Small molecule in situ resin capture provides a compound first approach to natural product discovery

Abstract

Culture-based microbial natural product discovery strategies fail to realize the extraordinary biosynthetic potential detected across earth's microbiomes. Here we introduce Small Molecule In situ Resin Capture (SMIRC), a culture-independent method to obtain natural products directly from the environments in which they are produced. We use SMIRC to capture numerous compounds including two new carbon skeletons that were characterized using NMR and contain structural features that are, to the best of our knowledge, unprecedented among natural products. Applications across diverse marine habitats reveal biome-specific metabolomic signatures and levels of chemical diversity in concordance with sequence-based predictions. Expanded deployments, in situ cultivation, and metagenomics facilitate compound discovery, enhance yields, and link compounds to candidate producing organisms, although microbial community complexity creates challenges for the later. This compound-first approach to natural product discovery provides access to poorly explored chemical space and has implications for drug discovery and the detection of chemically mediated biotic interactions.

Fig. 1. SMIRC deployment in a Zostera marina meadow (Mission Bay, San Diego), extract analysis, compound identification, and in situ microbial growth.

A From left to right: activated HP-20 resin before enclosing in Nitex mesh and embroidery hoop, SMIRC deployment, recovered resin before extraction, concentrated extract in MeOH. B UV₃₆₀ chromatogram of the resin extract and corresponding microfractionation bioassay (80 fractions collected over 20 min) with heatmap showing E. coli LptD4123 growth inhibition (OD₆₅₀, red = no growth, green = 100% growth relative to control). Blue arrow: UV spectrum of active peak (7.6 min). MS spectrum of the active compound shows loss of sulfate. C ¹H NMR (500 MHz) and structure of the flavonoid chrysoeriol, a degradation product of chrysoeriol sulfate. D UV₃₆₀ chromatogram of extract generated from resin embedded in agar (in situ cultivation) deployed at the same site revealed an additional peak (red arrow). E Structure and MS spectrum of aplysiopsene A isolated from the agar/resin peak (red arrow). F Pink colonies growing on agar/resin matrix. UV (ultraviolet), calcd (calculated), OD (optical density).

Fig. 2. SMIRC deployment at cabrillo state marine reserve (CSMR) and extract analysis.

A SMIRC deployed on rocky substrate. B Base peak chromatogram (BPC) of SMIRC extract and mass spectra of compounds targeted for isolation. ESI (electrospray ionization). C ¹H NMR spectrum of m/z 328.2485 compound cabrillostatin (1, 40 μg, 600 MHz, 1.7 mm cryoprobe, CD₃OD). Ppm (parts per million).

Fig. 3. Novel compounds isolated using SMIRC.

Structures of cabrillostatin (1) and cabrillospirals A (2) and B (3).

Fig. 4. Bioactivity of cabrillostatin (1) and cabrillospirals A (2) and B (3).

A Principal component analysis of high dimensional phenotypic profiles of 1–3 (red, green, yellow, respectively), reference compounds (gray), and DMSO (blue) in nine diverse cell lines: AsPC 1 (human pancreas), A549 (lung), DU145 (prostate), HCT116 (colon), HEPG2 (hepatoma), OVCAR4 (ovarian), U87MG (glioblastoma), WM164 (melanoma), 786-O (renal). DMSO (dimethyl sulfoxide). B Bioactivity expressed as significance (−log² p val) across all cell lines. Responses above the dashed line are considered significant (p < 10⁻⁶). P values for compound-to-DMSO distances were calculated based on the empirical null distribution of DMSO-DMSO distances (one-sided, no adjustments for multiple comparisons; Methods). C Representative Ca²⁺ transient traces of DMSO (top) and 1 (bottom). D Scatter plot showing beating frequency and mean peak amplitude of 1 (red), DMSO (blue), and anti-arrhythmic and anti-cancer drugs (gray).

Fig. 5. Chemical diversity across biomes.

A PCoA plot of metabolomes derived from four locations (colors). B New compounds identified in the molecular network include 1-2 and others that have yet to be structurally characterized. Colors as per A. Numbers within nodes (circles) indicate m/z values. C Additional cabrillostatins identified in DOM based on MS/MS fragmentation spectra. MF (molecular formula), calcd (calculated).