Small Molecule in situ Resin Capture - A Compound First Approach to Natural Product Discovery

Abstract

Microbial natural products remain an important resource for drug discovery. Yet, commonly employed discovery techniques are plagued by the rediscovery of known compounds, the relatively few microbes that can be cultured, and laboratory growth conditions that do not elicit biosynthetic gene expression among myriad other challenges. Here we introduce a culture independent approach to natural product discovery that we call the Small Molecule In situ Resin Capture (SMIRC) technique. SMIRC exploits in situ environmental conditions to elicit compound production and represents a new approach to access poorly explored chemical space by capturing natural products directly from the environments in which they are produced. In contrast to traditional methods, this compound-first approach can capture structurally complex small molecules across all domains of life in a single deployment while relying on Nature to provide the complex and poorly understood environmental cues needed to elicit biosynthetic gene expression. We illustrate the effectiveness of SMIRC in marine habitats with the discovery of numerous new compounds and demonstrate that sufficient compound yields can be obtained for NMR-based structure assignment. Two new compound classes are reported including one novel carbon skeleton that possesses a functional group not previously observed among natural products and a second that possesses potent biological activity. We introduce expanded deployments, in situ cultivation, and metagenomics as methods to facilitate compound discovery, enhance yields, and link compounds to producing organisms. This compound first approach can provide unprecedented access to new natural product chemotypes with broad implications for drug discovery.

Keywords: Biological sciences – biochemistry; Physical sciences – chemistry; drug discovery; environmental metabolomics; microbiomes; natural products.

Figure 1.

SMIRC deployments in Zostera marina meadow (Mission Bay, San Diego). A. From left to right: activated HP-20 resin before enclosing in Nitex, 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 minutes) with heatmap showing E. coli LptD4123 growth inhibition (red = no growth). Blue arrow: UV spectrum of active peak (7.6 min). MS spectrum of the active compound shows loss of a sulfate moiety. C. ¹H NMR (500 MHz) and structure of the isolated 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 that yielded aplysiopsene A.

Figure 2.

SMIRC deployment at Cabrillo State Marine Reserve (CSMR). A. SMIRC resin deployed on rocky substrate. B. Base peak chromatogram of a SMIRC extract and mass spectra (a-c) of compounds targeted for isolation. C. ¹H NMR spectrum of the m/z 328.2485 compound cabrillostatin (1, 40 μg, 600 MHz, 1.7 mm cryoprobe, CD₃OD).

Figure 3.

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

Figure 4.

Reproducibility of three target compounds at the CSMR site. In all cases, compound yields were highest for the longest deployments. Cabrillostatin (1), cabrillospiral A (2), unknown compound (m/z 757).

Figure 5.

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 (grey), and DMSO (blue) in nine diverse cell lines. B. Bioactivity expressed as significance (-log2pval) across all cell lines. 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 (grey).