Discovery of a New Phosphotyrosine Mimetic for PTP1B Using Breakaway Tethering

Abstract

Protein tyrosine phosphatases play important roles in many signaling cascades involved in human disease. The identification of druglike inhibitors for these targets is a major challenge, and the discovery of suitable phosphotyrosine (pY) mimetics remains one of the key difficulties. Here we describe an extension of tethering technology, "breakaway tethering", which is ideally suited for discovering such new chemical entities. The approach involves first irreversibly modifying a protein with an extender that contains both a masked thiol and a known pY mimetic. The extender is then cleaved to release the pY mimetic, unmasking the thiol. The resulting protein is screened against a library of disulfide-containing small molecule fragments; any molecules with inherent affinity for the pY binding site will preferentially form disulfides with the extender, allowing for their identification by mass spectrometry. The ability to start from a known substrate mimimizes perturbation of protein structure and increases the opportunity to probe the active site using tethering. We applied this approach to the anti-diabetic protein PTP1B to discover a pY mimetic which belongs to a new molecular class and which binds in a novel fashion.

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).…”

Discovery of an allosteric site in the caspases

“…Productive binding fragments are captured under equilibrium conditions by reversible thioldisulfide interchange (10). Fragments from Tethering have been used to nucleate and augment the identification of potent compounds that target both enzymes and protein-protein interfaces (10)(11)(12)(13)(14). We present this work on the caspases as an approach for the simultaneous discovery of an allosteric site and compounds that affect this site.…”

Kinetic Template‐Guided Tethering of Fragments

“…Erlanson et al applied disulfide tethering to identify a weak active site fragment binder of PTP1B. [61] The catalytic cysteine, at the bottom of the narrow active site channel in PTP1B, was considered inappropriate for disulfide tethering. The residues surrounding the active site pocket were considered crucial to maintain the finely tuned, delicate catalytic machinery.…”

“…[61] A cysteine was introduced far from the active site and subsequently modified with breakaway extender 16, composed of a reactive bromoacetamide and a pTyr mimic, Figure 8. The pTyr mimic binds to the active site pocket protecting the active site cysteine from alkylation and positioning the bromoacetamide close to the mutant cysteine for covalent modification, Figure 9.…”