Tethering

Abstract

IntroductionOne of the common challenges throughout this book is how to identify small chemical fragments that bind weakly to target biological molecules. Among fragment-based approaches, Tethering is unique in using a covalent, reversible bond to stabilize the interaction between a fragment and a target protein [1,2].The general process is outlined in Fig. 14.1. First, a cysteine residue is either coopted or introduced in 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 the individual fragments. In the absence of any affinity between a fragment and the protein, no fragment should bind more favorably than any other; a pool of fragments produces a statistical mixture of different protein-fragment complexes, as well as unmodified protein and cysteaminemodified protein. However, if a fragment has inherent affinity for the protein and binds near the cysteine residue, the fragment-protein conjugate is stabilized, and this complex predominates. A fragment thus selected can be easily identified by mass spectrometry of the equilibrium mixture, and if each fragment in a pool has a unique molecular weight, so do the resulting protein-fragment conjugates. While the identified fragments are often weak ligands, X-ray crystallography of the protein-fragment conjugates is often facilitated by the covalent bond. These captured fragments then serve as starting points for conversion to non-covalent ligands by chemical optimization and removal of the thiol functionality.In the following pages, we present an overview of the theory and uses of Tethering. After first considering the basis of the technique in thermodynamic terms (section 14.2), we show how the technology can be used in the active sites of enzymes to identify fragments (section 14.4), which can then be elaborated to more potent inhibitors. Section 14.5 considers how Tethering can be used to not only 285 Fragment-based Approaches in Drug Discovery. Edited

Current perspectives in fragment-based lead discovery (FBLD)

“…They used protein-observed NMR (see later) to identify fragments binding to two independent binding sites and linked these fragments together to make potent inhibitors. The approach was extended, first by Abbott, to use X-ray crystallography to identify the fragments [ 19 ], an approach that was rapidly exploited and developed by Astex [ 3 ] during the early 2000s, along with developments at Sunesis [ 4 ] and Vernalis [ 5 ]. Although a few larger companies did experiment with the approaches [ 20 ], it was the small companies that primarily developed the methods and demonstrated success, which by the mid-2000s led to the wider take-up of the methods across the pharmaceutical industry.…”

“…fragments that contain a reactive group that will form a covalent bond with the protein. The origins of these ideas were in the tethering work of the company Sunesis, who developed a library of fragments which would react with surface exposed cysteines [ 4 ]. An extension of this approach is to introduce a cysteine mutation close to the functional site [ 51 ] – an interesting example of this type of approach was also demonstrated for K-Ras where a particular mutation seen in some tumours (G12C) was exploited [ 48 ].…”

“…The first report of fragment-based discovery was in 1996 [ 2 ] (discussed later) and there was rapid development of the methods and their application in the late 1990s and early 2000s, particularly in small, structure-based discovery companies such as Astex [ 3 ], Sunesis [ 4 ] and Vernalis [ 5 ]. What was vital was that progress could be made with a small (approximately 1000 compounds) fragment library providing novel chemical matter to start a discovery project, and the companies were able to exploit high-throughput crystallographic methods to generate candidate compounds.…”

Simple One-Pot Synthesis of Disulfide Fragments for Use in Disulfide-Exchange Screening

“…One of the inherent difficulties of fragment screening is detection of the low affinity binding events. Disulfide-exchange screening or Tethering is a mass-detected screening method that involves equilibrium exchange of disulfide-containing fragments with native or introduced cysteine residues on a protein target of interest. , Those fragments that form effective noncovalent surface contacts in addition to the covalent disulfide bond are sufficiently stable to persist and be detected by mass spectrometry. This method has been used to identify drug leads and has helped to identify novel allosteric sites in therapeutically relevant targets such as caspases. − …”

Fragment-Based Discovery of Nonpeptidic BACE-1 Inhibitors Using Tethering

“…We have shown previously that the fragment-based discovery method, Tethering, can be utilized to identify small molecule fragments that can serve as starting points for the design of potent enzyme and protein−protein interaction inhibitors (Scheme ) , . Tethering entails the introduction of individual cysteine mutations surrounding a site of interest on the target protein surface, followed by screening against libraries of thiol linker-containing fragments.…”