Teicoplanin reprogrammed with the N-acyl-Glc pharmacophore at the

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Teicoplanin reprogrammed with the N-acyl-Glc pharmacophore at the penultimate residue of aglycone acquires broad-spectrum antimicrobial activities effectively killing Gram-(+/-) pathogens Chun-Man Huang, Syue-Yi Lyu, Kuan-Hung Lin, Chun-Liang Chen, Mei-Hua Chen, Hao-Wei Shih, Ning-Shian Hsu, I-Wen Lo, Yung-Lin Wang, Yi-Shan Li, Chang-Jer Wu, and Tsung-Lin Li ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00317 • Publication Date (Web): 01 Jan 2019 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Infectious Diseases

Teicoplanin reprogrammed with the N-acyl-Glc pharmacophore at the penultimate residue of aglycone acquires broad-spectrum antimicrobial activities effectively killing Gram-(/) pathogens Chun-Man Huang1,2, Syue-Yi Lyu1, Kuan-Hung Lin1, Chun-Liang Chen1, Mei-Hua Chen1, Hao-Wei Shih1, Ning-Shian Hsu1, I-Wen Lo1, Yung-Lin Wang1, Yi-Shan Li1, Chang-Jer Wu3, Tsung-Lin Li1,4* 1:

Genomics Research center, Academia Sinica, 128 Academia Road, Section 2, Nankang Taipei 11529, Taiwan 2: Department of Microbiology and Immunology, National Yang-Ming University, 155 Linong Street. Section 2, Beitou Taipei 11221, Taiwan 3: National Taiwan Ocean University, 2 Peining Road, Jhongjhong Keelung 20224, Taiwan 4: National Chung-Hsing University, 145 Xingda Road, South Taichung 402, Taiwan *: Corresponding author; email: [email protected] ORCID: Chun-Man Huang: 0000-0002-8358-8799 Tsung-Lin Li: 0000-0002-9739-9761 Lipoglycopeptide antibiotics, e.g. teicoplanin and A40926, are more potent than vancomycin against Gram-(+) drug-resistant pathogens, e.g. MRSA. To extend their therapeutic effectiveness on VRSA, the biosynthetic pathway of the N-acyl Glc pharmacophore at residue 4 (r4) of teicoplanin pseudoaglycone redirection to residue 6 (r6) was attempted. On the basis of crystal structures, two regio-selective biocatalysts Orf2*T (a triple-mutation mutant S98A/V121A/F193Y) and Orf11*S (a singlemutation mutant W163A) were engineered, allowing them to act on GlcNAc at r6. New analogs thereby made show marked antimicrobial activity against MRSA/VRSA by 23 orders of magnitude better than teicoplanin/vancomycin. The lipid side chain of the Tei-analogs armed with a terminal mono-/di-guanidino group extends the antimicrobial specificity from Gram-(+) to Gram-(), comparable to that of kanamycin. In addition to low cytotoxicity/high safety, the Tei analogs exhibit new modes of action as a result of re-sensitization of VRSA and Acinetobacter baumannii. The redirection of the biosynthetic pathway for the N-acyl Glc pharmacophore from r4 to r6 bodes well a large-scale production for selected r6,Tei congeners in an environment-friendly synthetic biology approach. KEYWORDS: lipoglycopeptide antibiotics, MRSA/VRSA, regioselectivity,

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guanidination, Acinetobacter baumannii, protein engineering

Emergence of antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and Acinetobacter baumannii (AB), has cast a serious public health threat of global concern.1-3 The glycopeptide antibiotics (GPAs) teicoplanin (Tei, 1) and vancomycin (Van, 2) were the drug of choice to treat severe Gram-(+) infections (Figure 1A).4-6 This type of compounds binds specifically to the D-Ala-D-Ala terminus of the pentapeptide branch of Lipid II disrupting cell-wall integrity of pathogens.7-8 Since lipid II is a chemical entity rather than a mutable protein, it takes a longer time for pathogens to morph into GPAs resistance (i.e. through modifications of the D-Ala-D-Ala terminus of a lipid II precursor to D-Ala-D-Lac or D-Ala-D-Ser). The genes that remodel cell wall polymers conferring VRE immune to vancomycin have horizontally passed to MRSA on several occasions, staging an even gloomy public health threat because of an augmented virulence of the pathogens.5, 9 Increasing incidences of infections caused by VRE, VISA (vancomycin-intermediate S. aureus) and VRSA (vancomycin-resistant S. aureus) have heralded an urgent need for new antibiotics that are able to shunt the drug resistance or eradicate the pathogens.9-11 Because of the unmatched preponderance, GPAs have been the choice of natural products to develop to more efficacious antibiotics in this drug-resistance battle.12-13 For example, three second-generation GPAs (telavancin, oritavancin, and dalbavancin) showed higher potency than their individual parent compounds because of introduction of new mode of action.14-17 Given the pressure of evolutionary selection, any new antibiotics that were deployed to clinics would eventually succumb to therapeutic failure.18-19 New antibiotics therefore need to be constantly developed in order to keep paces with development of drug resistance. Despite structural complexity of GPAs, recent advances on new GPAs faithfully reflect their value in therapeutics. Haldar and co-workers reported that vancomycin appended with a lipophilic cationic moiety at the carboxyl end of its peptide core provided an excellent VRE-killing activity, which is attributable to both disruption of cell membrane integrity and increased permeability of cell wall other than inhibition of cell wall biosynthesis.20-22 Boger and colleagues demonstrated that the combination of the lipid II binding-pocket modification ([Ψ[CH2NH]-Tpg4]vancomycin), the carboxylterminus quaternary ammonium salt functionalization and the peripheral (4chlorobiphenyl)methyl (CBP) modification (as oritavancin), improved the minimal inhibitory concentration (MIC) values to the levels of 0.01−0.005 μgmL-1 against VRE as a result of a synergetic effect by three independent modes of action, reinstated inhibition of transpeptidase, inhibition of transglycosylase, and stimulation of cell wall


