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Spiroaspertrione A, a Bridged Spirocyclic Meroterpenoid as a Potent Potentiator of Oxacillin against MethicillinResistant Staphylococcus aureus from Aspergillus sp. TJ23 Yan He, Zhengxi Hu, Weiguang Sun, Qin Li, Xiao-Nian Li, Hucheng Zhu, Jinfeng Huang, Junjun Liu, Jianping Wang, Yongbo Xue, and Yonghui Zhang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b00056 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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The Journal of Organic Chemistry

Spiroaspertrione A, a Bridged Spirocyclic Meroterpenoid as a Potent Potentiator of Oxacillin against Methicillin-Resistant Staphylococcus aureus from Aspergillus sp. TJ23 Yan He,†,§ Zhengxi Hu,†,§ Weiguang Sun,† Qin Li,† Xiao-Nian Li,‡ Hucheng Zhu,† Jinfeng Huang,† Junjun Liu,† Jianping Wang,† Yongbo Xue,*,† and Yonghui Zhang*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

ABSTRACT: Bioassay-guided isolation of cultures of Aspergillus sp. TJ23 yielded a novel terpene-polyketide hybrid spiromeroterpenoid, spiroaspertrione A (1), bearing a unique spiro[bicyclo[3.2.2]nonane-2,1′-cyclohexane] carbocyclic skeleton and a new bio-intermediate andiconin B (2). Their structures and absolute configurations were elucidated by spectroscopic analyses, singlecrystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compound 1 demonstrated potent resensitization of oxacillin against MRSA by lowering the oxacillin minimal inhibitory concentration (MIC) up to 32-fold from 32 µg/mL to 1 µg/mL.

INTRODUCTION Multidrug-resistant pathogens are a growing threat to human health with many infectious diseases effectively regressing to the pre-antibiotic era.1 Methicillin-resistant Staphylococcus aureus (MRSA) is one of these “superbugs” that expresses a remarkable array of resistance and virulence factors that contribute to its prominent roles in hospital and communityacquired infections.2 It acquires a series of drug-resistance gene mutations, as exemplified by mecA, and encodes penicillin-binding protein 2a (PBP2a) that produces inducible resistance to β-lactam antibiotics.3 Additionally, the rapid acquisition of resistance to other types of antibiotic (besides glycopeptides and lipopeptides) increasingly restricts the therapeutic options for MRSA.4 Potentiating the effect of existing antimicrobial agents may provide promising approaches to combating infections due to multidrug-resistant pathogens.5 Recently, researchers have been exploring the combined use of small molecules that are able to render MRSA sensitive to the effects of conventional β-lactam antibiotics.6 Combination therapy could mitigate the emergence of antibiotic resistance by preserving the usefulness of agents already available in the pharmacological armamentarium.6 In our efforts to identify structurally novel and bioactive natural products from fungi,7 approximately 1500 semi-pure fractions from fungal culture extracts were screened against MRSA (ATCC43300) in vitro. The results show that one semi-pure fraction produced by the endophytic fungus Aspergillus sp. TJ23 exhibited the best antibacterial activity against

MRSA with an MIC value of 16 µg/mL. Bioassay-guided isolation of the active fraction afforded two new terpenepolyketide hybrid meroterpenoids (Figure 1), spiroaspertrione A (1), representing the first spiromeroterpenoid with a unique spiro[bicyclo[3.2.2]nonane-2,1′-cyclohexane] carbocyclic skeleton, and a new bio-intermediate, andiconin B (2), wherein 1 was active against MRSA with an MIC value of 4 µg/mL. Additionally, a synergistic test revealed that 1 performed as an effective potentiator for oxacillin in suppressing MRSA growth by reducing the oxacillin MIC up to 32-fold, and the mode of the resensitization activity occurs via inhibition of PBP2a in MRSA. Herein, the isolation, structure elucidation, and plausible biosynthetic pathway as well as the biological evaluations of compounds 1 and 2 are presented.

Figure 1. Structures of spiroaspertrione A (1) and andiconin B (2).

