Hao-Wei Shih

Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli.

Drug-resistant bacteria have caused serious medical problems in recent years, and the need for new antibacterial agents is undisputed. Transglycosylase, a multidomain membrane protein essential for cell wall synthesis, is an excellent target for the development of new antibiotics. Here, we determined the X-ray crystal structure of the bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli in complex with its inhibitor moenomycin to 2.16-Å resolution. In addition to the transglycosylase and transpeptidase domains, our structure provides a complete visualization of this important antibacterial target, and reveals a domain for protein–protein interaction and a transmembrane helix domain essential for substrate binding, enzymatic activity, and membrane orientation.

A common glycan structure on immunoglobulin G for enhancement of effector functions

Significance Antibodies are important therapeutic agents and have been used for the treatment of many diseases, including infectious and inflammatory diseases, and cancer. The therapeutic efficacy of an antibody is usually determined not only by the selectivity and affinity toward the target but also by the Fc-glycan structure interacting with the Fc receptors on immune cells. This study describes the preparation of various antibodies with different Fc-glycan structures as homogeneous glycoforms for the investigation of their effector activities. During this study, it was discovered that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimal structure that is able to enhance the activities of antibodies against cancer, influenza, and inflammatory diseases. Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

High-throughput identification of antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase

To identify new transglycosylase inhibitors with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activities, a high-throughput screening against Staphylococcus aureus was conducted to look for antibacterial cores in our 2M compound library that consists of natural products, proprietary collection, and synthetic molecules. About 3600 hits were identified from the primary screening and the subsequent confirmation resulted in a total of 252 compounds in 84 clusters which showed anti-MRSA activities with MIC values as low as 0.1 μg/ml. Subsequent screening targeting bacterial transglycosylase identified a salicylanilide-based core that inhibited the lipid II polymerization and the moenomycin-binding activities of transglycosylase. Among the collected analogues, potent inhibitors with the IC(50) values below 10 μM against transglycosylase were identified. The non-carbonhydrate scaffold reported in this study suggests a new direction for development of bacterial transglycosylase inhibitors.

Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism

Bacterial transpeptidase and transglycosylase on the surface are essential for cell wall synthesis, and many antibiotics have been developed to target the transpeptidase; however, the problem of antibiotic resistance has arisen and caused a major threat in bacterial infection. The transglycosylase has been considered to be another excellent target, but no antibiotics have been developed to target this enzyme. Here, we determined the crystal structure of the Staphylococcus aureus membrane-bound transglycosylase, monofunctional glycosyltransferase, in complex with a lipid II analog to 2.3 Å resolution. Our results showed that the lipid II-contacting residues are not only conserved in WT and drug-resistant bacteria but also significant in enzymatic activity. Mechanistically, we proposed that K140 and R148 in the donor site, instead of the previously proposed E156, are used to stabilize the pyrophosphate-leaving group of lipid II, and E100 in the acceptor site acts as general base for the 4-OH of GlcNAc to facilitate the transglycosylation reaction. This mechanism, further supported by mutagenesis study and the structure of monofunctional glycosyltransferase in complex with moenomycin in the donor site, provides a direction for antibacterial drugs design.

Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl Moiety

The preparation of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.

Combinatorial approach toward synthesis of small molecule libraries as bacterial transglycosylase inhibitors

The development of iminocyclitol-based small molecule libraries against a bacterial TGase is described. An iminocyclitol was conjugated with a pyrophosphate mimic using either a 1,3-dipolar cycloaddition or reductive amination reaction, which was then condensed with a variety of lipophilic carboxylic acids in an amide bond coupling to generate a desired molecular library. With assistance of microtiter plate-based combinatorial chemistry and in situ screening, a potential inhibitor, the first potent iminocyclitol-based inhibitor against bacterial TGases was efficiently developed.

Modular synthesis of N -glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

A new synthetic approach toward bacterial transglycosylase substrates, Lipid II and Lipid IV.

A new synthetic approach toward the bacterial transglycosylase substrates, Lipid II (1) and Lipid IV (2), is described. The key disaccharide was synthesized using the concept of relative reactivity value (RRV) and elaborated to Lipid II and Lipid IV by conjugation with the appropriate oligopeptides and pyrophosphate lipids. Interestingly, the results from our HPLC-based functional TGase assay suggested Lipid IV has a higher affinity for the enzyme than Lipid II.

