Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.06.23.166686

Quantifying synergy in the bioassay-guided fractionation of natural product extracts

Dettweiler, M.·Marquez, L.·Bao, M.·Quave, C. L.
View original source ↗Export metadata ↓

Abstract

Mixtures of drugs often have greater therapeutic value than any of their constituent drugs alone, and such combination therapies are widely used to treat diseases such as cancer, malaria, and viral infections. However, developing useful drug mixtures is challenging due to complex interactions between drugs. Natural substances can be fruitful sources of useful drug mixtures because secondary metabolites produced by living organisms do not often act in isolation in vivo. In order to facilitate the study of interactions within natural substances, a new analytical method to quantify interactions using data generated in the process of bioassay-guided fractionation is presented here: the extract fractional inhibitory concentration index (EFICI). The EFICI method uses the framework of Loewe additivity to calculate fractional inhibitory concentration values by which interactions can be determined for any combination of fractions that make up a parent extract. The EFICI method was applied to data on the bioassay-guided fractionation of Lechea mucronata and Schinus terebinthifolia for growth inhibition of the pathogenic bacterium Acinetobacter baumannii. The L. mucronata extract contained synergistic interactions (EFICI = 0.4181) and the S. terebinthifolia extract was non-interactive overall (EFICI = 0.9129). Quantifying interactions in the bioassay-guided fractionation of natural substances does not require additional experiments and can be useful to guide the experimental process and to support the development of standardized extracts as botanical drugs.View Full Text

Source connections

Explore related subjects

pharmacology and toxicologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Dettweiler, M., Marquez, L., Bao, M., Quave, C. L.. 2020-06-23. Quantifying synergy in the bioassay-guided fractionation of natural product extracts. https://doi.org/10.1101/2020.06.23.166686

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A metastable GαS/β-arrestin complex sustains endosomal PTH1R signaling

G protein-coupled receptors (GPCRs) initiate cellular signaling by activating heterotrimeric G proteins at the plasma membrane and are silenced when beta-arrestin binds the phosphorylated receptor, preventing G protein binding. However, many receptors continue to generate cAMP after internalization, even while barr is bound, a paradox that lacked a molecular mechanism in live cells. A direct barrestin1-Gas interface in the PTH1R-barr1-Gas complex has recently been defined structurally, but whether the complex operates in endosomes, and how it behaves there, has not been established. Using intact cells, we examine the assembly at three spatial resolutions. Confocal imaging with a nanobody selective for nucleotide-free Gas places activated Gas, barr, and agonist-occupied PTH1R on the same endosomes. Single-molecule localization microscopy, with illumination modes that separate the plasma membrane from the endosomal membrane in single cells, shows that the agonist recruits Gas and barr at both membranes. FRET, however, reveals a specific interaction only on endosomes, where a Gas inhibitor restricted to that compartment abolishes the sustained cAMP phase without affecting receptor internalization. Weighted-ensemble-enhanced molecular simulations show that the complex is metastable, releasing Gas through a large rigid-body rotation away from barr while barr itself tightens its interactions with the receptor. These findings provide a molecular basis for endosomal Gs/cAMP signaling and open a new avenue for drug development targeting this pathway.

pharmacology and toxicology↗
bioRxiv2026

Advancing AOPs for genotoxicity through quantifying KERs linking DNA alkylation to increase in mutations and chromosome aberrations

Quantitative Adverse Outcome Pathways (qAOPs) help translate mechanistic knowledge into quantitative toxicity predictions; however, their development is constrained by the limited availability and heterogeneity of quantitative data. Here, we investigated the feasibility of developing a qAOP for genotoxicity by modeling quantitative relationships linking DNA alkylation to increased mutations and structural chromosome aberrations through intermediate biological key events (KEs). We compiled and harmonized published concentration-response data from human lymphoblastoid TK6 cells exposed to ethyl methanesulfonate, methyl methanesulfonate, or N-ethyl-N-nitrosourea, covering eight genotoxicity assays representing five KEs with well-established dependencies. We evaluated sources of variability, compared points of departure (PoDs), and quantified relationships between successive events using response-response modeling. Substantial between-study variability could to a limited extent be explained through differences in experimental design while batch effects for individual studies and researchers were substantial. PoD estimates that were calculated from combined results of independent studies with similar experimental design, were often uncertain due to insufficient concentration-response characterization or conflicting results between studies. Nevertheless, response-response modeling suggested a threshold effect for certain key-event relationships (KERs). Specifically, DNA adducts seemed to be triggered at lower concentrations of alkylating agents compared to the subsequent KEs of DNA strand breaks and chromosome aberrations. Furthermore, these fitted response-response models can be used for prediction of downstream KEs from earlier ones. The general approach we developed for BMC analysis followed by response-response modeling is applicable to other AOPs as well. These findings highlight both the feasibility and limitations of using existing data for qAOP development.

pharmacology and toxicology↗
bioRxiv2026

Denaturation-sensitive BRET probes enable intracellular thermal shift measurements across diverse protein targets and biomolecular complexes

Thermal shift assays have revolutionized drug discovery, but can be limited in sensitivity and scalability. Combining the unique attributes of differential scanning fluorimetry (DSF) and cellular thermal shift (CETSA), we report the methodological framework for the bioluminescence resonance energy transfer shift assay (BRETSA). BRETSA utilizes denaturation sensitive BRET probes, in combination with luciferase-tagged target proteins, to track and longitudinally record thermal shift in living cells. Denaturation profiles were successfully measured for over 500 globular proteins, spanning 24 target families in diverse cellular compartments. Among these, target engagement was confirmed for over 200 proteins; including validated drug targets as well as understudied members of the kinase subfamily. Leveraging orthosteric, allosteric, and covalent ligands in potency studies, this method offers improved sensitivity and scalability compared to other thermal shift methods. We extend the utility of BRETSA to interrogate target engagement at biomolecular complexes and query cooperative binding for molecular glues and emerging tricomplex drug modalities.

pharmacology and toxicology↗