Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site

Abstract

Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (␤-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via ␤-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery.The emergence of bacterial strains resistant to antibiotic therapy is a major public health threat (1). Of particular concern is Staphylococcus aureus, because this Gram-positive pathogen is the leading cause of infections in the bloodstream, lower respiratory tract, skin, and soft tissue in the United States (2). S. aureus strains exhibiting resistance against multiple antibiotics, such as methicillin-resistant S. aureus, are isolated in 30 -60% of community and Ͼ80% of hospital infections with this pathogen (3). Vancomycin or other glycopeptides are considered last-resort therapies for methicillin-resistant S. aureus; however, S. aureus strains with intermediate or full resistance to vancomycin can cause infections for which antimicrobial treatment may no longer be effective (4).Surface proteins of Gram-positive bacteria play important roles during pathogenesis (5). Sortases anchor these polypeptides to the bacterial cell wall envelope (6). For example, S. aureus sortase A recognizes proteins destined for the cell surface via an LPXTG motif in their C-terminal sorting signal (7). Following cleavage between the threonine and the glycine residues, an acyl-enzyme intermediate captures cleaved substrate at the active site thiol of sortase (8). Nucleophilic attack of the amino group of the peptidoglycan precursor lipid II (at the thioester intermediate resolves the acyl enzyme and forms an amide bond between the C-terminal threonine of surface protein and pentaglycine crossbridges (9). Lipid II-linked polypeptide is subsequently incorporated into the cell wall envelope of staphylococci (10). The final product of this pathway, protein linked to cell wall pentaglycine cross-bridges, is displayed on the bacterial surface and enables interactions between the pathogen and tissues of its host.Surface protein anchoring to the cell wall envelope is thought to be an essential strategy for bacterial survival during infection, because mutants lacking genes for one or more sortase enzymes are attenuated in virulence (11). Inhibition of sortases by small molecules may therefore function as a therapeutic strategy for bacterial infections. ...

Irreversible Inhibition of Epidermal Growth Factor Receptor Activity by 3-Aminopropanamides

“…The new 3-aminopropanamides, characterized by a quinazoline (5−7 and 11−13) or quinoline-3-carbonitrile (15−17 and 19−21) driving portion, showed inhibition of EGFR autophosphorylation in A549 cells after 1 h of incubation at 1 μM concentration, and the effect generally persisted up to 8 h after removal of the compounds from the reaction medium (Table 1). In principle, the long-lasting effect observed on EGFR autophosphorylation could be ascribed to different phenomena: (i) accumulation of the inhibitor in cells, as previously demonstrated for some reversible quinazolines; 66 (ii) conversion of the competitive inhibitor into an irreversible one at the active site of the enzyme (mechanism-based inhibition), as described for other β-aminocarbonyl compounds; 38 (iii) generation of the corresponding reactive acrylamide, as described for aryl β-aminoethyl ketones that have potential application as prodrugs of unsaturated ketones. 67 As previously described, 54,66 some reversible quinazoline EGFR inhibitors are sequestered in cells generating falsepositive results in the autophosphorylation assay based on the 8 h washout protocol.…”

“…38−40 These compounds possess a particular profile of reactivity, being nonreactive directly, but able to covalently modify their biological target after bioconversion via β-elimination to the corresponding α,β-unsaturated carbonyl compound. Maresso and co-workers 38 reported aryl β-aminoethyl ketones as irreversible inhibitors of the bacterial enzyme sortase, a target for the development of antibacterial therapeutics. They demonstrated through mass spectrometry and X-ray analysis that enzyme inhibition proceeds via a reactive olefin intermediate, generated in situ by amine elimination, which forms a covalent adduct with a specific cysteine residue within the protein active site.…”

Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase

“…Earlier work used in vitro (inhibition of fluorogenic substrate cleavage) and virtual screening of compound libraries to identify sortase inhibitors (15,(52)(53)(54)(55)(56). Although these studies identified both competitive and noncompetitive inhibitors (57,58), isolated compounds have not yet been shown to inhibit in vivo sortase activity in staphylococci, i.e., the cleavage of sorting signals or the assembly of surface proteins into the bacterial cell wall (54,(59)(60)(61)(62). Many of the isolated compounds diminish or block staphylococcal growth, indicating that they cannot function as selective inhibitors of S. aureus sortase (53,61,63,64).…”

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

“…Other sortase enzymes have also been shown to possess a similar overall structure, including SrtB from S. aureus (27,35), SrtB from Bacillus anthracis (27,36), SrtA from S. pyogenes (37), and the SrtC-1 and SrtC-3 enzymes from S. pneumoniae (38). However, the molecular basis of substrate recognition remains poorly understood, because all of the structures reported to date have not contained a sorting signal bound to the enzyme.…”