.

Fig. 10.6 Concept of multitarget affinity specificity screening (MASS). Macromolecular targets (typically structured RNA constructs or proteins) in nondenaturing buffers are mixed in solution with a collection of potential ligands derived from natural product fractions, combinatorial libraries, or other diverse compound collections. The mixture is analyzed by ESI-MS under gentle desolvation conditions that preserve noncovalent complexes; the exact molecular weight of compounds binding to targets (which can be used to derive an elemental composition), their relative (or absolute) affinity, and binding specificity of compounds are derived from the mass spectra.

which the complexity and range of functional groups is not known; it might represent a series of engineered compounds derived from carefully synthesized variants of known high affinity ligands; or it might represent a random combinatorial library or subset of a master compound collection. The target (or collection of targets) is allowed to interact in solution, under nondenaturing buffer conditions, with the compound library. Ligands which have an affinity for a given target will bind in solution with a compound-specific dissociation constant (Kd) and specificity.

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