Effect of the Peptide Moiety of Lipid II on Bacterial Transglycosylase

The rise of antibiotic-resistant bacteria, such as VRE (vancomycin resistant Enterococcus), MRSA (methicillin resistant Staphylococcus aureus), MDR-TB, and XDR-TB (multidrug resistant and extensively drug-resistant tuberculosis) has stimulated the development of new antibiotics and new strategies to tackle the problem of antibiotic resistance. In bacterial cell-wall biosynthesis, the bifunctional enzyme on the surface, such as E. coli PBP1b, possesses two catalytic domains for transpeptidase and transglycosylase activity. The transpeptidase responsible for the cross-linking of peptidoglycans has long been a target for antibiotic discovery and development. 2] In contrast, no antibiotics, except moenomycin used in animal feeds, have been developed to target the transglycosylase (TGase), which catalyzes the polymerization of Lipid II to form a peptidoglycan (Figure 1). Because TGase is essential for bacteria and does not have a eukaryotic counterpart, it is thought to be an attractive target for antibiotic discovery and development. Lipid II (1) consists of a disaccharide, a pyrophosphate, an undecaprenol lipid tail, and an oligopeptide (d-lactyl-lalanyl-g-d-glutamyl-meso-diaminopimelyl (or l-lysyl-dalanyl-d-alanine) moieties (Figure 1). Notably, the first GlcNAc linked to lactic acid to form a ubiquitous component in cell walls is also called MurNAc (N-acetylmuramic acid), which is only found in bacteria. Currently, little information is available regarding the interaction of Lipid II with TGase, presumably because of difficulties in the synthesis and modification of Lipid II (1). In this report, we systematically study how the various Lipid II constituents interact with TGases. Although the peptide moiety in Lipid II is for the transpeptidation process, the last cross-linking step between neighboring peptidoglycan chains catalyzed by transpeptidase, the function of the peptide moiety in Lipid II towards TGase is still unclear. We have recently reported a method for the synthesis of Lipid II and derivatives, and the X-ray crystal structure of MRSA TGase in complex with a Lipid II analogue to elucidate the mechanism of Lipid II polymerization. Herein, we describe the preparation of Lipid II analogues with varying peptide moieties for evaluation as TGase substrates, as an effort toward development of new antibiotics. Five analogues 1–5 were designed (Figure 2). Compounds 1–3 contain a peptide chain (R) prepared by sequential removal of the two amino acids from the end of the peptide stem; 4 has no peptide chain, but a methyl group instead; and 5 was prepared from 1 by adding the fluorophore, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino]hexanoyl (NBD-X), to the e-NH2 group of the lysine residue. Our synthetic strategy for 1–3 is depicted in Scheme 1. The lactyl group was installed at the C3 hydroxy group of 6 to obtain 7 in 72 % yield. The anomeric thiol group in 7 was removed and the product subjected to phosphorylation to give 8 as a single diastereomer. Deprotection of the C4 OTBS group in 8, followed by ester hydrolysis gave the acid intermediate, which was individually coupled with peptides and subsequently debenzylated to give 12. Finally, conjugation of 12 with an undecaprenyl phosphate (C55P), and global deprotection gave the corresponding 1–3 in 31–48% yields over two steps. Compound 4 was directly prepared from 6 Figure 1. Formula of Lipid II (1) for bacterial transglycosylase (TGase) and transpeptidase (TPase).

Total synthesis of polyprenyl N-glycolyl lipid II as a mycobacterial transglycosylase substrate.

A feasible synthetic approach toward the Mycobacterium tuberculosis (Mtb) N-glycolyl lipid II-like molecule 1 is described. Compound 1 bears pendant undecaprenol and l-lysin moieties instead of the naturally occurring decaprenol and meso-diaminopimelic acid, which are not readily available. Functionalization of 1 with a fluorophore on the peptide side chain gave 14, which was found to be recognized as an Mtb TGase substrate. This result suggests it has tremendous utility for mechanistic studies, the characterization of mycobacterial enzymes, and mycobacterial TGase inhibitor evaluation.