Development of transglycosylase inhibitors

TPase and TGase are two important enzymes that catalyze the formation of three-dimension backbone of peptidoglycan. The TPase inhibitors such as vancomycin and penicillin have been used in clinical treatment for decades. With the more and more resistant cases of TPase inhibitors, scientists now shift the goal to this appealing target for several reasons:

i. TGase is located on the external of cell membrane, so inhibitors can reach the target without entering cytoplasm.

ii. Polysaccharide backbone retains intact in wild-type and resistant bacterial strains.

iii. TGase is unique in prokaryote and does not exists in eukaryotic counterparts.¹¹ Natural products that inhibit TG can be divided into two types: 1) substrate binder which binds to lipid II, and 2) enzyme binder that directly inhibits TGase.¹¹ The most salient TGase substrate binder is a lantibiotics, nisin (Figure 12) which coordinates the pyrophosphate moiety of lipid II by the N-terminal backbone amides to form a cage structure to interfere with the biosynthesis of peptidoglycan. Moenomycin A, a member of moenomycin family, is the most prominent TGase binder. It shows better affinity to TGase than the growing glycan chain, so moenomycin A can directly interact with the active site of TGase to inhibit the polymerization of peptidoglycan.^5d

Figure 12. Structures of nisin and moenomycin A

Moenomycin A is composed of three components, including a pentasaccharide, a phosphoglycerate and a C25 moenocinol lipid tail which is attached to C1 of the F ring via phosphoglycerate. The minimal inhibitory concentration (MIC) of moenomycin A against several Gram-positive bacteria ranges from 1 to 100 ng/mL. However, the poor pharmacokinetic properties render moenomycin A inadequate for treatment to humans.

Moenomycins have been legally added to animal feed as the growth promoters under the

trademarks Flavomycin and Flavophospholipol. The issue of using antibiotic additives in animal nutrition has been debated for a long time, and an ultimate decision is made in 2006 to forbid the use of antibiotic additives on all animal feed in entire European Union.¹⁹

Welzel and co-workers have reported the structure activity relationships of moenomycin A derivatives with TGase inhibition. It is shown that the lipid chain of

moenomycin A first anchors to the cytoplasmic membrane and the sugar part

subsequently binds to the donor binding site of TGase to compete with the growing glycan chain rather than lipid II. The -[1,2] junction between the E and F rings of moenomycin differs from the -[1,4] connection of growing glycan chain, and thus prevents the 4-OH

of lipid II GlcNAc from attacking the phosphate group of the F sugar.^{5d, 20} In contrast, rings A and D of moenomycin A, which resemble the pentapeptide of MurNAc, project from the planar polysaccharide scaffold and thus make fewer contacts to TGase.¹⁵

The degradation studies of moenomycin have shown that the rings E and F are crucial for activity and the modifications of the substituents on these rings decrease the biological activity. Particularly, the carbamoyl group at C3, the hydroxyl group at C4 and the carbamoyl groups at C3 and C5 of ring F, the acetyl group at C2 of ring E as well as the carboxyl group of phosphoglycerate are the pivotal functional groups that are exposed to the surface of TGase.^{20, 21}

In 2010, Walker, Kahne and coworkers have reported that the carboxylate group of phosphoglycerate and the isoprenyl lipid in moenomycin A are important to TGase activity (Table 1). The inhibitory activity of moenomycin A decreases when the carboxylate group is replaced by carboxamide group, acylsulfonamide group or hydrogen atom. This results indicate that the carboxylate group binds to target by ionic interactions

with the positively charged side chains of enzyme, and the interactions help to dispose the lipid chain to attach the hydrophobic position of active site to cell membrane. In addition, when the isoprenyl chain is altered by neryl chain, the inhibitory activity also decreases. It indicates that the C10 chain is too short to maintain the biological activity and the C25 chain is crucial to interact with the hydrophobic cell membrane.²²

Table 1. TGase inhibitors reported by Walker, Kahne and coworkers²²

Compound

Due to the low quantities and difficult isolation of lipid II in nature, the development of artificial TGase inhibitors progresses slowly.²³ In 1999, Sofia et al. have applied a

solid-phase combinatorial approach to construct a series of disaccharides mimicking the E and F rings of moenomycin A (Figure 13). The resin containing photocleavable linker is removed after the derivatization of disaccharides. A library of 1300 disaccharides is prepared and compounds 4‒9 are shown to inhibit bacterial growth. Therefore compounds

4‒9 were resynthesized and purified to show the IC50 values of 8‒10 g/mL for TGase and 3.12‒12.5 g/mL of MIC values.