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permeability.23-26 The same group further explored the N-terminus modification, which showed comparable activity with the C-terminus modification but lack of membrane permeabilization.27 Huang and colleagues independently reported that vancomycin derivatives with an extra sugar moiety to the phenyl ring of the terminal residue gained extra therapeutic merits on enhanced efficacy, optimal pharmacokinetics and lower cytotoxicity.28 Cooper and colleagues recently reported that vancomycin conjugated with a positively charged electrostatic effector sequence in ligation to an N-terminal lipophilic membrane-insertive element (vancapticin) showed superlative activity against MRSA and VRE.29 Wright and co-workers reported a synthetic biology approach to expand antibiotic chemical diversity to a greater extent through in-cell GPA modification.30 The same group meanwhile discovered a new GPA, pekiskoycin, via screening for antibiotic resistance.31 A sizable chemoenzymatic library of vancomycin had been made by Thorson and co-workers as one of the first reports of glycorandomization.32 We, on the other hand, reported that di-lipidated GPAs can be innovatively prepared using the flavin-containing hexose oxidase (Dbv29) or acylCoA-dependent acyltransferase (Orf11*) to produce a cohort of teicoplanin analogs with an additional amidated, aminated or esterified lipid chain at a relatively intractable chemical space (C6-OH of glucosamine at r4).33-34 These di-lipid derivatives presented an exceptional antimicrobial activity versus a collection of VREs, likely by enhancing the dual effects of membrane anchoring and compromise of membrane integrity resulting in cell wall/membrane dysfunction. Peculiarly, these newly developed GPAs are more effective against VRE rather than MRSA. Lipidation is a proven strategy for expanding GPA biological effectiveness particularly useful against drug-resistant pathogens.6, 29, 33-36 We hypothesized that lipidation on chemical spaces other than those reported positions should greet distinctive biological functionalities and resistant profiles (e.g. effective against MRSA), thus allowing one to get paces ahead of resistant development upon new drugs introduced into clinic settings. Despite the structural complexity of Tei that assures a formidable challenge, reprograming the N-acyl Glc pharmacophore from residue 4 (r4) to residue 6 (r6) may gain extra merits other than those reported. Three genes orf10*, orf2* and orf11* in the tei biosynthetic gene cluster respectively code for a glycosyltransferase, a deacetylase and an acyltransferase in a row to decorate Tei aglycone with the N-acyl Glc pharmacophore at r4 (Figure 1B; Figure 2A a-d).37-41 These enzymes in conjunction with three other enzymes Orf1, Dbv27 and NahK (see below) in their cognate or engineered states lend an opportunity to form new Tei analogs (Figure 2A e-f),39-40, 42 in which more efficacious analogs ought to exist. Our endeavor stemmed from using Orf1, a glycosyltransferase in the tei gene cluster, to add GlcNAc from UDP-GlcNAc to r6 of the Tei aglycone (Figure 2A f). The amino group



on the sugar moiety was unleashed and re-acylated by using a deacetylase (Orf2*) and an acyltransferase (Orf11*), respectively, where the regioselectivity at r6 was evolved via structure-based protein engineering (Figure 2A h-i). The N-terminal primary amino group was protected using the methyltransferase Dbv27 encoded in the dbv gene cluster prior to r6-glucosamine acylation (Figure 1C). The functionality of the r6-lipidation was further expanded by adding a positively charged mono-/di-guanidino group at the terminus of the lipid chain. These regio-specific (guanidino)-lipidated Tei analogs subjected to biological examinations show exceptional lower cytotoxicity, broader antimicrobial activity, and a drug-resistant/sensitive Gram-(+/)-pathogens killing effect. RESULTS AND DISCUSSION Enzymatic synthesis of r6,N-acyl Glc Tei analogs. To achieve the goal set, we first took advantage of Orf1 because it was straightforward given if glucosamine (Glm) in place of GlcNAc can be added on Tei aglycone. While UDP-Glm can be quantitatively synthesized using NahK (to form Glm-1-phosphate) along with GlmU (to form UDPGlm) (supplementary information, Figure S1),42 our attempt failed because the substrate of Orf1 is strictly limited to UDP-GlcNAc (Figure 2A f). A structure-based protein engineering for Orf1 was practiced. This endeavor was not successful either, as neither native nor ligand-bound Orf1 structures were obtained with myriad conditions screened. Orf1 was alternatively subjected to homology modeling using the Phyre2 protein fold recognition server with the solved crystal structures GtfA (PDB ID: 1PNV), GtfB (PDB ID: 1IIR) or chimerical GtfAH1 (PDB ID: 3H4I) as the model templates (Figure S2AB).43-45 Site-directed mutants were made on the basis of the generated model; chimeras were also made through swapping the sugar recognition domain of Orf1 with that of Orf10*/GtfA/B (Figure S2C).45 Unfortunately, these attempts remained unsuccessful because of no anticipated activity (wherein several mutants/chimeras were insoluble). We then exploited the reversible glycosyltransferase-catalyzed reactions reported by Thorsen et al. in a hope to transfer epi-vancosamine from chloroeremomycin to Tei aglycone.46 This approach still did not work here. We finally took a stepwise strategy outlined in Figure 1C: Orf1 adds GlcNAc to r6 of the Tei aglycone, where the N-acetyl group at r6 was removed using an engineered Orf2* prior to lipidation with a long aliphatic chain catalyzed by an engineered Orf11* to form the representative 3 (see below). Orf2* is a deacetylase that specifically hydrolyzes the N-acetyl group off GlcNAc at r4 of Tei-pseudoaglycone. On the basis of the solved crystal Orf2* complex, protein engineering for residues in the substrate binding site was practiced, whereby a triple mutant (Orf2*T: S98A/V121A/F193Y) was generated. The mutant was able to unleash


