Discovery and structure–activity relationship analysis of Staphylococcus aureus sortase A inhibitors

Abstract

Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-infective agents as this enzyme attaches virulence factors to the cell wall. Using highthroughput screening, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure-activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione analogs that inhibit SrtA with IC 50 values in the sub-micromolar range. Many of these molecules also inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) Pyruvate Kinase as a Target for Bis-indole Alkaloids with Antibacterial Activities

“…Using allelic replacement mutagenesis, PK has recently been identified as an absolutely essential gene for survival of other bacteria such as Haemophilus influenzae, Streptococcus pneumoniae, and Mycobacterium tuberculosis (51)(52)(53)(54). Importantly, specific StrA inhibitors did not impair microbial growth in cell culture (55), suggesting that in vitro antibacterial activity of bis-indole may be rather due to the inhibition of MRSA PK enzymatic activity. Taken together, our findings showing that StrA is not an essential gene, coupled with the direct inhibition of MRSA PK enzymatic activity by bis-indole alkaloids suggests that MRSA and MSSA PK are the likely targets of these marine compounds leading to bacterial cell death.…”

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal

“…All conformers possess good covalent geometries and have no NOE, dihedral angle, or scalar coupling violations greater than 0.5 Å, 5°, or 2 Hz, respectively. In the ensemble, backbone atoms of amino acids Asp 57 -Lys 210 in the protein, and all residues in the bound peptide are well defined; the backbone and heavy atom coordinate root mean square deviation (r.m.s. deviation) to the average structure are 0.42 Ϯ 0.06 and 0.89 Ϯ 0.06 Å, FIGURE 1.…”

“…Mutants, wild-type Ba SrtA (amino acid residues Asp 57 -Lys 210 ), and wild-type Ba SrtA ⌬64 (amino acid residues Asp 65 -Lys 210 ) were purified as described previously (23). In vitro substrate hydrolysis assay was performed as previously described (57). Briefly, the cleavage of a self-quenched fluorescent peptide, Abz-LPETG-Dap(Dnp)-NH 2 (Peptide 2.0 Inc., Chantilly, VA), was monitored by excitation at 335 nm and recording emission at 420 nm on an Infinite M1000 PRO spectrofluorometer (Tecan, San Jose, CA).…”

“…The N-terminal appendage contacts the active site histidine residue and is poised to hydrogen bond to the backbone of the bound signal via the side chain of Ser 59 . The appendage spans residues Asp 57 to Val 79 and contains helix H1. In the structure of the apo-form of the enzyme, the portion preceding helix H1 packs against the imidazole ring of His 126 via contacts from the side chains of Ile 61 and Pro 64 .…”

The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

“…The data from the backbone amide nitrogen atoms of 59 residues could be fit using model 1 (S 2 only), 4 residues fit model 2 (S 2 and e ), 13 residues fit model 3 (S 2 and R ex ), 3 residues fit model 4 (S 2 , e , and R ex ), and 12 residues, in addition to the side chain indole N⑀ atom of Trp 171 could be fit using model 5 (S 2 f , S 2 s , and e ). Enzyme Kinetics Measurements-Substrate cleavage reaction was performed as previously described (52). The cleavage of the substrate, o-aminobenzoyl-LPETG-2,4-dinitrophenyl (abz-LPETG-DNP), was monitored by excitation at 335 nm and recording emission at 420 nm on a SpectraMax M5 spectrofluorometer (Molecular Devices).…”