Natural products as chemical probes

Abstract

Natural products have evolved to encompass a broad spectrum of chemical and functional diversity. It is this diversity, along with their structural complexity, that enables nature's small molecules to target a nearly limitless number of biological macromolecules and to often do so in a highly selective fashion. Because of these characteristics, natural products have seen great success as therapeutic agents. However, this vast pool of compounds holds much promise beyond the development of future drugs. These features also make them ideal tools for the study of biological systems. Recent examples of the use of natural products and their derivatives as chemical probes to explore biological phenomena and assemble biochemical pathways are presented here.

Figure 1

Early examples of natural products that were utilized as chemical probes. a) A derivative of trapoxin was used in studies to identify the first histone deacetylase. Trapoxin was immobilized on a matrix, resulting in the conjugate called K-trap, to enable enrichment of its binding partners. The electrophilic epoxyketone in trapoxin mimics the acetylated lysine substrates of these enzymes and promotes covalent modification of this natural product’s protein targets. b) Rapamycin and FK506 have similar structures but influence T cell signal transduction by distinct mechanisms. c) Rapamycin binds to FKBP forming a complex that subsequently interacts with TOR through the FKBP-rapamycin-binding (FRB) domain. d) Rapamycin can be used to dimerize two proteins by conjugation of the target proteins to FKBP and FRB. Treatment with rapamycin enables temporal control over association of the target proteins or promotes interactions between two proteins that do not ordinarily associate.

Figure 2

A diversity of natural products have been used as chemical probes to understand processes ranging from mitotic spindle assembly to amyloid formation. a) Identification of the target of diazonamide A revealed the identity of an enzyme not previously known to be involved in mitosis, ornithine δ–amino transferase. b) The role of fellutamide B in NGF secretion was elucidated following the recognition that this natural product resembled MG-132, a known proteasome inhibitor. The electrophilic aldehyde in these compounds is involved in their inhibitory activity. c) A combination of EGCG and DAPH-12 targets myriad mechanisms of prionogenesis. d) Methylene blue and myricetin both inhibit Hsp70 ATPase activity resulting in tau degradation demonstrating that this protein may be a viable target for the treatment of neurodegenerative diseases.

Figure 3

Development of a high-throughput screen enabled the functional annotation of a cancer-associated protein. a) Enzyme inhibitors were identified using a fluorescence polarization (FP) assay. Each compound was incubated with RBBP9 followed by addition of a reactive probe that specifically labels the active site of the serine hydrolase. Active compounds prevented the probe from tagging the enzyme leaving it free in solution where it tumbles quickly, causing light depolarization (top). Inactive compounds do not bind the enzyme leaving it susceptible to probe labeling. The bound probe tumbles more slowly than the free compound yielding a lower depolarization response (bottom). b) Using the devised FP assay, emetine was identified as a selective inhibitor of RBBP9. Examination of ~75 structurally related compounds showed that this binding interaction is highly specific, even the dehydroemetine analog, which differs by only one unit of unsaturation, was not bound by RBBP9.

Figure 4

Natural product-based compounds often yield highly selective chemical probes. a) Pateamine A targets eIF4A-based translation activity, but does not affect other steps in translation. b) A suite of natural product-inspired probes was developed for profiling of penicillin binding protein (PBP) activity in vivo. The alkyne-functionalized natural product analogs are depicted here. Together, these probes targeted both the anticipated PBPs and a pool of virulence- and resistance-associated enzymes.

Figure 5

Chemical probes were developed to facilitate profiling of PKS and NRPS enzymes. a) Carrier proteins were targeted with a coenzyme A precursor analog (CP probe) while thioesterase domains were tagged with a fluorophosphonate probe (TE probe) known to be specific for serine hydrolases. b) The OASIS method. Isolated proteomes were tagged with either the CP or TE probe. Proteome labeled with the CP probe were conjugated to the biotin tag using copper catalyzed click chemistry. The biotin-labeled proteins were enriched on avidin beads and subjected to on-bead trypsin digestion. The cleaved peptides were analyzed by liquid chromatography-mass spectrometry (LC/LC-MS/MS) to yield protein identifications. Utilization of these two probes enabled global analysis of PKS and NRPS enzymes. Note that some peptides were in common between the two probes while many differed.