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the amine group of GlcNAc at r6,Tei-pseudoaglycone (Figure 2A h).37 ITC analysis revealed that the binding affinity of r6,GlcNAc-Tei-pseudoaglycone versus Orf2*T (Kd = 0.125 ± 0.014 mM) is higher than that of r6,GlcNAc-Tei-pseudoaglycone versus Orf2* (Kd = 1.418 ± 1.098 mM) (Figure 2B). The overall catalytic specificity Orf2*T versus r6,GlcNAc-Tei-pseudoaglycone was assessed to be kcatKm-1 = 45.28 s-1mM-1 well in a typical enzymatic scope (Figure S3), albeit it is about 8-fold less specific than Orf2* versus the native r4 counterpart (kcatKm-1 = 377.3 s-1mM-1). The crystal structure of Orf2*T was solved (Figure 3A; the diffraction parameters and refinement statistics are summarized in Table S1),37, 47 while the r6,Glm-Tei-pseudoaglycone-complexed structure was not obtained due to undefined electron density under a higher molecular oscillation. The capping loop region (residues 110-120), which acts as a lid governing the substrate entry, cannot be built likely due to the r6,Tei-pseudoaglycone-induced conformational disorder. The low root-mean-square deviation (RMSD) of 0.44 Å for 207 Cα atoms on superposition of both Orf2* and Orf2*T structures suggested that there are no significant conformational changes except the side chains of the residues that were mutated in Orf2*T (Figure S4). In brief, S98 may assist r4,GlcNAc of Teipseudoaglycone in alignment toward the reaction center by interacting with C6-OH; V121 may help anchor the aglycone scaffold through hydrophobic interactions with the backbone amide bonds of r1 and r2; the phenyl ring of F193 is also hydrophobically interacting with the m-chlorine substituted phenyl ring of r4. Given the triple mutant S98A/V121A/F193Y, r6,Tei-pseudoaglycone is allowed to rotate ~90 in a trajectory suitable for the deacetylation reaction to proceed by one order of magnitude less efficient than that of r4,Tei-pseudoaglycone versus WT (kcatKm-1 = 377.3 s-1mM-1) (Figure S3). Given r4,r6-di-GlcNAc-Tei, the one at r4 is still dominant for the deacetylation reaction, likely because of a better overall fitness of the original conformation to the binding site. Having deacetylated, the primary amine group of r6,Tei-pseudoaglycone can be lipidated by several handy organic reactions, such as acylation or alkylamination. One, however, need to be noted that there is another primary amine group at the N-terminus (r1). Chemical protection is required at the r1 amino group in order to ensure the regioselective lipidation at r6 of Tei-pseudoaglycone. Dbv27, predicted to be an Sadenosylmethionine (SAM)-dependent methyltransferase at the N-terminus in the maturation of A40926 (Figure S5),40 may lend an opportunity to selectively protect the amino group at r1. While A40926 contains no sugar moiety at r6, either r6,GlcNAc- or r6,Glm-Tei-pseudoaglycone can serve as a substrate for Dbv27 allowing methylation to take place at the N-terminal amino group (Figure 2A g). ITC analysis revealed that Dbv27 binds strongly with SAM (Kd = 10 M) or r6,Glm-Tei-pseudoaglycone (Kd = 5 M) (Figure 2C). Interestingly, increasing the molar ratio of SAM versus Tei-



pseudoaglycone (2:1, pH 8) the N-terminal amino group can be doubly methylated to form r1,Me2-r6,Glm-Tei-pseudoaglycone in a fully protected manner (Figure 2A g). Despite that r1,Me2-r6,N-acyl-Glc-Tei analogs can now be subjected to chemical acylation or alkylamination, an integrated biochemical approach was further attempted as a foundation of synthetic biology for large-scale production. Orf11* that catalyzes acylation of r4,Glm-Tei-pseudoaglycone was co-opted to complete this task. The advantage of using Orf11* is that the substrate r6,Glm-Tei-pseudoaglycone is a close regioisomer to its cognate substrate r4,Glm-Tei-pseudoaglycone, and the ternary structures of Orf11* in complex with the r4,Glm-Tei-pseudoaglycone acceptor and the acyl-CoA donor are available.34-35 The Tei-pseudoaglycone binding site is located at the colossal junction between the N- and C-terminal domains, where W163 in the Nterminal domain serves as the anchoring base with the bulky indole moiety fitting well into the concave of the aglycone core to align r4,Glm toward the reaction center (Figure 3B, left panel). W163 was therefore mutated to W163A to disrupt the binding fidelity, so that r6,Glm may head to the reaction center for the acyl-transfer reaction to take place. As expected, W163A is capable of transferring an acyl side chain from a corresponding Co-A derivative to r6,Glm forming r1,Me2-r6,N-acyl-Glc-Teipseudoaglycone in an acceptable enzymatic scope despite not so ideal (kcat = 3.85 ± 0.23 min-1, Km = 0.3 ± 0.03 mM, kcatKm-1 = 12.92 min-1mM-1) (Figure 2A i; Figure S6). The binding affinity of r6,Glm-Tei-pseudoaglycone (Km = 0.3 mM) is about 5-fold less than that of r4,Glm-Tei-pseudoaglycone (Km = 0.07 mM) in line with catalytic specificity (kcatKm-1 = 12.92 and 87.49 min-1mM-1 for r6,Glm-Tei-pseudoaglycone and r4,Glm-Tei-pseudoaglycone, respectively). In analogy to Orf2*T, r4,Glm still outruns r6,Glm, as r4,Glm-Tei-pseudoaglycone as a whole is more fit to the substrate bindingsite. The crystal structure of W163A was solved (Figure 3B, right panel), while the ternary complex soaked with r1,Me2-r6,Glm-Tei-pseudoaglycone remained unobtainable because of an unclear electron density map for the aglycone core due to a larger extent of molecular oscillation (the diffraction parameters and refinement statistics are summarized in Table S1). Structural superposition of Orf11* over W163A shows a low root-mean-square deviation (RMSD) of 0.286 Å for 295 Cα atoms, suggesting there is no significant conformational changes between Orf11* and W163A except the designated mutation (Figure S7AB). The removal of the anchoring indole is, however, competent allowing Tei-pseudoaglycone to rotate ~90 for the primary amine of r6,Glm in alignment with the reaction center to proceed the acyltransfer reaction (Figure 3B, right panel). Though there is still room for further improvement for both Orf2*T and W163A (now termed as Orf11*S) in views of reaction efficiency/overall yield, the current lineup, nevertheless, has enabled us to synthesize a cohort of r6,N-acyl-Glc Tei analogs