RESULTS AND DISCUSSION Spiroaspertrione A (1) was isolated as colorless crystals (in a mixture of CHCl3–MeOH) and was determined to have a molecular formula of C25H28O5 based on a [M + Na]+ ion at m/z 431.1832 in the HRESIMS analysis, which is indicative of 12 degrees of unsaturation. A detailed analysis of 1H NMR data

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(Table 1) demonstrated the presence of five singlet methyls at δH 1.15 (H3-14), 1.18 (H3-15), 1.39 (H3-13), 1.48 (H3-10′), and 2.36 (H3-9′), one oxygenated methylene at δH 4.22 and 5.03 (both d, J= 11.6 Hz, H2-1′), and four olefinic protons at δH 7.49 (d, J= 10.3, H-1), 5.94 (d, J= 10.3, H-2), 5.69 (s, H-11), and 6.76 (s, H-6′). The 13C NMR data (Table 1) with the help of a DEPT spectrum revealed the presence of 25 carbon resonances corresponding to five sp3 methyls, four methylenes (including one oxygenated), five methines (including four olefinic), and eleven quaternary carbons (including three ketos, one ester carbonyl, and two olefinic). Four carbonyls (δC 210.2, 202.5, 193.0, and 167.0) and three double bonds, accounting for seven out of 12 degrees of unsaturation, were present in the molecule and required that 1 possessed a pentacyclic ring system. Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data for 1 and 2 (δ in ppm, J in Hz). 1 (in CDCl3) 2 (in CD3OD) no. δHa δC δHa δC 1 7.49 d (10.3) 152.6 6.57 d (10.2) 157.5 2 5.94 d (10.3) 126.5 5.83 d (10.2) 125.8 3 202.5 206.4 4 44.7 45.3 5 1.81 m 50.3 1.43 m 39.6 6 1.85 m; 1.97 m 18.4 1.73 m; 1.82 m 19.3 7 1.64 m; 1.67 m 35.6 1.83 m; 2.06 m 24.9 8 62.7 46.6 9 210.2 1.56 m 56.5 10 51.2 39.8 11 5.69 s 125.8 1.30 m; 1.71 m 39.7 12 2.01 m; 2.09 m 41.7 1.12 m; 1.59 m 51.6 13 1.39 s 20.5 1.28 s 23.0 14 1.15 s 21.9 1.05 s 17.3 15 1.18 s 27.0 1.12 s 23.9 1′ 4.22 d (11.6); 71.3 4.17 d (9.4); 70.2 5.03 d (11.6) 4.34 d (9.4) 2′ 56.3 58.2 3′ 161.4 56.5 4′ 193.0 219.4 5′ 54.1 51.1 6′ 6.76 s 139.1 4.02 d (7.1) 73.3 7′ 137.0 3.24 d (7.1) 42.4 8′ 167.0 176.5 9′ 2.36 s 26.7 1.12 s 16.3 10′ 1.48 s 22.7 1.06 s 21.8 a “m” means overlapped or multiplet with other signals.

The planar structure of 1 was elucidated on the basis of 2D NMR spectra including 1H–1H COSY and HMBC correlations (Figure 2). Two proton sequences, H-1/H-2 and H-5/H2-6/H27, were disclosed by the 1H–1H COSY spectrum as well as the observed HMBC correlations from H-2 to C-4; from H2-7 to C-8 and C-9; from H3-13 to C-1, C-5, C-9, and C-10; from H315 to C-3, C-4, C-5, and C-14 established part A of 1. Additionally, the main HMBC correlations from H2-12 to C-2′, C-4′, and C-6′; from H-11 to C-4′ and C-5′; from H-6′ to C-12 and C-2′; from H3-9′ to C-2′, C-3′, and C-11; and from H3-10′ to C-4′, C-5′, C-6′, and C-12 indicated the presence of a unique bicyclo[3.2.2]nonane carbocyclic core with two methyl substituents at C-3′ and C-5′, a keto group at C-4′, and two double bonds at C-6′/C-7′ and C-3′/C-11. A five-membered lactone moiety was incorporated into the bicyclo[3.2.2]nonane core at C-2′ and C-7′, which was confirmed by HMBC correlations from H2-1′ to C-2′, C-7′, C-8′, and C-8 and from H-6′ to C-8′. Accordingly, part B was established as shown in Figure 2. Moreover, the HMBC correlations of H2-7 with C-8, C-

