Publications

2017

Sabulski M, Pidgeon S, Pires M. Immuno-targeting ofsurface remodeling complexes. Chem Sci. 2017;8(10):6804–6809. doi:10.1039/c7sc02721d
Agents with novel mechanisms of action are needed to complement traditional antibiotics. Towards these goals, we have exploited the surface-homing properties of vancomycin to tag the surface of Gram-positive pathogens with immune cell attractants in two unique modes. First, vancomycin was conjugated to the small molecule hapten 2,4-dinitrophenol (DNP) to promote bacterial opsonization. Second, we built on these results by improving the tagging specificity and mechanism of incorporation by coupling it to a sortase A substrate peptide. We demonstrated, for the first time, that the surface of () can be metabolically labeled in live hosts. These constructs represent a class of promising narrow-spectrum agents that target for opsonization and establish a new surface labeling modality in live host organisms, which should be a powerful tool in dissecting features of host-pathogen interactions.
Pidgeon S, Pires M. Cell Wall Remodeling of Staphylococcus aureus in Live Caenorhabditis elegans. Bioconjug Chem. 2017;28(9):2310–2315. doi:10.1021/acs.bioconjchem.7b00363
Peptidoglycan (PG) scaffolds are critical components of bacterial cell walls. They counter internal turgor pressure to prevent lysis and protect against external insults. It was recently discovered that various types of bacteria release large quantities of PG building blocks (d-amino acids) into their surrounding medium. Contrarily, cultured bacteria were also found to incorporate d-amino acids (both natural and synthetic) from the medium directly into their PG scaffold. These two processes may potentially function, in concert, to metabolically remodel PG in live host organisms. However, demonstration that bacteria can decorate their cell surfaces with exogenous d-amino acids was limited to in vitro culture conditions. We present the first evidence that bacteria remodel their PG with exogenous d-amino acids in a live host animal. A tetrazine click partner was conjugated onto the side chain of a d-amino acid to capture incorporation into the bacterial PG scaffold using a complementary click-reactive fluorophore. Staphylococcus aureus infected Caenorhabditis elegans treated with exogenous d-amino acids readily revealed in vivo PG labeling. These results suggest that extracellular d-amino acids may provide pathogens with a mode of late-stage in vivo cell-surface remodeling.

2016

Sarkar S, Libby E, Pidgeon S, Dworkin J, Pires M. In Vivo Probe of Lipid II-Interacting Proteins. Angew Chem Int Ed Engl. 2016;55(29):8401–4. doi:10.1002/anie.201603441
β-Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin-binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.
Fura J, Pidgeon S, Birabaharan M, Pires M. Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment. ACS Infect Dis. 2016;2(4):302–309. doi:10.1021/acsinfecdis.6b00007
The number of antibiotic-resistant bacterial infections has increased dramatically over the past decade. To combat these pathogens, novel antimicrobial strategies must be explored and developed. We previously reported a strategy based on hapten-modified cell wall analogues to induce recruitment of endogenous antibodies to bacterial cell surfaces. Cell surface remodeling using unnatural single d-amino acid cell wall analogues led to modification at the C-terminus of the peptidoglycan stem peptide. During peptidoglycan processing, installed hapten-displaying amino acids can be subsequently removed by cell wall enzymes. Herein, we disclose a two-step dipeptide peptidoglycan remodeling strategy aimed at introducing haptens at an alternative site within the stem peptide to improve retention and diminish removal by cell wall enzymes. Through this redesigned strategy, we determined size constraints of peptidoglycan remodeling and applied these constraints to attain hapten-linker conjugates that produced high levels of antibody recruitment to bacterial cell surfaces.
Yu Y, Sabulski M, Schell W, Pires M, Perfect J, Regen S. Simple Strategy for Taming Membrane-Disrupting Antibiotics. Bioconjug Chem. 2016;27(12):2850–2853. doi:10.1021/acs.bioconjchem.6b00629
A strategy has been devised for increasing the cellular selectivity of membrane-disrupting antibiotics based on the attachment of a facially amphiphilic sterol. Using Amphotericin B (AmB) as a prototype, covalent attachment of cholic acid bound to a series of α,ω-diamines has led to a dramatic reduction in hemolytic activity, a significant reduction in toxicity toward HEK293T cells, and significant retention of antifungal activity.

2015

Fura J, Kearns D, Pires M. D-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling. J Biol Chem. 2015;290(51):30540–50. doi:10.1074/jbc.M115.683342
Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural D-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural D-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural D-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural D-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural D-amino acids into bacterial peptidoglycan.
Sabulski M, Wang Y, Pires M. PAD2 Activity Monitored via a Fluorescent Substrate Analog. Chem Biol Drug Des. 2015;86(4):599–605. doi:10.1111/cbdd.12526
The post-transitional modification of peptidyl arginine to citrulline by PAD2 can affect the inherent biophysical properties of the citrullinated protein. Furthermore, dysregulation of PAD2 activity has been implicated in a number of human diseases. Inhibition of these enzymes by small molecules can serve as essential probes in establishing a link to pathogenesis. Herein, we describe a profluorescent substrate analog that reports on the activity and the inhibition of PAD2 in a robust assay. Most noteworthy, we expect future drug discovery efforts based on PAD2 inhibition can be pursued via this assay.
During the past few decades there has been a rapid emergence of multidrug resistant bacteria afflicting human patients. At the same time, reduced output from pharmaceutical industry in this area precipitated a sharp decrease in the approval of new antibiotics. The combination of these factors potentially compromises the ability to effectively combat bacterial infections. While traditional drug discovery efforts continue in the pursuit of small molecule agents that disrupt bacterial growth, non-traditional efforts could serve to complement antimicrobial strategies. We recently demonstrated our ability to remodel the surface of bacterial cells using unnatural D-amino acids displaying the antigenic dinitrophenyl (DNP) handle. These immune stimulant D-amino acids derivatives were metabolically incorporated onto the peptidoglycan of bacteria via a promiscuous surface-anchored transpeptidase. The covalent modification of DNP moieties onto the peptidoglycan led to the anti-DNP antibody opsonization of the bacterial cell surface. Herein, we show that the amidation of the C-terminus to generate DNP-displaying D-amino carboxamide drastically improves antibody recruitment. Antibody opsonization using the D-amino carboxamide agent is observed at lower concentrations than the D-amino acid counterpart. In addition, the recruitment of endogenous antibodies in pooled human serum to the DNP-modified bacterial cell surface is demonstrated for the first time. We envision that the C-terminus amidation of DNP-conjugated D-amino acids could potentially facilitate translation of these results to in vivo animal disease models.
Pidgeon, Pires. Metabolic remodeling of bacterial surfaces via tetrazine ligations. Chem Commun (Camb). 2015;51(51):10330–3. doi:10.1039/c5cc01693b
Bioorthogonal click ligations are extensively used for the introduction of functional groups in biological systems. Tetrazine ligations are attractive in that they are catalyst-free and display favorable kinetics. We describe the efficient remodeling of bacterial cell surfaces using unnatural d-amino acids derivatized with tetrazine ligation handles. The metabolic incorporation of these unnatural d-amino acids onto bacterial cell surfaces resulted in a site-selective installation of fluorophores.
Pidgeon S, Fura J, Leon W, Birabaharan M, Vezenov D, Pires M. Metabolic Profiling of Bacteria by Unnatural C-terminated D-Amino Acids. Angew Chem Int Ed Engl. 2015;54(21):6158–62. doi:10.1002/anie.201409927
Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.