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in quantity (at the level of >20 mg for each) as shown in Figure 4A (Figure S9 for LC/MS spectra). Two representative compounds 12 and 14 were further subjected to 1D and 2D NMR analysis for structural validation (Table S3 and Figure S10-S14, Table S4 and Figure S15-S19, respectively). Antimicrobial activity of r6,N-acyl-Glc Tei analogs. Having synthesized r6,N-acylGlc-Tei analogs, the minimal inhibition concentrations (MICs) for compounds 13-27 (Figure 4A, Figure S9A i-v) alongside r4,di-acyl-Glm-Tei (28) (effective in killing VREs) were assessed against a collection of drug-sensitive Enterococci/Staphylococcus aureus (VSE/MSSA) and drug-resistant VRE/MRSA/VISA/VRSA pathogens in comparison with Tei (1)/Van (2). As shown in Table 1, in terms of drug sensitivity 1 is more potent than 2 by one order of magnitude against the selected VRE strains (except ATCC 700221, which is a vanA-type strain resistant to both 1 and 2). In contrast, 1 is marginally more potent than 2 against the selected MRSA strains, in which ATCC 700698 and ATCC 700789/ATCC 700787 are vancomycin intermediate (VISA) and resistant (VRSA) strains, respectively. The MIC values shown here are by and large in agreement with those reported previously.33-34 While r4,N-acyl-Glm-Tei analogs (28 in particular) are relatively more potent than r6,N-acyl-Glc counterparts (13-19) versus VREs,34 the r6,N-acyl-Glc Tei analogs, strikingly, outperforms 1, 2, or 28 by 1-3 orders of magnitude (as low as 0.01 gmL-1) versus VISA/VRSA (ATCC 700698/ ATCC 700789 and ATCC 700787). It is clear that r4,N-acyl-Glc (28) or r6,N-acyl-Glc analogs (13-19) each has a distinctive pharmaceutic effect on VRE or MRSA, respectively. In brief, the r6,N-acyl-Glc-Tei analogs with or without methyl groups at r1 make no apparent difference on MIC, suggesting the N-terminal methyl groups (10, 20) have little or no role on the growth inhibition effect. Similarly, an extra mannose at r7 (26) makes not much improvement as opposed to Van derivatives reported by Huang et. al.28 These modifications, nevertheless, may be crucial in vivo in view of pharmacokinetics/pharmacodynamics (e.g. methylation at r1 may resist hydrolysis from endogenous proteases; an extra sugar may enhance drug solubility). Better potency can be expected for an analog with a longer lipid chain. A phenyl ring (15, 16, 18), a substituent on the phenyl ring (18), a hydroxyl group (19, 21), or an amino group (22) at the terminus of the lipid chain shows no better activity than the linear fatty acid one, concluding that lipidation on r6,Glm is the most critical factor for the pathogen growth inhibition effect on both drug-sensitive and -resistant S. aureus. Coupling r4 and r6,N-acyl-Glm Tei analogs. Having learned that Tei analogs with lipidation at r4 or r6 award each analogs a selective bactericidal effect respectively on VRE or MRSA/VISA/VRSA, we hence wondered whether such antimicrobial



selectivity could be expanded by integrating these two features as a whole. To test this idea, a bifunctional short chemical linker was used to latch r4,- and r6,N-acyl-Glm Teipseudoaglycone together through a r1-r1 linkage. To minimize unpredicted variables one fixed length linker (C10) was selected, which also rendered a higher yield in the coupling reaction. Three combinations include two r4, N-acyl-Glm Teipseudoaglycone 29, two r6,N-acyl-Glm Tei-pseudoaglycone 30, and one r4 and one r6 N-acyl-Glm Tei-pseudoaglycone 31 (Figure 4B, Figure S9A w-y,), which were subjected to antimicrobial assessment (Table 1). On the basis of MIC values, the antimicrobial activity, however, was not as broad/sensitive as anticipated. The overall performance is no better than each standalone against selected bacteria. The lack of an additive effect may be attributable to two bulky components, which limit their spatial freedom while exerting their individual antimicrobial effects. The fact is that both separate r4 and r6 analogs (14 and 28) added together did show a synergistic effect (low-dose inhibition, where the quantity of each analog is halved) on both drug-resistant enterococcus and staphylococcus (extended antimicrobial spectrum) particularly effective against VRSA (both ATCC 700787 and ATCC 700789) and vanA-type VRE (ATCC 700221) (Table 1). Antimicrobial activity against Gram-(–) bacteria. Given a cationic trimethyl moiety attached to the terminus of a lipid side chain at the r7 carboxyl group of A47934/Van (dalbavancin/the Van analogs modified by the Haldar’s group), the specific Gram-(+) antimicrobial spectrum expanded to some selected Gram-(–) strains, such as E. coli.16, 48-50 The effectiveness may be attributable to both disruption of cell membrane integrity and/or enhancement of cell membrane permeability in addition to block the lipid II precursors/intermediates during the cell wall biosynthesis. We applied this seminal concept to r6,N-octyl-Glc Tei (14) by adding a hydroxyl (21), amine (22), trimethylamine (23), guanidine (24), or di-guanidine (25) functional group at the terminus of the lipid side chain at r6,Glm and assess their antimicrobial activity against the notorious nosocomial strain A. baumannii (AB). First, each terminal substituted octanoic acid was chemically converted to a corresponding Nacetylcysteamine thioester, which then transesterified with CoA to form a given CoA derivative. Compounds 21, 22 and 23 were enzymatically synthesized using Orf11*S in the presence of a corresponding CoA derivative (Figure S9A q-s). For synthesis of 24 and 25, compound 22 was subjected to guanidination using 1-guanyl-pyrazole hydrochloride (Figure S9A t-u). These modified analogs are effective against MRSA/VRE with antimicrobial activity comparable to 14. Compound 21 shows a marginal antimicrobial activity (152.84 gmL-1) on AB, while 23 slightly improves its MIC value by 2-fold reduction (78.52 gmL-1). The MIC can be further improved by


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another 2-fold reduction (39.24 gmL-1) with introduction of a terminal mono- (24) or di-guanidino (25) group (the latter is slightly better than the former) (Table 2, Table S2). The terminal guanidino group at r4 (27, Figure S9A v) is relatively less potent than that at r6, suggesting that the regio-location is somewhat related to the inhibition activity. The MIC values of 24/25 (in molarity) is comparable to that of kanamycin, an aminoglycoside antibiotic used in standard medications. Compound 14/24 was further examined for its intrinsic bactericidal activity (MBC), where the MBC/MIC ratios are 4 and 1 for MRSA and AB, respectively (Table 3). The modes of action apparently are multifaceted, including enhancement of the binding affinity with phospholipid components of cell membranes, facilitation of the cell-membrane permeation to interact lipid II precursors, as well as disruption of cell membrane integrity (see below). Cell wall morphology by TEM. To assess what damages the r4, or r6,N-acyl-Glm Tei analogs impose on MRSA, VRE or AB, the testing bacteria treated with a given analog were subjected to thin-section transmission electron microscopy (TEM) analysis (Figure 5). The morphologies of VRE/MRSA and AB respectively display typical cocci and coccobacillus structures in the absence of compounds (untreated control), where stratificated structures of an intact cell envelop (cell wall and cell membrane) can be distinguished on TEM images. In contrast, the well-defined structures of VRE or MRSA treated with 1, 2, 14, or 28 morphed into rather distinct phenotypes, whereon shape deformations and abnormalities showed extensive alterations, particularly, when cells underwent division. Upon treatment with 2 (32 gmL-1), 1 (32 gmL-1), 28 (4 gmL-1) or 14 (0.064 gmL-1), the dense and well-defined cell wall structure of MRSA (ATCC 700787) underwent systemic swelling to various extents manifested as floppy, loose, disintegrated, or cell wall damage toward full cell lysis in a progressive manner (Figure 5A). The cell wall/membrane apparently is the targets, while the extent of damage depends on what compound imposes on the strain. Compound 14 displays a lowest effective dosage. Cell wall thickening has been a common phenotype in clinical MRSA isolates, which may well be a phenotypic determinant for VRSA. The systemic cellwall damage may account for the cell-wall biosynthesis damaged everywhere on the cell surface in cell-wall thickening strains, thus leading cells to lysis as a result of the osmotic pressure of cytoplasm. The superlative antimicrobial activity of 14 is attributed to the r6,N-acyl-Glm pharmacophore, where the regio-position/direction of the lipid chain is different from 1 (Tei) and those modified at the C-terminus (r7) (Figure 3C, Figure S7CD), effecting on lipid II or nascent peptidoglycan precursors with a higher binding affinity and/or a better inhibition trajectory.



