Fig. 1.  The recently determined structure of SrtA covalently bound to an analog of the LPXTG sorting signal12. (A) Chemical structure of the Cbz-LPAT* peptide analog attached via a disulfide bond to the thiol of C184. (B) The first thio-acyl intermediate that forms immediately after SrtA cleavage of the sorting signal. (C) Stereo image showing the ensemble of 20 lowest energy structures of the SrtA-LPAT* complex. The protein backbone heavy atoms (blue) and the covalently linked peptide (red) are shown. Yellow spheres represent calcium ions. (D) Ribbon drawing of the structure of the SrtA-LPAT* complex (ref 12).

Inhibitor Development and Mechanistic Studies of Sortase Enzymes

Cell Surface Protein Anchoring: Mechanism and Drug Development

During an infection, bacteria use an array of surface-attached proteins to adhere to specific organ tissues, resist phagocytosis, invade host cells and acquire essential nutrients. In Gram-positive bacteria, many surface proteins are covalently anchored to the cell wall peptidylglycan by sortase enzymes (11, 20, 21). Sortases catalyze a transpeptidation reaction between a cell wall sorting signal that is located in their protein substrate and a cross-bridge peptide nucleophile that resides within the cell wall. In a collaborative research effort with Professor Mike Jung's group at UCLA we are investigating the molecular basis of sortase mediated protein anchoring reaction in S. aureus and other pathogens (4-15). We are also developing inhibitors of this process that could function as therapeutically useful anti-infective agents (13).

Fig. 2. Newly discovered pyridazinone molecules that inhibit the sortase enzymes from S. aureus and B. anthracis (ref 13). The molecules were discovered using high-throughput screening and structure activity relationship analyses. Left, the pyridazinone scaffold. Right, model of compound 2-35 bound to sortase via Induced-Fit Docking.

Mechanistic Studies:

An understanding of the molecular basis of protein anchoring is poorly understood because the intermediates of catalysis are short-lived. In recently published work, we overcame this problem by synthesizing a peptide analog of the sorting signal that forms a covalent complex with sortase (Fig. 1) (8, 12). The structure of the SrtAΔN59-LPAT* covalent complex mimics a key enzyme-protein thioacyl intermediate that sheds light onto the mechanism of transpeptidation and the role of highly conserved active site residues. It also reveals how binding of the LPXTG sorting signal triggers major changes in the structure and dynamics of the enzyme that facilitate substrate recognition and direct catalysis towards product formation. Ongoing research is using structural, computational, and biochemical approaches to investigate other aspects of the mechanism of catalysis.

Drug-Development:

Because S. aureus is a leading cause of morbidity and sortase is required for its virulence, we have used a high-throughput fluorescence assay to search for small molecule sortase inhibitors (13). This work led to several promising inhibitors that have recently been published (Fig. 2) (13). The in vivo efficacy of these molecules will be tested using a mouse model. In ongoing collaborative work with Professor Mike Jung's group we are also optimizing several other lead compounds using structure activity relationship analyses and rational design approaches.

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