Introduction

Pressure to keep early phase pipelines filled with drug leads has heightened interest in developing innovative technology to discover drug-like molecules. One alternative and effective approach for generating small-molecule inhibitors is ''fragment-based drug discovery'', a process which identifies one or more low-affinity, low molecular weight, drug-like ''fragments'' and subsequently elaborates or combines them to make compounds that are analogous to a high throughput screening (HTS) hit [1-3]. Fragment-based discovery has an advantage over traditional HTS because it samples more chemical ''diversity space'' with significantly fewer molecules (thousands for fragment-based approaches compared to millions in traditional HTS formats) [4].

One pervasive challenge in fragment-based discovery is how to identify small chemical fragments that bind only weakly to target biological molecules. Currently, several different screening techniques are used for discovering such fragments: functional binding assays [5], NMR-based screening [6-8], crystallography-based screening [9-10] and mass spectrometry-based methods [11-15]. All have unique advantages and limitations.

For mass spectrometry, modern ionization methodologies such as electrospray ionization (ESI) [16] and matrix-assisted laser desorption ionization (MALDI) [17], along with advances in current mass spectrometry platforms, would seem ideal for the rapid discovery of fragments, but detecting molecules possessing millimolar binding affinities is not trivial. Mass spectrometry-based ligand binding assays such as non-covalent mass spectrometry and the myriad of front-end, affinity-based mass spectrometry techniques (such as AS-MS [18]) are not ideal for detecting such low affinity ligands. To overcome these barriers, we developed a discovery technology, Tethering [19], centered on detecting fragment-protein conjugates by LC/MS. Among fragment-based approaches, Tethering is unique in using a covalent, reversible bond to stabilize the interaction between a fragment and a target protein. The bond forms is stable only when there is inherent

Fig. 9.1 Tethering schematic. A fragment will be selected if it has inherent affinity for the protein and binds in the vicinity of the cysteine residue. An example disulfide-containing fragment is shown below, illustrating the variable portion, the linker, and the cysteamine piece that is lost when the fragment forms a disulfide bond with the protein.

Fig. 9.1 Tethering schematic. A fragment will be selected if it has inherent affinity for the protein and binds in the vicinity of the cysteine residue. An example disulfide-containing fragment is shown below, illustrating the variable portion, the linker, and the cysteamine piece that is lost when the fragment forms a disulfide bond with the protein.

affinity between the ligand and the protein target. The fragment is then rapidly identified using electrospray mass spectrometry to detect the modified, intact protein.

The general process of Tethering is outlined in Fig. 9.1. First, a cysteine residue is either co-opted or introduced into a target protein. Metaphorically, the cysteine residue serves as a fishing line to capture fragments (fish) that bind near the cysteine. The protein is incubated with pools of thiol-containing small molecule fragments which are conjugated to a common, hydrophilic thiol (such as cysteamine) for improved water solubility. By controlling the redox conditions in the experiment with exogenous reducing agents, equilibria can be established so that the cysteine residue in the protein reversibly forms disulfide bonds with individual fragments. If no fragments have affinity for the interrogated area of the protein, no fragment should bind more favorably than any other, and a pool of fragments will produce a statistical mixture of different protein-fragment complexes, plus unmodified and cysteamine-modified protein. However, if a fragment has inherent affinity for the protein and binds near the cysteine residue, the fragmentprotein conjugate will be stabilized, and this complex will predominate. A fragment thus selected can be easily identified through mass spectrometry of the equilibrium mixture: if each fragment in a pool has a unique molecular weight, so will the resulting protein-fragment conjugates. These captured fragments then serve as starting points for conversion to non-covalent ligands by removal of the thiol functionality and chemical optimization.

In the following pages, we present an overview of the theory, practice, and uses of Tethering. First we examine the experimental nuances of the screening meth odology. Next we demonstrate how the technology can be used in the active sites of enzymes to identify fragments, which can then be elaborated to more potent inhibitors. The final section considers how Tethering can be used not only to identify fragments but also to link these fragments to more rapidly identify starting points for drug discovery.

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