Upon treatment with the same analogs to VRE (ATCC700221, vanA type) a distinct phenotype was shown in Figure 5B. The cell wall was almost intact but interspersed with holes, where cell fluids outflew leading cells to death. VRE is immune to 1 or 2 (>256 gmL-1), median sensitive to 14 (32 gmL-1), but highly sensitive to 28 (4 gmL-1). The effectiveness of 28 may well be ascribed to new mode of action as manifested by the phenomenal bursting effect, where local cell membrane integrity and cell wall permeability are severely injured as a causation of the r4,N-di-acyl-Glc pharmacophore wreaking on VRE at specific foci on cell surface. AB (ATCC 19606), a Gram-(–) bacterium, features a distinctive outer membrane visible on TEM images. This species is insensitive to glycopeptide antibiotics (1 and 2, >187.97 gmL-1), medium sensitive to kanamycin (14.56 gmL-1), but highly sensitive to colistin (1.98 gmL-1) (Figure 5C). Interestingly, AB is medium sensitivity to 24 or 25 in contrast to 1 and 2 (re-sensitization by >8 folds). As shown in the TEM images, the intactness of cell membrane/cell wall was severely damaged upon treatment with 24 (50 M) or colistin (3.2 M), causing outflow of cell fluids and thus leading cells to death. In contrast, AB treated with kanamycin remained enclosed by the generally intact cell membrane/wall structures, while the area of cytoplasm was severely damaged. A phenomenal synergistic effect with the combination of a half dose of 24 (25 M) and kanamycin (25 M) was observed, where massive transudates gush out of the damaged cell membrane/wall of AB. As a result, the effectiveness of 24 or 25 that outdoes the primary (22) or quaternary amine (23) counterparts may be ascribed to the conjugated positive charge on the guanidino group with a higher affinity to the membrane phospholipid. The lipid effector may exploit its hydrophobicity to perturb cell membrane, so that Tei pesudoaglycone aptly captures lipid II precursors/intermediates and thereby impedes cell-wall biosynthesis. Safety/cytotoxicity and cell-membrane permeability of r6,N-acyl-Glc Tei analogs. The Alamar Blue (AB) assay,51 which contains a redox indicator in response to metabolic activity, was used to evaluate mammalian cell cytotoxicity in vitro. The cytotoxicity of 1, 14, 18, 24 alongside daptomycin was individually examined against a human embryonic kidney cells (HEK293T) cell line at two different time points 2 and 24 h (Figure 6A). These selected compounds did not exhibit any observable toxicity to HEK293T cells up to 100 μgmL-1 as opposed to daptomycin that showed a significant extent of cell toxicity. The developed analogs were also subjected to drug-induced hemolysis examination,52-53 which is rare but a serious toxicity. Similarly, all the test compounds except daptomycin showed little or no hemolysis (Figure 6B). The capability of the developed compounds that permeabilize the cytoplasmic membrane of bacteria was examined using propidium iodide (PI) dye.54-55 In principle,


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PI should pass across the membrane of compromised bacterial cells and fluoresce upon binding to bacterial DNA. Teicoplanin (1), compounds 14 and 24 showed a median inner membrane permeability on MRSA when compared to colistin (a positive control) and water (a negative control) (Figure 6C). Compound 24 is relatively more potent than 14 due to the terminal guanidino group in addition to the unique r6,N-acyl-Glc geometry, for which 1-guanidino octanoic acid alone did not show any activity on disruption of cell wall/membrane. The same phenomenon can be seen in AB, where 24 showed a median inner membrane permeability (equivalent to Triton X-100) in contrast to 14, which had no activity (Figure 6D). In the 1-N-phenylnaphthylamine (NPN) permeabilization assay,55-56 14 and 24, however, did not show any outer membrane permeabilization on MRSA (without outer membrane) or AB (with outer membrane) (Figure S8) in agreement with the inherent barrier of outer membrane for lipoglycopeptide antibiotics. Though 24 and 25 are effective against AB, there is no a clear-cut answer at the moment with respect to how these compounds reach the inner membrane. In vivo efficacy of r6,N-acyl-Glc Tei analogs. The sensitivity in vitro of MRSA and AB to r6,N-octyl-Glc-Tei analogs prompted us to examine their efficacy in vivo. The effectiveness on bacterial clearance by 14 and 24 was respectively evaluated against MRSA and AB in a sepsis infection model. For MRSA, mice were inoculated intraperitoneally (IP) with 0.3 mL of MRSA ATCC 700787 suspension (~107 CFUmL-1),57 The treatment started 1 h post-challenge with a single dose of 14 (20 mgkg-1), 1 (20 mgkg-1) and saline through IP, where the latter two respectively served as positive and negative control. To assess the density of bacteraemia, 1 mL peritoneal fluid (PF) was collected from each mouse at 24 h postinoculation. For the saline treated control, bacterial count was calculated (∼7.5 log CFUmL-1, left panel). The bacterial load was reduced by 1.5 and 2 log CFUmL-1 with the treatment of 1 and 14, respectively, where 14 outperforms 1 in agreement with in vitro assays (Figure 7A). For AB (ATCC 19606), female C57BL/6 mice were first anesthetized by intraperitoneal injection of zoletil and then intranasally inoculated with 50 μL of bacteria in a PBS solution (~107-108 CFUmL-1).58 The treatment started 1 h postinfection with a single dose of 24 (30 mgkg-1), 1 (30 mgkg-1), amikacin (15 mgkg-1), or kanamycin (30 mgkg-1), where saline/1 and amikacin/kanamycin respectively served as negative and positive control. To assess the density of bacteraemia, the lungs of mice were lavaged to collect 0.6 mL bronchoalveolar lavage (BAL) fluid from each mouse at 24 h post-inoculation. For saline/1 treated controls, the bacterial load was counted (∼4 log CFUmL-1, right panel), where the bacterial load was reduced by 0.5 or 1 log



