Inhibiting AMPylation: A Novel Screen To Identify the First Small Molecule Inhibitors of Protein AMPylation

Abstract

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahaemolyticus, catalyzes the transfer of AMP on to the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization based high-throughput-screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g. calmidazolium, GW7647 and MK886) with Kis ranging from 6–50 µM and upwards of 30-fold selectivity versus HYPE, the only known human AMPylator.

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

“…A threonine/AMP-specific mouse serum was used to confirm VopS-mediated AMPylation of Cdc42 and Rac1 and to study HypE-mediated BiP AMPylation (21). In addition, a set of small molecules inhibiting VopS in in vitro assays has been characterized (22). However, such tools are few in number.…”

“…A recent study characterized small molecule inhibitors of VopS; however, these molecules failed to rescue VopS-induced cell rounding and subsequent cell death in tissue culture assays (22). There are other examples of inhibiting or neutralizing V H Hs (35), but only a few V H Hs have shown stimulatory activity, such as a pair of V H Hs capable of increasing the zymogen activity of the carboxypeptidase TAFI (38).…”

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

“…This method has identified many times more candidate substrates compared with all previous methods 10,14,15,17–19 ; (2) High throughput . Thousands of unique proteins can be tested simultaneously.…”

HDX-MS guided drug discovery: small molecules and biopharmaceuticals

“…Correlating deuterium incorporation patterns from several small molecule ligands with functional assays has proven to be an effective approach to develop structure activity relationship and delineate functional selectivity between closely related compounds [10-13]. HDX-MS also provides a means to identify allosteric small molecule binding sites [2, 14, 15], which are often challenging to locate but desirable for the development of agents with improved selectivity.…”