Titration-Based Screening for Evaluation of Natural Product Extracts: Identification of an Aspulvinone Family of Luciferase Inhibitors

Abstract

The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography.

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

“…18,19 The production of aspulvinones, as revealed in a previous study, is a general stress response of A. terreus . 20 The biological functions of aspulvinones, and what role they play in the biology of A. terreus , remain elusive.…”

Leveraging the Promise of Chemical Genomics

“…One emerging solution is to use natural products extracts, mixtures of compounds that are isolated from microorganisms (5). But producing extracts that are clean enough and homogenous enough for screening is complicated.…”

Matching the power of high throughput screening to the chemical diversity of natural products

“…However, the principles derived from these studies and from more empirical assessment of extracts have been applied via careful reagent, detection, configuration, and process development to successful HTS assays employing natural product extracts. As a result, in the last 10-12 years, HTS of natural product extracts has identified modulators of a number of enzymatic targets including: E3 ubiquitin ligase MDM2, 57 luciferase, 101 proteases, 102 AKT kinase, 103 asparaginyl-tRNA synthetase, 109 PPARgamma, 110 aromatase, 100 influenza neuraminidases, 111,112,113 tyrosinase, 114 acetylcholinesterase, 115 phosphodiesterase, 116,117 HDAC and SIRT enzymes, 118 MRSA pyruvate kinase 119 and lipases. 116 Protein-protein interaction screens have included HIV gp41 binding 120 and Hes1 dimerization.…”