CFUmL-1 with the treatment of amikacin/kanamycin or 24, respectively. Given the better in vivo therapeutic efficacy, 24 that is more effective than amikacin/kanamycin in the animal study is well perceived (Figure. 7B). CONCLUSION The previously developed r4,di-acyl Glc Tei analogs (e.g. 28) exhibited a superb VREkilling effect by 1-3 orders of magnitude better than Tei (1)/Van (2), while the antimicrobial activity against MRSA is no better than that of Tei (1)/Van (2). The r6repositioned isomers developed herein, however, demonstrated marked effectiveness against MRSA/VRSA, while their activity against VRE is relatively less than that of the r4 counterparts. These two types of analogs covalently linked together did not gain an additive antimicrobial effect, while each in conjunction with a second type of antibiotics (e.g. kanamycin, colistin) did show a synergistic effect and a broadened antimicrobial spectrum. Reprograming the pharmacophore on the glycopeptide core at chemically intractable spaces other than N- or/and C-terminus appears to be a valuable and practical drug-development strategy in the development of new antibiotics on resensitization of drug-resistant pathogens or reestablishment of a distinct drug-resistant profile in the combat against drug-resistant pathogens. The lipidated pharmacophore positioned at r4 or r6 further denotes an intriguing structure-activity relationship, delineating delicate physiological discrepancies between two major types of Gram-(+) drug-resistant pathogens in cell-wall architecture and/or biosynthesis. The tunable modifications of the lipid side chain of Tei (1), particularly the terminal guanidination of r6,N-acyl-Glc Tei (e.g. 14), considerably extends the antimicrobial specificity crossing the genera from Gram-(+) to Gram-(–). Their potency against a broader spectrum of Gram-(–) species can be anticipated, for example, by virtue of introducing multivalent guanidinium groups at both r4 and r6 positions in the Tei pseudoaglycone. These new types of Tei analogs appear to exert new modes of action in addition to a showcase of high safety, broad antimicrobial spectrum, superlative synergistic killing effect, and low-dose efficacy. Importantly, the redirected biosynthetic route bodes well a large-scale production of selected r6,Tei congeners in an environment-friendly synthetic biology approach.

ASSOCIATED CONTENT Supporting Information Protein expression, purification and confirmation of purity were performed according standard protocols. The molecular replacement (MR) method was used to solve structures of Orf2*T and Orf11*S. Mutants were made by using QuikChange®.


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Biochemical analyses for WT and mutants were performed using LC-PDA-MS. Tei analogs were isolated and purified by column chromatography/HPLC. Biological assays were performed following standard protocols. Detailed experimental procedures, compound synthesis, data processing and structure reﬁnement statistics, and supplementary ﬁgures and tables are given in the Supporting Information (SI) online. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Accession codes The coordinates have been deposited in the Protein Data Bank under accession number. Orf2*T: 6IIX and Orf11*S: 6IIY.

Corresponding Author Correspondence and requests for materials should be addressed to T.L.L. ([email protected]) Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by funds from the Ministry of Science and Technology (MOST), Taiwan (105-2311-B-001-050, 106-0210-01-15-02 and 107-0210-01-19-01), and Academia Sinica intramural funding. Portions of this research were carried out at the National Synchrotron Radiation Research Center (NSRRC), a national user facility supported by MOST of Taiwan, ROC. We thank both NSRRC of Taiwan and SPring8 of Japan for beam time allocations at beam lines 13C, 13B, 05A, 15A, and 44XU. We also thank the Mr. Huang, Electron Microscope Facility, ICOB, Academia Sinica, for thin-section transmission electron microscopy (TEM) services.



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54. Jenkins, M. B.; Anguish, L. J.; Bowman, D. D.; Walker, M. J.; Ghiorse, W. C., Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts. Appl Environ Microb 1997, 63 (10), 3844-3850. 55. Hoque, J.; Konai, M. M.; Gonuguntla, S.; Manjunath, G. B.; Samaddar, S.; Yarlagadda, V.; Haldar, J., Membrane active small molecules show selective broad spectrum antibacterial activity with no detectable resistance and eradicate biofilms. J Med Chem 2015, 58 (14), 5486-5500. DOI: 10.1021/acs.jmedchem.5b00443. 56. Helander, I. M.; Mattila-Sandholm, T., Fluorometric assessment of Gram-negative bacterial permeabilization. J Appl Microbiol 2000, 88 (2), 213-219. 57. Domenech, A.; Ribes, S.; Cabellos, C.; Taberner, F.; Tubau, F.; Dominguez, M. A.; Montero, A.; Linares, J.; Ariza, J.; Gudiol, F., Experimental study on the efficacy of combinations of glycopeptides and beta-lactams against Staphylococcus aureus with reduced susceptibility to glycopeptides. J Antimicrob Chemother 2005, 56 (4), 709-716. DOI: 10.1093/jac/dki294. 58. Harris, G.; Kuo Lee, R.; Lam, C. K.; Kanzaki, G.; Patel, G. B.; Xu, H. H.; Chen, W., A mouse model of Acinetobacter baumannii-associated pneumonia using a clinically isolated hypervirulent strain. Antimicrob Agents Chemother 2013, 57 (8), 3601-3613. DOI: 10.1128/aac.00944-13. 59. Hanwell, M. D.; Curtis, D. E.; Lonie, D. C.; Vandermeersch, T.; Zurek, E.; Hutchison, G. R., Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminformatics 2012, 4. DOI: 10.1186/1758-2946-4-17. 60. Rappe, A. K.; Casewit, C. J.; Colwell, K. S.; Goddard, W. A.; Skiff, W. M., Uff, a full periodic-table force-field for mlecular mechanics and molecular-dynamics simulations. J Am Chem Soc 1992, 114 (25), 10024-10035. DOI: 10.1021/ja00051a040.