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12, and C-2′ and of H2-12 with C-7, C-8, and C-9 revealed that parts A and B first gathered at C-8 and generated a unique spiro[5.6]dodecane ring system. Thus, the planar structure of 1 was determined.

Figure 2. Key 2D NMR correlations of 1 and 2.

The relative configuration of 1 was partially ascertained by the examination of its NOESY data (Figure 2). The correlations of H-5 with H3-15 and H-7α but without H3-13 indicated that H-5 and H3-13 were α- and β-configured, respectively. Moreover, the characteristic correlations of H3-13 with H-12α and H-12β, of H-12β with H-6β and H-7β, of H-7α with H-1′β, and of H-1′α with H3-9′ suggested that two planes of the rings of parts A and B were vertically arranged and that C-12 was on the upside of part A. However, due to the absence of certain evidence, the orientation of the “CH-6′–C-7′” bridge was left unassigned. Fortunately, after repeated recrystallization by various solvent systems, 1 furnished a high-quality crystal in a mixture of CHCl3–MeOH (10:1) at room temperature, which was successfully subjected to single-crystal X-ray diffraction using Cu Kα radiation (Figure S3, Supporting Information) with a Flack parameter of 0.03(3) (CCDC 1518652),8 which enabled us to confirm its absolute configuration as 5R, 8R, 10S, 2′S, and 5′S. To further support this conclusion, the calculated ECD spectra of 1 and ent-1 were performed using time-dependent density functional theory (TDDFT) of which the calculated spectrum of 1 at the B3LYP/6–31G(d,p) level showed an excellent coincidence with the experimental ECD curve (Figure S27), which undoubtedly corroborated its absolute structure. Andiconin B (2) was isolated as colorless crystals (in MeOH). Its HRESIMS data disclosed a positive molecular ion peak at m/z 435.2148 ([M + Na]+, calcd for 435.2147), which corresponded to a molecular formula of C25H32O5. Analysis of the NMR data (Table 1) of 2 suggested that its structural features were closely similar to those of the known compound andiconin, an intermediate originating from the heterologous expression of the Emericella variecolor gene cluster in Aspergillus oryzae,9 except that a carbonyl group at C-6′ was replaced by a hydroxy group at the same position in 2 that resulted in the H-7′ signal resonating as a doublet at δH 3.24 (d, J = 7.1 Hz) rather than a singlet. This was further verified by the 1 H–1H COSY correlation of H-6′/H-7′ and HMBC correlations of H3-10′ with C-4′, C-5′, and C-6′. The vicinal coupling constant of H-6′ and H-7′ was 7.1 Hz, which indicated a cisrelationship between them10 and was supported by the NOESY interactions of H-6′, H-7′, and H-12β (δH 1.59). Thus, OH-6′ was determined as an α-direction. A further 2D NMR spectra


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confirmed its planar structure and relative configuration as shown in Figure 2. Furthermore, the absolute configuration of 2 was established by a single-crystal X-ray diffraction analysis (Figure S5, Supporting Information) with a Flack parameter of 0.02(11) (CCDC 1520159),8 which demonstrated that the chiral centers in 2 were 5R, 8S, 9S, 10R, 2′R, 3′R, 5′S, 6′R, and 7′R. This conclusion was subsequently supported by the comparison of experimental and DFT-calculated ECD spectra of 2 (Figure S28, Supporting Information). It is noteworthy that 2 was reported as the second case in nature with the identical carbocyclic skeleton of andiconin. Scheme 1. Plausible Biosynthetic Pathway for 1.

