*4.6. Isolation*

Crude ethanolic extract of *P. aurata* (200 mg) was dissolved in MeOH. HPLC separation was conducted on a Thermo Electron Betasil C18 column (5 μm, 21.2 × 150 mm) at a flow rate of 9 mL/min: using gradient solvent system (10–100% MeOH in 50 min, 100% MeOH from 50–60min) and 60 fractions were collected by minutes. Fraction 33 and 34 were further purified on the same column using gradient solvent from 30–80% MeOH to a fford polycarpine.

Polycarpine was obtained as yellow powder. HRMS (positive mode): m/z 469 [M + H] + (calculated for C22 H25 N6O2S2, 469.1475). 1H NMR (CD3OD, 800MHz) δ 7.49 (2H, d, *J* = 8.98Hz, H-7/11), 7.03 (2H, d, *J* = 8.98Hz, H-8/10), 3.91 (3H, s, 12-CH3), 3.25 (3H, s, 13-CH3). Carbon chemical shift was confirmed from 2D NMR, δ 109.8 (C-1), 148.1 (C-3), 137.6 (C-5), 117.6 (C-6), 127.7 (C-7/11), 113.7 (C-8/10), 161.4 (C-9), 54.4 (C-12) and 27.8 (C-13).

### *4.7. Dose-Response Data Analysis*

The relative abundances of protein-ligand complex to total protein in the mass spectra correlated to the relative equilibrium concentrations of ligand to total protein in solution. The pseudo *K*D of polycarpine with Rv1466 was determined using the following equations [18]:

$$\frac{\Sigma \text{ I} (\text{P} - \text{L})^{\text{n} +} / \text{n}}{\Sigma \text{ I} (\text{P})^{\text{n} +} / \text{n} + \Sigma \text{ I} (\text{P} - \text{L})^{\text{n} +} / \text{n}} = \frac{\text{[P} - \text{L}]}{\text{[P}]\_{\text{t}}} \tag{1}$$

$$\frac{\Sigma \operatorname{I}(\mathbf{P} - \mathbf{L})^{\mathbf{n} +} / \mathfrak{n}}{\Sigma \operatorname{I}\left(\mathbf{P}\right)^{\mathbf{n} +} / \mathfrak{n} + \Sigma \operatorname{I}\left(\mathbf{P} - \mathbf{L}\right)^{\mathbf{n} +} / \mathfrak{n}} = \frac{[\mathbf{P}]\_{\mathfrak{t}} + [\mathbf{L}]\_{\mathfrak{t}} + \mathbf{K}\_{\mathbf{D}} - \sqrt{([\mathbf{P}]\_{\mathfrak{t}} + [\mathbf{L}]\_{\mathfrak{t}} + \mathbf{K}\_{\mathbf{D}})^2 - 4[\mathbf{P}]\_{\mathfrak{t}}[\mathbf{L}]\_{\mathfrak{t}}}}{2[\mathbf{P}]\_{\mathfrak{t}}} \tag{2}$$

Experimental relative ratios of protein-ligand complex and total protein ion abundances were plotted against the total concentration of ligand. The pseudo *K*D could be obtained as a parameter of a nonlinear least-squares curve fitting.

**Author Contributions:** Conceptualization, R.J.Q. and M.L.; methodology, Y.X., A.D.C., T.M., Y.F., G.W.B. and P.J.M.; resources, R.J.Q. and Y.F.; data curation, M.L.; writing—original draft preparation, Y.X.; writing—review and editing, R.J.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bill and Melinda Gates Foundation (OPP1035218, OPP1174957). This research was supported by an Australian Research Council Discovery and Linkage Projects funding (DP160101429, LP120100485) and equipment support (LE120100170, LE140100119). This research was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under Federal Contract number HHSH27220120025C, HHSN272200700057C, and HHSN272201700059C.

**Acknowledgments:** We acknowledge the NatureBank biota repository that is housed at the Gri ffith Institute for Drug Discovery, Gri ffith University (www.gri ffith.edu.au/gridd). We thank Vasanthi Ramachandran, Sreevalli Sharma, Supreeth Guptha, Sunita DeSousa from the former AstraZeneca Bangalore for the *M. tuberculosis* H37Rv assay. Part of the research was conducted at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by U.S. Department of Energy's O ffice of Biological and Environmental Research (BER) program located at Pacific Northwest National Laboratory (PNNL). Battelle operates PNNL for the U.S. Department of Energy.

**Conflicts of Interest:** The authors declare no conflict of interest.
