Application of Bioactive Thermal Proteome Profiling to Decipher the Mechanism of Action of the Lipid Lowering 132-Hydroxy-pheophytin Isolated from a Marine Cyanobacteria.

Ana Carrasco Del Amor,Olatz Fresnedo,Susana Cristobal,Ralph Urbatzka,Sara Freitas

doi:10.3390/md17060371

Ana Carrasco Del Amor, Olatz Fresnedo + Show 3 more

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https://doi.org/10.3390/md17060371

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Abstract

The acceleration of the process of understanding the pharmacological application of new marine bioactive compounds requires identifying the compound protein targets leading the molecular mechanisms in a living cell. The thermal proteome profiling (TPP) methodology does not fulfill the requirements for its application to any bioactive compound lacking chemical and functional characterization. Here, we present a modified method that we called bTPP for bioactive thermal proteome profiling that guarantees target specificity from a soluble subproteome. We showed that the precipitation of the microsomal fraction before the thermal shift assay is crucial to accurately calculate the melting points of the protein targets. As a probe of concept, the protein targets of 132-hydroxy-pheophytin, a compound previously isolated from a marine cyanobacteria for its lipid reducing activity, were analyzed on the hepatic cell line HepG2. Our improved method identified 9 protein targets out of 2500 proteins, including 3 targets (isocitrate dehydrogenase, aldehyde dehydrogenase, phosphoserine aminotransferase) that could be related to obesity and diabetes, as they are involved in the regulation of insulin sensitivity and energy metabolism. This study demonstrated that the bTPP method can accelerate the field of biodiscovery, revealing protein targets involved in mechanisms of action (MOA) connected with future applications of bioactive compounds.

Highlights

The identification of protein targets from novel bioactive compounds is one of the biggest challenges of the field of biodiscovery
To demonstrate the applicability of bioactive thermal proteome profiling (bTPP) to the field of biodiscovery, we studied the protein targets of 132 -hydroxy-pheophytin a, a chlorophyll derivative with novel lipid-reducing activities that has recently been isolated from a marine cyanobacteria [14]
thermal proteome profiling (TPP)-based method toto identify the the protein targets that interact with a bioactive compound

Summary

Introduction

The identification of protein targets from novel bioactive compounds is one of the biggest challenges of the field of biodiscovery. The function of those proteins would define the MOA of any bioactive compound, predicting the mode of action at the cellular level, as well as possible secondary or harmful effects. Phenotypic screening was the principal strategy for drug and bioactive compound discovery until the 80s. This methodology has attracted renewed interest in connection with biodiscovery programs for terrestrial natural sources [1]. Mar. Drugs 2019, 17, 371; doi:10.3390/md17060371 www.mdpi.com/journal/marinedrugs

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