Page 20 of 31

Table 1. The minimal inhibition concentrations (MICs, gmL-1) for compounds 13-27, r4,di-acyl-Glm-Tei 28 and coupled compounds 29-31 against selected pathogens. Compds

SA

MRSA

MRSA

MRSA

MRSA

MRSA

MRSA

MRSA

EF　

VRE

VRE

VRE

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

ATCC

29213

43300

BAA-44

BAA-38

BAA-39

700789

700698

700787

29302

700221

700802

700425

VRSAa

hVISAb

VRSAc

(vanA)

(vanB)

(vanC)

1

0.25

0.25

0.5

0.25

0.5

4

4

8

0.125

>128

0.5

1

2

1

1

2

1

1

16

4

16

2

>128

64

16

9

0.25

0.25

0.125

0.25

0.125

2

1

0.5

2

>128

4

4

10

0.25

0.25

0.125

0.25

0.125

2

1

0.5

2

>128

4

4

13

0.063

0.125

0.031

0.063

0.063

1

0.063

0.063

0.5

>16

4

4

14

0.016

0.063

0.016

0.016

0.016

0.5

0.016

0.016

0.25

>16

0.5

1

15

0.031

0.031

0.016

0.016

0.016

0.5

0.016

0.031

0.25

>16

0.5

1

16

0.125

0.063

0.016

0.031

0.063

0.5

0.016

0.125

0.25

>16

0.25

1

18

0.063

0.031

0.016

0.016

0.063

0.5

0.016

0.125

0.25

>16

0.25

1

19

0.016

0.063

0.016

0.031

0.016

0.25

0.016

0.016

0.25

>16

0.5

1

20

0.016

0.063

0.016

0.016

0.016

0.5

0.016

0.016

0.25

>16

0.5

1

21

0.125

ND

ND

ND

ND

ND

0.063

0.125

1

>16

8

8

22

0.031

ND

ND

ND

ND

ND

0.125

0.063

1

>16

1

2

23

0.031

ND

ND

ND

ND

ND

0.031

0.031

0.25

>16

1

2

24

0.5

ND

ND

ND

ND

ND

0.031

0.016

0.5

>16

4

8

26

0.016

0.125

0.016

0.125

0.016

0.5

0.031

0.5

0.5

>16

1

1

28

0.25

0.25

0.25

0.25

0.25

0.5

0.5

1

0.5

1

0.5

0.5

29

>16

>16

>16

>16

>16

>16

16

>16

>16

>16

>16

>16

30

0.25

0.5

0.125

0.5

0.25

4

0.25

4

4

>16

4

4

31

4

4

2

4

4

4

4

>16

8

>16

8

4

14 + 28

0.016

0.16

0.016

0.016

0.016

0.0625

0.016

0.0625

0.0625

1

0.125

0.125

SA: S. aureus; EF: E. faecalis ND: not detected VRSAa: ATCC 700789 was defined as a VISA strain but it exhibited high resistance to vancomycin in this study. hVISAb: The strain has apparent vancomycin MICs in the susceptible range; only clinical failure with vancomycin despite susceptible-range MICs suggests the possibility of an hVISA infection. VRSAc: vancomycin MIC ≥16 μgmL-1 according to the document 2015 CLSI M100-S25.


Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. The minimal inhibition concentrations (MICs) for selected compounds against A. baumannii ATCC 19606. A. baumannii ATCC 19606 (MICs, μM/μgmL-1) 1

100/187.97

24

25/39.24

2

200/297.15

25

25/40.29

10

>200/>285.67

27

50/96.73

14

50/74.22

28

50/100.18

21

100/152.84

kanamycin sulfate

25/14.56

22

25/38.19

streptothricin F

12.5/6.28

23

50/78.52

colistin sulfate

1.56/1.98



Page 22 of 31

Table 3. The minimal inhibition concentrations (MICs, μgmL-1) and the minimal bactericidal concentrations (MBCs, μgmL-1) for 14 and 24 versus MRSA (ATCC 700787) and AB (ATCC 19606). MRSA ATCC 700787

AB ATCC 19606

Compounds

MIC

MBC

MBC/MIC

MIC

MBC

MBC/MIC

14 24

0.016 0.016

0.063 0.031

4 2

74.22 39.24

74.22 39.24

1 1


Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A r6

r2

r4

r5 r7

r3 r1 1

2

B UDP-GlcNAc

UDP

acetic acid deacylase (Dbv21/Orf2*)

glycosyltransferase (Dbv9/Orf10*) 4

5

acyl-CoA

CoA

SAM

acyltransferase (Dbv8/Orf11*) 6

SAH

methyltransferase (Dbv27) 7

8

C UDP-GlcNAc UDP

SAM

glycosyltransferase (Orf1) 4

SAH

methyltransferase (Dbv27) 9

acetic acid

acyl-CoA

deacylase (Orf2*T)

10

CoA

acyltransferase (Orf11*S) 11

3

Figure 1. Chemical structures and biosynthetic pathways of lipoglycopeptide antibiotics. (A) Chemical structures of Tei 1 and Van 2. (B) Biosynthetic pathway of the N-acyl-Glc pharmacophore at r4 of Tei pseudoaglycone. (C) Redirected biosynthetic pathway for relocation of the N-acyl-Glc pharmacophore at r6 of Tei pseudoaglycone. The genes orf1, orf2*, orf10* and orf11* in the tei biosynthetic gene cluster respectively code for a glycosyltransferase, a deacetylase, a glycosyltransferase, and an acyltransferase; the genes dbv8, dbv9, dbv21 and dbv27* in the dbv biosynthetic gene cluster respectively code for an acyltransferase, a glycosyltransferase, a deacetylase, and a methyltransferase. Orf2*T and Orf11*S respectively are triple and single mutants developed in this study.