To the best of our knowledge, spiroaspertrione A (1) represented a novel bridged spirocyclic meroterpenoid with a unique spiro[bicyclo[3.2.2]nonane-2,1′-cyclohexane] carbocyclic core. A biosynthetic origin for 1 was proposed with the co-isolated 2 as a bio-intermediate (Scheme 1). Derived from farnesyl pyrophosphate (FPP) and DHDMP,9 a series of alkylation, intramolecular cyclization, and redox reactions led to formation of the key intermediate andiconin, in accordance with its complete biosynthetic pathway.9 On this basis, a key reduction reaction at C-6′ could create the biogenetic intermediate 2. Afterwards, the crucial oxidative cleavage and rearrangement11 reactions constructed a new seven-membered ring of the backbone rooting in that carbon C-11 of the sesquiterpenoid moiety was blended into the polyketide moiety, which finally yielded 1. Antibacterial activities against MRSA were observed for 1 and 2 with MIC values of 4 µg/mL and 16 µg/mL, respectively. A checkerboard assay was employed to determine the synergistic activity of 1 and 2 with oxacillin and other anti-MRSA antibiotics.12 Compound 1 was found to work synergistically with oxacillin with a ∑FIC value12 of 0.28 (Table S1, Supporting Information). However, it did not show any synergistic effects with other antibiotics, such as chloramphenicol and vancomycin, against MRSA. Noticeably, when co-dosed with 1 (1 µg/mL), the MIC value of oxacillin against MRSA was markedly reduced from 32 µg/mL to 1 µg/mL (32-fold), thus dropping the oxacillin MIC below the breakpoint for clinical resistance. Time-kill curves for MRSA in the presence of 1 and/or oxacillin were further constructed (Table S2, Supporting Information). Compound 1 (4 µg/mL) inhibited the growth of bacteria entering exponential phase and resulted in nearly constant bacterial counts, which indicate that 1 is bacteriostatic in nature. A combination of 1 (1 µg/mL) with oxacillin (1 µg/mL) also showed bacteriostatic effects with a slight reduc-

tion in colony counts after 2 h. In addition, this combination resulted in log CFU reductions of 3.70 and 2.11 when using oxacillin (1 µg/mL) or 1 (1 µg/mL) alone, which further confirmed their synergistic effects. Because it is important as a potential antibiotic adjuvant, 1 was tested for hemolytic activity using mechanically defibrinated sheep blood.12 As a result, 1 exhibited little effect on the eukaryotic cell membranes, and less than 2% lysis was observed compared to Triton as a positive control at as high as 16 µg/mL (4-fold MIC). To further investigate the antibacterial mechanism of 1, in silico target confirmation assays7d,13 were employed to forecast its possible targets. Therefore, 1 was screened against all five PBPs (PBP2a, PBP1, PBP2, PBP3, and PBP4) encoded in the MRSA core genome in silico, which were considered to be important or possible targets for staphylococcal β-lactam resistance.3,14 The calculated results of the docking scores (Table S3, Supporting Information) predicted that PBP2a showed significantly higher binding affinity for 1 than other PBPs.15 From the generated docking model (Figure 3), 1 is extended in the allosteric site of PBP2a, and hydrogen bonds are predicted between the C-3, C-9, and C-4′ carbonyl oxygens of 1 with Lys316, Tyr105, and Asn297, respectively. Additionally, ring A of the sesquiterpenoid moiety of 1 forms a π-π stacking interaction with Tyr297. Based on the molecular docking results, 1 performs as a potential substrate of PBP2a; importing hydrophilic group substituents placed at the C6-position is a worthy target for further structural modification.

A

B Figure 3. The 3D docking pose showing interaction for 1 in the binding site of the PBP2a crystal structure from MRSA (PDB ID: 4CJN). (A) An overlay of one monomer of the MRSA PBP2a and 1 binary complex. (B) A detailed view of the substrate binding pocket of 1.