Figure 13. TGase inhibitors reported by Sofia and coworkers²⁴

In 2004, Walker, Vederas and coworkers have reported a series of mono- and di-saccharides C-phosphonates containing curtailed alkyl chains to mimic the active portion of moenomycin A and lipid II (Table 2). The activity assay against E. coli PBP1b is performed based on the radioactive labeling. Compounds 10a and 10b which are prepared to test the distance between the noncleavable C-phosphonate and the sugar, are shown to have 17% and 0% inhibition at 100 M. In addition, compound 11a‒11c with extended

linkage which are prepared to examine the effect of the first sugar of moenomycin, show inhibition of 25%, 10% and 25% at 100 M, respectively.These results suggest that the

introduction of properly extended linkage between mono- or di-saccharides and phosphate are needed for TGase inhibition.²⁵

Table 2. The reported TGase inhibitors by Walker, Vederas and coworkers²⁵

Compound

In 2008, Wong, Ma, Cheng and coworkers have used a fluorescence anisotropy (FA)-based assay as a new strategy to search TGase inhibitors. The high-throughput screening (HTS) of 57,000 molecules reveals compounds 12‒14, that have IC50 values of 34.0, 3.7 and 9.3 M, respectively (Table 3). The structures of these compounds all contain numerous planar hydrophobic groups and negative charge properties, which are

speculated to compete the pyrophosphate binding site with the fluorescent moenomycin derivatives.²⁶

Table 3. TGase inhibitors reported by Wong, Ma, Cheng and coworkers²⁶

Compound IC50 (M) MIC (M)

In 2010, Wong and coworkers have again conducted an HTS of nearly 2 M compound library, and further narrowed down to 252 hit compounds. These hits are tested by FA assay for TGase inhibition, and then verified by TGase-catalyzed lipid II polymerization.

Among all the synthesized compounds, 15‒18 with the salicylanilide-based core structure (Table 4) have the best inhibitory activity at 100 M against TGase. It is concluded that

the third aryl group on the anilide is necessary for TGase inhibition.²⁷

Table 4. TGase inhibitors with salicylanilide-based core structure²⁷ azasugars-based structure, which is conjugated with a truncated pyrophosphate lipid chain and lipophilic carboxylic acids (Table 5). The azasugars are designed to mimic the charge and shape of the oxonium ion transition state during the TG reaction. In order to enhance the stability of the inhibitors, the O-glycoside was replaced by C-glycoside to connect the azasugar and phosphate. The potential hit compounds are identified by the

FA-based assay. The best inhibitor 19 shows 100% inhibition at 1 mM and 80% inhibition at 100 M against E. coli PBP1b. In comparison, the pentapeptide-containing lipid I and

lipid II-based inhibitors (22 and 23), which are synthesized by walker and Vederas et al.

show worse inhibition than compound 19.²⁸

Table 5. TGase inhibitors reported by Wong, Cheng and coworkers²⁸

In 2015, Zuegg and coworkers have reported the novel compounds with non-planar pyranose scaffold and benzimidazole moiety to replace the phosphoglycerate group in moenomycin (Table 6), in order to reduce the molecular weight and hydrophobicity.

Compounds 24 and 25 possess good in vitro antibacterial activity against several Gram-positive bacteria. In addition, the in vitro docking experiment suggests that the two compounds are able to bind at both the donor and acceptor sites. The in vivo experiments

show that both compounds can be tolerated in mice up to a dose of 100 mg/kg, and have good metabolic stability in rats.²⁹

Table 6. TGase inhibitors reported by Zuegg and coworkers²⁹

Compound inhibition

MIC (g/mL)

S. aureus E. faecium E. faecalis S. pneumoniae E. coli

24 200 g/mL 4 16 4 4 > 64

25 200 g/mL 4 2 8 8-16 > 64