3.10.2. Griess Assay and Cytotoxicity of Compound **1** in RAW 264.7 Cells

RAW 264.7 murine macrophages (ATCC TIB-71) were seeded at 5 <sup>×</sup> 104 cells in 96-well plates in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% endotoxin-low FBS (HyClone, characterized, Endotoxin: ≤ 25 EU/mL), 190 μL/well, and incubated for 24 h at 37 ◦C. Compound **1** at concentrations of 55, 28, 14, or 7 μM was applied in triplicate, and after 1 h lipopolysaccharide (LPS from Escherichia coli 026:B6, =10,000 EU/mg, Sigma-Aldrich, Oakville, ON, Canada) was added (0.5 or 1.5 μg/mL) to all wells except those for the LPS-free controls and those for evaluating the pro-inflammatory effects of compound **1**. LPS alone was used as a negative control, whereas the same LPS concentration with 1% DMSO served as the positive control in the Griess assay. After 24 h, Griess reactions (Section 3.10.3) were used to assess NO generation as a proxy for inflammation, and MTT staining (Section 3.10.1) was used to assess cell viability. Doxorubicin at 3.3 μg/mL was used as a positive control for assessing cell viability. Cell survival was calculated as a percentage compared to wells with 1% or 1.5% EtOH and no LPS. A NO concentration standard curve was prepared in Microsoft Excel based on eight serial dilutions of a nitrite standard (0–100 μM) with DMEM. One-way ANOVA and Tukey's method were used to test for significance in the cell survival results from the assay; high mortality in certain conditions made statistical analyses of the inflammation data inappropriate. Statistical analyses were applied using GraphPad Prism version 8.0.0 for Windows. Batch variability in LPS potency and RAW 264.7 murine macrophage sensitivity, as well as limited availability of compound **1** necessitated using differing reagent concentrations across the biological replicates.

#### 3.10.3. Griess Reaction

Supernatant from each sample well (50 μL) was added to the experimental wells in triplicate. A 1:1 mixture of 1% sulfanilamide solution in 5% phosphoric acid and 0.1% *N*-1-napthylethylenediamine dihydrochloride (100 μL) was added to each well and the plate was incubated in the dark for 20 min. Optical density was measured at 570 nm on a SpectraMax M2 microplate reader. The raw data were exported to a Microsoft Excel work sheet and the concentration of nitrite in the samples was determined by comparison to the standard curve using regression analysis.

#### 3.10.4. In silico Antibiotic Screening

The simplified molecular-input line-entry system (SMILES) structures of compound **1**, proposed analogs **2**–**10**, C12-tertramic acid, and C14-tetramic acid were submitted to Chemprop Predict (http: //chemprop.csail.mit.edu/predict) [79], using the Antibiotics model checkpoint.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/10/515/s1, Figure S1: Molecular network of *M. bouillonii* crude extracts; Figure S2: Predicted 13C shifts for candidate structure **1a**; Figure S3: Predicted 13C shifts for candidate structure **1b**; Figure S4: Compound **1** derived 2-methyoctanoic acid compared to standards; Figure S5: Compound **1** derived lysine compared to standards; Figure S6: Doscadenamide A (**1**) consensus MS2 spectrum; Figure S7: Molecular network cluster of compound **1** and analogs, highlighting *m*/*z* 168 frag. peak; Figure S8: Structure of compound **1** with structure proposals for analogs (**2–10**); Figure S9: Doscadenamide B (**2**) consensus MS2 spectrum; Figure S10: Doscadenamide B (**2**) proposed fragmentation; Figure S11: Doscadenamide C (**3**) consensus MS2 spectrum; Figure S12: Doscadenamide C (**3**) proposed fragmentation; Figure S13: Doscadenamide D (**4**) consensus MS2 spectrum; Figure S14: Doscadenamide D (**4**) proposed fragmentation; Figure S15: Doscadenamide E (**5**) consensus MS2 spectrum; Figure S16: Doscadenamide E (**5**) proposed fragmentation; Figure S17: Doscadenamide F (**6**) consensus MS2 spectrum; Figure S18: Doscadenamide F (**6**) proposed fragmentation; Figure S19: Doscadenamide G (**7**) consensus MS2 spectrum; Figure S20: Doscadenamide G (**7**) proposed fragmentation; Figure S21: Doscadenamide H (**8**) consensus MS2 spectrum; Figure S22: Doscadenamide H (**8**) proposed fragmentation; Figure S23: Doscadenamide I (**9**) consensus MS2 spectrum; Figure S24: Doscadenamide I (**9**) proposed fragmentation; Figure S25: Doscadenamide J (**10**) consensus MS2 spectrum; Figure S26: Doscadenamide J (**10**) proposed fragmentation; Figure S27: Representative structures from compound families similar to the doscadenamides; Figure S28: Results of compound **1** in Griess assay—biological replicate 1; Figure S29: Results of compound **1** in Griess assay—biological replicate 2; Figure S30: Results of compound **1** in Griess assay—biological replicate 3; Figure S31: UV/Vis absorbance spectrum (200–400 nm) for compound **1**; Figure S32: IR spectrum for compound **1**; Figure S33: 1H NMR spectrum for