Prompted by these results, the protein level of PBP2a was analyzed by Western blotting. As shown in Figure 4, 1 alone could attenuate PBP2a expression, whereas 1 together with oxacillin markedly lowered the expression of PBP2a protein in MRSA, which suggests that 1 could suppress the PBP2a protein level and that the synergism between 1 with oxacillin in suppressing MRSA growth might occur via PBP2a inhibition.16



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Screening the in-House Semi-Pure Natural Product Library against MRSA: The in-house semi-pure Natural Product Library was established by Professor Jianping Wang in Tongji Medical College, Huazhong University of Science and Technology. 128 fungal strains were cultured in two different media. All the cultured mediums were fractionated by Diaion HP-20 (Mitsubishi Chemical Co.) into six fractions to get approximately 1500 semipure fractions. Then, the obtained samples at a fixed concentration (100 µg/mL) were screened against MRSA ATCC43300. Five semi-pure fraction exhibiting potential anti-MRSA activities were further tested using broth microdilution method to determine the MICs. The results showed that one semi-pure fraction (50% CH3OH) produced by an endophytic fungus Aspergillus sp. TJ23 exhibited the best antibacterial activity against MRSA with an MIC value of 16 µg/mL. Figure 4. PBP2a production treatment with oxacillin (32 µg/mL) and 1 (4 µg/mL) alone (lanes 2 and 3, respectively) and in combination (1 µg/mL 1 and 1 µg/mL oxacillin). The control (Con) was used without treatment drugs (lane 1). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

In summary, our research demonstrates that spiroaspertrione A (1), with an intriguing bridged spirocyclic structure, has anti-MRSA activity and resulted in potent activity in suppression of oxacillin resistance in MRSA by reducing the oxacillin MIC to 1 µg/mL. Moreover, the synergism of the anti-MRSA effect is associated with the inhibition of PBP2a expression. For oxacillin, CLSI17 established a susceptibility breakpoint of ≤ 2 µg/mL against MRSA. The above results suggest that 1 has the potential to preserve the usefulness of the conventional β-lactam oxacillin against MRSA. Because MRSA resistance to antibiotics is a growing public health threat of broad concern, we believe that the disclosed bioactivity of 1 may provide a promising lead compound for the development of a combination treatment regime against MRSA.

EXPERIMENTAL SECTION General Experimental Procedures: Optical rotations were measured with a Perkin-Elmer 341 polarimeter. UV, CD, and FTIR spectra were measured using a Varian Cary 50, a JASCO-810 CD spectrometer, and a Bruker Vertex 70 instruments, respectively. NMR spectra were recorded on a Bruker AM-400 spectrometer, and the 1H and 13C NMR chemical shifts were referenced to the solvent or solvent impurity peaks for CD3OD (δH 3.31 and δC 49.0) and CDCl3 (δH 7.26 and δC 77.16). High-resolution electrospray ionization mass spectra (HRESIMS) were recorded in the positive ion mode on a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer. Semi-preparative HPLC was carried out on an Agilent 1200 quaternary system with a UV detector or a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 autosamper injector, and an Ultimate 3000 DAD detector controlled by Chromeleon software (version 6.80), using a reversed-phased C18 column (5 µm, 10 × 250 mm). Column chromatography were performed using silica gel (100–200 and 200– 300 mesh) and Sephadex LH-20. Thin-layer chromatography (TLC) were performed with silica gel 60 F254 and RP-C18 F254 plates. Fungal Material: The fungus Aspergillus sp. TJ23 was isolated from the leaves of plant Hypericum perforatum collected from the Dragon Rack of Hubei Province, China. The sequence data for this strain have been submitted to the DDBJ/EMBL/GenBank under accession no. KY346978. A voucher sample has been deposited in the culture collection of Tongji Medical College, Huazhong University of Science and Technology.

