Screening methods for bioactive metabolites

 Screening methods for bioactive metabolites are essential in drug discovery and natural product research. These methods help identify biologically active compounds from complex mixtures, like plant extracts, microbial cultures, or other biological sources. Below are some common screening methods, along with suitable examples

1. Bioassay-Guided Fractionation

This method involves fractionating a crude extract and testing the fractions for biological activity. Active fractions are further purified and tested until the bioactive metabolite is isolated.

• Example:
  • Artemisinin was isolated from Artemisia annua using bioassay-guided fractionation based on its antimalarial activity. Initial crude extracts were tested in a malaria assay, leading to the identification of the active compound.

2. Metabolomics

Metabolomics is the comprehensive analysis of metabolites within a biological system. It involves high-throughput techniques such as liquid chromatography-mass spectrometry (LC-MS) or nuclear magnetic resonance (NMR) to identify and quantify metabolites.

• Example:
  • Anticancer drug discovery from marine organisms: LC-MS was used to screen marine sponges, leading to the discovery of Halichondrin B, a potent anticancer compound that formed the basis for the drug Eribulin.

3. High-Throughput Screening (HTS)

HTS involves screening large libraries of compounds for biological activity using automated techniques. It allows the rapid identification of bioactive metabolites from large compound libraries.

• Example:
  • HTS was used to discover Paclitaxel (Taxol) from the Pacific yew tree (Taxus brevifolia). Paclitaxel's anti-cancer activity was identified through a screening process involving hundreds of plant extracts.

4. Dereplication

Dereplication involves identifying known compounds early in the screening process to avoid redundant discovery of previously characterized metabolites. Techniques like LC-MS or NMR are used for rapid comparison with databases of known compounds.

• Example:
  • In fungal metabolite screening, dereplication was employed to avoid rediscovery of common antibiotics like Penicillin, focusing instead on novel antifungal agents.

5. Genomic and Metagenomic Approaches

These methods involve sequencing the DNA of microorganisms or environmental samples to identify biosynthetic gene clusters that may produce bioactive metabolites. Synthetic biology techniques can be used to express these genes in heterologous hosts.

• Example:
  • The discovery of novel polyketides and nonribosomal peptides from soil metagenomes using gene cluster analysis led to the identification of bioactive compounds with antibiotic properties.

6. Microbial Fermentation

Fermentation is a traditional method where microorganisms like bacteria, fungi, or actinomycetes are cultured, and their secondary metabolites are tested for biological activity.

• Example:
  • Streptomyces species produce various antibiotics through fermentation. Streptomycin, the first antibiotic used to treat tuberculosis, was discovered using this method.

7. Affinity-Based Screening

In this approach, target biomolecules (like enzymes or receptors) are immobilized on a solid support, and potential bioactive compounds from an extract are allowed to bind. Active metabolites are then eluted and identified.

• Example:
  • Affinity-based screening identified Captopril, an angiotensin-converting enzyme (ACE) inhibitor, from snake venom. The compound was discovered based on its ability to inhibit the ACE enzyme, leading to its use as an antihypertensive drug.

8. Activity-Directed Fractionation Using Fluorescence or Luminescence

This method utilizes fluorescence or luminescence-based assays to detect biological activity (e.g., enzyme inhibition) in fractions of an extract. These assays can be sensitive and specific to particular bioactivities.

• Example:
  • Luciferase-based screening was used to discover compounds inhibiting luciferase activity, which led to the identification of potential antibacterial agents from marine organisms.

These methods provide a structured and effective approach to discovering new bioactive metabolites that can be further developed into therapeutic agents.