Page 24 of 31

B

A

4

6

Kd = 1.418 ± 1.098 mM

5 7

b 8

9

c

SAM

d 10(Me)

11

Kd = 0.125 ± 0.014 mM

a

e

10(Me)2

C

f 3

g h i

10 11 12 13 14 15 16 17 18 19 20 21 Time (min) Kd = 0.0101 ± 0.0007 mM

Kd = 0.0051 ± 0.0003 mM

Figure 2. LC traces and isothermal titration calorimetry (ITC) thermographs. (A) LC traces of enzymatic reactions for the N-acyl-Glc pharmacophore at r4 of Teipseudoaglycone: a. the reaction substrate Tei-aglycone 4; b. the product r4,GlcNAc Tei-pseudoaglycone 5 in the enzymatic reaction catalyzed by Orf10*; c. the product r4,Glm Tei-pseudoaglycone 6 in the enzymatic reaction catalyzed by Orf2*; d. the product r4,N-acyl-Glc Tei-pseudoaglycone 6 in the enzymatic reaction catalyzed by Orf11*; e. the product r1,Me-r4,N-acyl-Glc Tei-pseudoaglycone 7 in the enzymatic reaction catalyzed by Dbv27; f. the product r6,GlcNAc Tei-pseudoaglycone 9 in the enzymatic reaction catalyzed by Orf1; g. the product r1,Me2-r6,GlcNAc Teipseudoaglycone 10 in the enzymatic reaction catalyzed by Dbv27; h. the product r1,Me2-r6,Glm Tei-pseudoaglycone 11 in the enzymatic reaction catalyzed by Orf2*T; i. the product r1,Me2-r6,N-acyl-Glc Tei-pseudoaglycone 3 in the enzymatic reaction catalyzed by Orf11*S. (B) Binding affinity of Orf2*T vs. r4,GlcNAc Teipseudoaglycone 6 (left panel) or r6,GlcNAc Tei-pseudoaglycone 9 (right panel). (C) Binding affinity of Dbv27 vs. S-adenosylmethionine SAM (Kd = 10 M) (left panel) or r6,Glm-Tei-pseudoaglycone (Kd = 5 M) (right panel).


Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

Zn2+ r4,N-acyl-Glc F193Y

Zn2+ r6,GlcNAc

S98A V121A

S98A V121A

F193Y

B

Acyl-CoA

r6,GlcNAc

Acyl-CoA

r4,Glm r6,Glm r4 W163 W163A

C

r6,N-acyl-Glc r4,N-acyl-Glc

r6,N-acyl-Glc r7-acylation

Figure 3. Comparisons of crystal structures and lapidated GPA analogs. (A) Superposition of Orf2*/Orf2*T, in which teicoplanin is colored yellow in the left panel and r6,Glm-Tei-pseudoaglycone is colored grey in the right panel. The designated mutations allow the substrate rotated ~90º for the deacylation reaction to take place. (B) Superposition of Orf11*/Orf11*T, in which deacylated teicoplanin is colored grey in the left panel and r6,Glm-Tei-pseudoaglycone is colored grey in the right panel. The



mutation of W163 (colored cyan) to W163A (colored grey) allows the substrate rotated ~90º for the acylation reaction to take place at r6. (C) Superimposition of r6,N-acyl-Glc Tei-pseudoaglycone (colored green) versus r7-lipidated GPA (colored cyan, left panel) or Tei (1 (with N-acyl-Glc at r4) colored grey, right panel), where the lipid side chain points to a regio-dependent direction in each individual compound (also see Figure. S7CD for views from different angles). The structures of r6,N-acyl-Glc Teipseudoaglycone and r7-lipidated GPA were generated by structural editing from the PDB-id 2XAD and 1C0R, respectively, using Avogado59 and subjected to geometry optimization under a universal force field.60 The structure of Tei was selected from 4MFQ.


Page 26 of 31

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A

5

6

7

8

23 14 4

17

3

10

H

9

24 15

18

20

11

16

19

21

22

12

13

27

26

25

28

29

B

30

31

Figure 4. Chemical structures of novel lipoglycopeptides developed in this study. (A) Chemical structures for r6,Tei-analogs, which were synthesized by the redirected biosynthetic pathway. (B) Chemical structures of r4-r4, r6-r6, and r4-r6 conjugated Tei analogs through the r1-r1 linkage. Refer to Figure S9A w-y for LC traces and Figure S9B for mass spectra.



Figure 5. Morphological changes of bacteria upon treatment with antibiotics recorded by the thin-section transmission electron microscopy (TEM). (A) The morphological changes of MRSA (ATCC 700787) upon: a. no treatment, b. treated with 2, c. treated with 1, d. treated with 14, e. treated with 28. (B) The morphological changes of VRE (ATCC 700221) upon: a. no treatment, b. treated with 2, c. treated with 1, d. treated with 14, e. treated with 28. (C) The morphological changes of AB (ATCC 19606) upon: a. no treatment, b. treated with 24, c. treated with kanamycin, d. treated with kanamycin + 24, e. treated with colistin. See text for explanations. IM: inner membrane; OM: outer membrane; CW: cell wall.


Page 28 of 31

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Figure 6. Safety/cytotoxicity and cell-membrane permeability of r6,N-acyl-Glc Tei analogs. (A) The Alamar Blue (AB) assay: the cytotoxicity for 1, 14, 18, 24 and daptomycin was examined against a human embryonic kidney cells (HEK293T) cell line at two different time points 2 and 24 h. (B) The drug-induced hemolysis examination. (C) The permeabilization capability of selected compounds on the cytoplasmic membrane of MRSA ATCC 700787 was examined using the propidium iodide (PI) dye assay. (D) The permeabilization capability of selected compounds on the cytoplasmic membrane of AB ATCC 19606 was examined using the propidium iodide (PI) dye assay.



Figure 7. In vivo therapeutic efficacy of r6,N-acyl-Glc Tei analogs. (A) Peritoneal fluid (PF) was collected from each MRSA-infected mouse after treatment with a given testing compound. (B) The lungs of AB-infected mice were lavaged to collect 0.6 mL bronchoalveolar lavage (BAL) fluid after treatment with a given testing compound. Statistical analysis was performed using the Student’s t test: *P < 0.05, **P < 0.01.


Page 30 of 31

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Table of Contents (TOC)

OH O

OH O

HO HO O

NH

O

H

HN

O

Cl H N

O

O

N H H

O

O

N H

Cl

H N HN

N

CH 3 CH 3

O

OH OH

HO

O O

HO

HO 2C H

HO

OH OH OH O

HO HO

O

O

NH O O

HN HO 2C H HO

H

O

O

Cl H N

O

N H H

O

N H HO

OH OH

Cl

O

OH HO HO H N O

O

O

HN

HN

NH 2

HO 2C H

O

HO

HO

OH O

OH O

HO HO

NH O

O

O

O HN

HO 2C H HO

H

O

Cl H N

N H H

O

O

N H HO

OH OH

H

N H H

HN N

N H HO

OH OH

O O

O

O

Cl

H N

O

Cl H N

O

NH 2 O

O

CH 3 CH 3

O HO


Cl

H N

O O

HN

O HO

N

CH 3 CH 3

Teicoplanin reprogrammed with the N-acyl-Glc pharmacophore at the

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