The scaled-up fermentation, extraction, isolation, and purification of compounds 1 and 2: The Aspergillus sp. TJ23 strain was cultured on potato dextrose agar (PDA) at 28 °C for 8 days in stationary phase to prepare the seed culture. Then the Agar plugs were cut into small pieces and inoculated into 25 Erlenmeyer flasks (1 L), previously sterilized by autoclaving, each containing 200 g rice and 200 mL distilled water. All flasks were incubated at 28 °C for 30 days. Following incubation, the growth of cells was stopped by adding 300 mL EtOAc to each flask, and the culture was homogenized. Then the suspension was extracted by ultrasonic extraction with EtOAc for five times at room temperature. The EtOAc was removed under reduced pressure to yield a brown extract (52.7 g) and then separated by HP-20 eluted with MeOH-H2O (10%, 30%, 50%, 70%, 90%, 100%) to afford six fractions (Fr.1–Fr.6). Fr.3 was further purified on a silica gel column eluted with CH2Cl2/MeOH (50:1–0:1), Sephadex LH-20 (MeOH), and followed by repeated semi-preparative HPLC (MeCN-H2O, 55%) to yield 1 (10.5 mg) and 2 (50.0 mg). ECD Calculation: The theoretical calculations of compounds 1 and 2 were performed using Gaussian 09.18 Conformational analysis was initially carried out using Maestro in Schrödinger 2010 conformational searching, together with the OPLS_2005 molecular mechanics methods. The optimized conformation geometries and thermodynamic parameters of all conformations were provided. The OPLS_2005 conformers were optimized at B3LYP/6-31G(d) level. The theoretical calculation of ECD was performed using time dependent Density Functional Theory (TDDFT) at B3LYP/6-311++G** level with PCM model in MeOH. The calculated ECD curves were generated using SpecDis 1.51. Rvel.19 Spiroaspertrione A (1). Colorless crystals; [α]20 –22.5 (c = 0.52, D MeOH); UV (MeOH) λmax (log ε) = 236 (3.64) nm; IR vmax = 3449, 2966, 1763, 1708, 1665, 1623, 1440, 1372, and 1243 cm–1; CD (MeOH) λmax (∆ε) = 216 (–1.6), 240 (+11.5), and 301 (–5.7) nm; For 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS (ESI/TOF-Q) m/z: [M + Na]+ Calcd for C25H28O5Na 431.1834; Found 431.1832. Andiconin B (2). Colorless crystals; [α] 20 –22.7 (c = 0.54, D MeOH); UV (MeOH) λmax (log ε) = 236 (3.70) nm; IR (KBr) vmax = 3505, 2969, 2929, 1768, 1732, 1668, 1460, 1377, and 1155 cm– 1 ; CD (MeOH) λmax (∆ε) = 207 (–0.83), 236 (+7.3), and 300 (–3.1) nm; For 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS (ESI/TOF-Q) m/z: [M + Na]+ Calcd for C25H32O5Na 435.2147; Found 435.2148. Crystal data for spiroaspertrione A (1): C25H28O5, M = 408.47, a = 11.1054(3) Å, b = 13.0801(4) Å, c = 14.4592(4) Å, α = 90°, β = 92.4620(10)°, γ = 90°, V = 2098.40(10) Å3, T = 100(2) K, space group P21, Z = 4, µ(Cu Kα) = 0.722 mm–1, 22571 reflections measured, 7044 independent reflections (Rint = 0.0318). The final R1 values were 0.0265 (I > 2σ(I)). The final wR(F2) values were 0.0674 (I > 2σ(I)). The final R1 values were 0.0265 (all data).


