Affinity selection-mass spectrometry screening techniques for small molecule drug discovery

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Affinity selection-mass spectrometry (AS-MS) techniques assess the binding of candidate molecules to immobilized or soluble receptors, and these methods are gaining acceptance in high throughput screening laboratories as valuable complements to traditional drug discovery technologies. A diversity of receptor types have been evaluated by AS-MS, including those that are difficult to screen using traditional biochemical approaches. AS-MS techniques that couple liquid chromatography-MS with size-based separation methods, such as ultrafiltration, gel permeation, or size-exclusion chromatography, are particularly amenable to the demands of MS-based screening and have demonstrated the greatest success across a broad range of drug targets. MS measurements of receptor function have many of the same advantages as AS-MS screening and are increasingly used for drug discovery as well.

Introduction

Mass spectrometry (MS) is one of the most powerful analytical techniques in modern science, and it plays a key role in nearly every stage of the drug development process [1]. Recent advances in MS instrument design, especially the development of electrospray ionization (ESI), have extended the reach of MS to the lead discovery stages of drug development as well. Modern pharmaceutical discovery relies increasingly on target-based screening techniques to identify new lead compounds for receptors that have known or suspected involvement in a disease pathway. High-throughput screening (HTS) methods that use MS as the detection system are of particular interest because of the exquisite sensitivity and unique selectivity possible with MS. In some screening formats, the selectivity achieved by MS enables direct analysis of compound mixtures such as combinatorial libraries and unpurified natural products extracts – an advantage that is not easily achieved using other analytical methods. MS-based screening is primarily implemented in two ways: by monitoring the functional output of a receptor-dependent biochemical reaction, or by using affinity-based methods that directly assess binding of a candidate molecule to its target receptor. Because of their expanding popularity, both of these styles of MS-based drug discovery have been the subject of several timely reviews, special issues of topical journals, and the focus of at least two recent books [2, 3••, 4, 5, 6, 7]. Herein we report on the recent contributions to the field of MS-based drug discovery, with special emphasis on lead identification methods that couple an affinity selection step with MS confirmation of receptor–ligand binding.

Section snippets

Affinity selection-MS: direct detection of receptor–ligand complexes

Affinity selection-MS (AS-MS) methods directly or indirectly measure binding of small molecules to their biomolecular target, and all varieties of AS-MS include the following steps: (i) an affinity selection stage, where the protein is equilibrated with one or more potential ligands, allowing the protein to form a complex with any compound capable of binding; (ii) the resulting receptor–ligand complexes are separated from non-binding mixture components; and (iii) ligands are identified by MS or

Immobilized ligand or receptor

In part to enable the use of non-volatile buffers in the receptor–ligand binding reaction, indirect AS-MS screening methods have been developed that couple a separation technique with ESI-MS detection. The simplest variants are those where one binding partner, either the receptor or the small molecule ligand, is immobilized on a solid support. Familiar non-MS methods infer receptor–ligand interactions by measuring changes in surface plasmon resonance (Biacore) or wavelength shift (Corning's

MS measurements of receptor function

MS-based techniques that monitor functional assays have advantages that parallel those of AS-MS. Target-based functional assays identify lead compounds by measuring the outcome of a biomolecule-mediated transformation, such as production or depletion of the starting materials or products of an enzymatic reaction, or displacement of a labeled marker compound from a biomolecular receptor. These measurements are traditionally done using radiochemical methods, fluorescence readouts, or UV

Conclusions and future directions

As shown in this report, the pharmaceutical industry is enthusiastically using MS-based HTS methods to discover and characterize new lead compounds, and we anticipate these applications will continue and expand in the future. Presently, no one-size-fits-all AS-MS solution is expected to satisfy all future screening demands, and no commercial product specifically tailored for AS-MS screening is available to interested researchers. Rather, a diversity of methods is implemented by different

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Satish Jindal, Jerry Shipps, Arshad Siddiqui, and our colleagues at SPRI for critical review of this manuscript.

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