compound **1**; Figure S34: 13C NMR spectrum for compound **1**; Figure S35: 1H-1H COSY spectrum for compound **1**; Figure S36: 1H-13C HSQC spectrum for compound **1**; Figure S37: 1H-13C HMBC spectrum for compound **1**; Figure S38: 1H-13C HSQC-TOCSY spectrum for compound **1**; Figure S39: 1H-13C Long-range HSQMBC spectrum for compound **1**; Table S1: Known compounds isolated from *M. bouillonii*; Table S2: Average relative abundances and feature selection scores for top 10 Saipan MS1 features; Table S3: Putative identifications for top 30 MS1 features in the *M. bouillonii* crude extract dataset; Table S4: In silico antibiotic screening results for the doscadenamides and tetramic acids; Table S5: *M. bouillonii* crude extract sample metadata; Table S6: ORCA parameter set for MS1 feature dendrogram; Table S7: ORCA parameter set for GNPS MS2 feature presence/absence dendrogram; Table S8: ORCA parameter set for MS1 feature selection; Table S9: GNPS parameter set for *M. bouillonii* crude extract molecular network; Table S10: GNPS parameter set for *M. bouillonii* crude extract MS2 feature bucket table; Table S11: GNPS parameter set for Saipan and Guam crude extracts and fractions molecular network; Table S12: VLC fractionation solvent systems; and Table S13: 1H and 13C NMR data for doscadenamide A (**1**) in CDCl3.

**Author Contributions:** Conceptualization, C.A.L. and W.H.G.; methodology, C.A.L., C.B.N., L.K., J.A. and E.J.E.C.-D.; software, C.A.L.; validation, C.A.L.; formal analysis, C.A.L.; investigation, C.A.L., C.B.N., L.K., J.A., E.J.E.C.-D. and E.G.; resources, C.A.L., C.B.N., J.A., E.J.E.C.-D., E.G., V.J., T.P.S., A.J.R., J.S.B., T.L., Y.Y., S.H., X.Y. and W.H.G.; data curation, C.A.L.; writing—original draft preparation, C.A.L. and W.H.G.; writing—review and editing, C.A.L., C.B.N., L.K., J.A., E.J.E.C.-D., E.G., V.J., T.P.S., A.J.R., J.S.B., T.L., Y.Y., S.H., X.Y. and W.H.G.; visualization, C.A.L.; supervision, W.H.G.; project administration, C.A.L.; funding acquisition, W.H.G. and C.B.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Institutes of Health (NIH) (grant GM107550 to W.H.G.) and the Gordon and Betty Moore Foundation (grant GBMF7622 to W.H.G.). This study was further supported in part by the National Key Research and Development Program of China, funded through MOST (the Ministry of Science and Technology of China; grant 2018YFC0310900 to X.Y. and C.B.N.), NSFC (the National Natural Science Foundation of China; grant 81850410553 to C.B.N.), and Ningbo STI (Ningbo Science and Technology Bureau; grant 010-20171JCGY01172 to C.B.N.). A portion of this study was supported by the University Grants Commission, Government of India under Indo-US 21st Century Knowledge Initiative Project (Grant No. 194-1/2009(IC) dated 7/2/2015) to V.J. and T.P.S. C.A.L. was supported by the UCSD Regents Pre-Doctoral Fellowship, the Robert L. Cody Memorial Pre-Doctoral Fellowship, the Kaplan Trust CMBB Pre-Doctoral Fellowship, and the NIH Training Program in Marine Biotechnology (T32GM067550). L.K. was supported by the Deutsche Forschungsgemeinschaft (Grant KE 2172/3-1 and KE 2172/4-1).

**Acknowledgments:** We acknowledge F.M. Brunner and C.P. Kubiak (UCSD Department of Chemistry & Biochemistry) for facilitating acquisition of IR data, and Y. Su, and L. Gross for HiRes mass spectrometry support at the UCSD Molecular Mass Spectrometry Facility. We thank B. Duggan and A. Mrse for NMR support, and M.P. Christy (Scripps Institution of Oceanography, UCSD) for support with the synthesis of standards. We also acknowledge the government of Sansha city, China, for permission to collect and study several of the marine samples used in this research and the sample collection assistance from L. Zhenhua, L. Daning and H. Da of the Xisha Marine Science Comprehensive Experimental Station, South China Sea Institute of Oceanology, Chinese Academy of Sciences. We acknowledge the support of I.S. Bright Singh of the National Centre for Aquatic Animal Health, Cochin University of Science and Technology, India, and the Department of Science and Technology, Kavaratti, Lakshadweep Islands, India for the required research permits. C.L. acknowledges travel support from the LBG foundation.

**Conflicts of Interest:** William H. Gerwick declares a competing financial interest as a cofounder of NMR Finder LLC. Otherwise, the authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