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The final wR(F2) values were 0.0675 (all data). The goodness of fit on F2 was 1.060. Flack parameter = 0.03(3). Crystal data for andiconin B (2): 3(C25H32O5)•2(CH4O), M = 1301.60, a = 7.3400(15) Å, b = 15.440(3) Å, c = 16.480(3) Å, α = 114.57(3)°, β = 97.75(3)°, γ = 94.55(3)°, V = 1663.9(7) Å3, T = 100(2) K, space group P1, Z = 1, µ(Cu Kα) = 0.731 mm–1, 41413 reflections measured, 10684 independent reflections (Rint = 0.0603). The final R1 values were 0.0648 (I > 2σ(I)). The final wR(F2) values were 0.1746 (I > 2σ(I)). The final R1 values were 0.0672 (all data). The final wR(F2) values were 0.1779 (all data). The goodness of fit on F2 was 1.047. Flack parameter = 0.02(11). The crystallographic data for 1 (deposition No. CCDC 1518652) and 2 (deposition No. CCDC 1520159) have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data could be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK (fax: +44-1223-336-033; or E-Mail: [email protected]). Biological Assay Protocols Strains, media, and antibiotics: The tested MRSA strain was obtained from the ATCC: MRSA (ATCC 43300). The reference compounds for the tests were recommended by the National Committee for Clinical Laboratory Standards:17 Oxacillin (Sigma, cat # 1481000); Piperacillin (Sigma, cat # 1541500); Chloramphenicol (Sigma, cat # 1107300); Vancomycin (Sigma, cat # 861987). The investigated compounds 1 and 2 were ≥ 95% pure (HPLC, wavelength = 210 nm). All compounds were dissolved in DMSO as 20 mg/mL stock solutions. Determination of Minimum Inhibitory Concentrations (MIC): Determination of the MICs were conducted according to our previously reported broth microdilution method.7a In brief, the inoculum was standardized to approximately 5 × 105 CFU/mL. The plates were incubated at 37 °C for 16 h, and MIC values were recorded as the lowest concentration of antibiotic at which no visible growths of bacteria were observed. Each experiment was performed three times. Combination Susceptibility Test against MRSA: Broth microdilution checkerboard assay was used for the determination of combination susceptibility.5 MHB was inoculated with MRSA (5 × 105 CFU/mL), and 100 µL were distributed into each well of a 96-well plate except well 1A. Inoculated MHB (200 µL) containing test compound (at 2 × MIC) was added to well 1A, and 100 µL of the same sample were placed in each of wells 2A-12A. Column A wells were mixed 6 to 8 times, and then 100 µL were withdrawn and transferred to column B. Column B wells were mixed 6 to 8 times, followed by a 100 µL transfer to column C. This procedure was repeated to serially dilute the rest of the columns of the plate up to column G (column H was not mixed to allow the MIC of antibiotic alone to be determined). Inoculated media (100 µL) containing antibiotic at two-times of MIC was placed in wells A1-H1 (row 1) and serially diluted in the same manner to row 11. The plates were incubated for 16 h at 37 °C. The MIC values of both compound and antibiotic in the combination were recorded, as well as the MIC values of compound alone (row 12) and antibiotic alone (column H). The ∑FIC values were calculated as follows: ∑FIC = FICCompd + FICAntibiotic, where FICCompd is the MIC of the compound in the combination/MIC of the compound alone, and FICAntibiotic is the MIC of the antibiotic in the combination/MIC of the antibiotic. The combination is considered synergistic when the ∑FIC value is ≤ 0.5, accumulative or indifferent when the ∑FIC value is > 0.5 but < 2, and antagonistic when the ∑FIC is ≥ 2.12 Time-kill curves of compound 1 and/or oxacillin against MRSA: MRSA ATCC 43300 was grown in MHB overnight and this culture was used to inoculate fresh MHB (5 × 105 CFU/mL). Inoculated media was aliquoted (3 mL) into culture tubes and

