In situ assembly of enzyme inhibitors using extended tethering

Abstract

Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.

Small-Molecule Inhibitors of IL-2/IL-2R: Lessons Learned and Applied

“…Because the binding of the fragment is stabilized by the reversible disulfide bond, tethering is especially powerful for identifying very weak interactions and enables the exploration of binding sites not easily accessible to NMR, SPR, or functional screening approaches. Sunesis used the disulfide-trapping (Erlanson et al 2003a;Yang et al 2009;Cancilla et al 2008) and also reported examples in which tethering identified previously unknown sites that allosterically regulated protein activity (Erlanson et al 2003b;Hardy and Wells 2009).…”

Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors

“…The exploration of the approaches to construct molecules from fragments leads to the generation of in situ fragment assembly techniques, such as click chemistry, 15 dynamic combinatorial library design, 16 and tethering with extenders. 17 Fragment-based screening offers a number of attractive features compared to HTS. First, whereas compounds from HTS libraries are more restricted in their rotational degrees of freedom, and thus less able to adapt to a given target site, a high proportion of the atoms of a fragment hit are directly involved in the desired receptor-ligand interaction, which allows for optimal positioning within the receptor pocket.…”

Binding of small molecules to an adaptive protein–protein interface

“…To further dissect the binding site, we screened for novel small molecule ligands by using a site-specific fragment-discovery method called tethering. The tethering method identifies fragments (M r typically Ͻ200) that are selected through disulfide exchange on the basis of their noncovalent association with the protein (6,23). A library of small drug-like fragments containing a common disulfide is allowed to reach equilibrium with a set of single-cysteine mutants of the target protein (Scheme 2).…”