Journal of Natural Products 10% CH3CN−H2O + 0.1% formic acid to 100% CH3CN + 0.1% formic acid to give pure 1 (2.0 mg), 2 (0.9 mg), and 3 (1.8 mg). Pseudonocardone A (1): colorless solid (2.0 mg); [α]20D −11 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 310 nm (3.11), 279 (sh), 271 nm (3.59); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD), see Table 1; (+)-HRESI m/z 497.1438 [M + H]+ (calcd for C26H25O10, 497.1448). Pseudonocardone B (2): yellow solid (0.9 mg); [α]20D −2 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 377 nm (3.52), 305 nm (3.87); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD), see Table 1; (+)-HRESI m/z 495.1295 [M + H]+ (calcd for C26H23O10, 495.1291). Pseudonocardone C (3): orange solid (1.8 mg); [α]20D −3 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 403 nm (3.53), 314 nm (3.96); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) see Table 1; (+)-HRESI m/z 511.1229 (calcd for C26H23O11, 511.1240). Antibiotic Assays. The appropriate test organism was grown in a 5 mL culture overnight in either LB medium (for E. coli and B. subtilis) or YPD medium (for C. albicans and S. cerevisiae) at 30 °C. In each case, the overnight culture was diluted with additional sterile medium (LB or YPD) to an OD600 of 0.01. Compounds 1−5 were dissolved in DMSO to give a concentration of 5 mg/mL and 2-fold serially diluted. These solutions (1 μL) were added to the wells of a 96-well plate, followed by the diluted culture of the test organism (99 μL) to give a ﬁnal compound concentration ranging from 50 to 0.1 μg/mL. The cultures were allowed to grow for 24 h at 30 °C before the OD600 was measured using a plate reader. The MIC was deﬁned as the lowest concentration that gave less than 5% of the maximum OD600. Each antibiotic assay was performed in duplicate. Dynemicin A was used as a positive control and gave MIC values against E. coli, B. subtilis, C. albicans, and S. cerevisiae of 313, 0.16, 156, and 156 ng/mL, respectively. Cytotoxic Assay. Compounds were tested for activity against HepG2 human hepatoma cells (ATCC) that were maintained in DMEM (Invitrogen), 10% FBS (Sigma), and 1% antibiotic− antimycotic (Invitrogen) in a standard tissue culture incubator (37 °C, 5% CO2). For assays, compounds 1−5 (in DMSO) were added in triplicate to 15 000 cells in a 384-well plate. The ﬁnal concentration of DMSO was 1%, and compounds varied from 0 to 50 μg/mL. Cells were incubated with the compounds for 2 days at 37 °C, and then liver cell viability was assessed with CellTiter-Glo (Promega). The relative signal intensity of each sample was evaluated with an EnVision (PerkinElmer) system. Liver-Stage P. berghei Assay. Liver-stage P. berghei assays were performed using a luciferase-expressing sporozoite strain of P. berghei ANKA. Parasites were obtained from dissection of Plasmodium- infected Anopheles stephensi mosquitoes (New York University Langone Medical Center Insectary). Malaria parasites (4000 sporozoites) were used to infect HepG2 cells (15 000 cells) in a 384-well plate in the presence of compounds 1−5 in triplicate. The ﬁnal concentration of DMSO was 1%, and compounds varied from 0 to 50 μg/mL. Cells were incubated with the compounds for 2 days at 37 °C, and then relative parasite load was determined after addition of Bright-Glo (Promega). Data analysis for HepG2 toxicity and liver- stage malaria activity was carried out using GraphPad Prism, and curves were ﬁt with a standard inhibition dose−response curve to generate an IC50 value. All statistical results are the mean IC50 value averaged from two independent experiments. Atovaquone was used as a positive control and gave an IC50 in blood stage assays of 0.3 nM. ■ ASSOCIATED CONTENT *S Supporting Information HPLC-MS traces for compounds 1−3 and NMR spectra for compounds 1−5. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *Tel: (617) 432-2845. Fax: (617) 432-6424. E-mail: jon_ clardy@hms.harvard.edu. Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS Note This work was supported by NIH RC4GM096347 (C.R.C. and J.C.), GM086258 (J.C.), and NSF CAREERDEB-747002 (C.R.C.), and a Ruth L. Kirschstein National Research Service Award F32GM093510 (E.R.D.). ■ REFERENCES (1) Cafaro, M. J.; Currie, C. R. Can. J. Microbiol. 2005, 51, 441−446. (2) Currie, C. R.; Scott, J. A.; Summerbell, R. C.; Malloch, D. Nature 1999, 398, 701−704. (3) Oh, D.-C.; Poulsen, M.; Currie, C. R.; Clardy, J. Nat. Chem. Biol. 2009, 5, 391−393. 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