varied concentration of compound 1 and/or oxacillin were added, untreated inoculated media served as the control. Tubes were incubated at 37 °C with shaking. Samples were taken at 2, 4, 6, 8, 12, and 24 h time points, serially diluted in fresh MHB and plated on tryptic soy agar. Plates were incubated at 37 °C overnight and the number of colonies enumerated.12 To confirm the results, the time-kill assays for each experiment were performed at least three times; the data are represented as mean data or standard deviation (SD). Hemolysis assay: Hemolysis assays were performed on mechanically difibrinated sheep blood (Hemostat Labs: DSB100). Difibrinated blood (1.5 mL) was placed into a microcentrifuge tube and centrifuged for 10 min at 10,000 rpm. The supernatant was then removed and then the cells were resuspended in 1 mL of phosphate-buffered saline (PBS). The suspension was centrifuged, the supernatant was removed and cells were resuspended two additional times. The final cell suspension was then diluted 10fold. Test compound solutions were made in PBS in small culture tubes and then added to aliquots of the 10-fold suspension dilution of blood. PBS was used as a negative control and a zero hemolysis marker. Triton X (a 1% sample) was used as a positive control serving as the 100% lysis marker. Samples were then placed in an incubator at 37 °C while being shaken at 200 rpm for one hour. After one hour, the samples were transferred to microcentrifuge tubes and centrifuged for 10 min at 10,000 rpm. The resulting supernatant was diluted by a factor of 40 in distilled water. The absorbance of the supernatant was then measured with a UV spectrometer at a 540 nm wavelength. Molecular Docking: In addition to PBP2a, MRSA has four other PBPs (PBPs 1–4), which are encoded in the bacterial core genome.14 Although PBP2a was considered directly caused oxacillin resistance of MRSA, other four PBPs might be possible media for staphylococcal β-lactam resistance. With the aim of understand the possible antibacterial mechanism of 1, we employed molecular docking method to predict the targets. For the inverse docking, crystal structures of docking targets (five PBPs in MRSA, Table S3) was obtained from the Protein Data Bank (http://www.rcsb.org). The docking was performed by using Surflx-Dock module of the Sybyl softare.13 Molecule was built with Chemdraw and optimized at molecular mechanical and semiempirical level by using Open Babel GUI.13 The crystallographic ligands were extracted from the active site and the designed ligands were modelled. All the hydrogen atoms were added to define the correct ionization and tautomeric states, and the carboxylate, phosphonate and sulphonate groups were considered in their charged form. In the docking calculation, the default FlexX scoring function was used for exhaustive searching, solid body optimizing and interaction scoring. The pose with the most favorable score was remained. Western Blot: The MRSA (ATCC 43300) strain was grown at OD600 of 0.5 in MHB for Western blot analysis. Strain cells were treated with varying concentrations of 1 and oxacillin for 24 h. The cellular protein extracts were prepared from the bacterial harvest cells collected after 30 min of the treatment. The harvested cells from the bacterial culture were suspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 5 mM phenylmethanesulfonyluoride (PMSF), 1 mM dithiolthreitol (DTT) containing 1% Triton X-100), and separated soluble protein was extracted by centrifugation 12,000 rpm for 15 minutes. The supernatant was collected, the total protein concentration was determined using Bradford reagent (Bio-Rad, USA). The proteins were then dissolved in SDS sample buffer and denatured. Proteins were separated using 12% SDS–PAGE, and then transferred to polyvinylidene fluoride (PVDF, Millipore, USA) membranes. The membranes were blocked with 5% non-fat milk for 1 h and then incubated with monoclonal mouse anti-PBP2a primary antibody



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(diluted 1:500; Biopike Inc.) at 4 °C overnight, and was reprobed with anti-mouse immunoglobulin G secondary antibody (diluted 1:1000; Sigma, cat # F9137). Loading differences were normalized with monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (diluted 1:500; Sigma, cat # G6019). Statistical analysis: Statistical analysis of the data was performed using Graph Pad Prism 5.0 software. The data were expressed as the means ± SD. Values were analyzed using SPSS version 12.0 software by one-way analysis of variance (ANOVA), and p < 0.05 was considered statistically significant.

ASSOCIATED CONTENT Supporting Information Experimental details, spectroscopic data including NMR, HRESIMS, UV, and IR spectra of 1 and 2, and X-ray crystallographic data of 1 and 2 (CIF). The materials are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y.X.). *E-mail: [email protected] (Y.Z.).

Author Contributions §

Y.H. and Z.H. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is financially supported by the Program for New Century Excellent Talents in University, State Education Ministry of China (NCET-2008-0224) and the National Natural Science Foundation of China (Nos. 81573316, 31370372, 31270395, 81573316, 31570361, 21502057, and 81641129).

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