Mar 4 2019
Canadian scientists, working in collaboration with Brazilian colleagues, have created 13 bioluminescence sensors for use in testing the efficacy of new medical drugs in the lab.
The research, published in the journal Science Signaling, makes the way for novel drugs to be tested and defined. The biosensors are founded on the action of G protein-coupled receptors (GPCRs), which are membrane-bound proteins involved in the communication among cells.
The scientists did not choose to explore GPCRs accidentally. Between one-third and half of all currently-available commercial drugs are projected to target these receptors.
“These proteins are targets of drugs used to treat a wide array of disorders and diseases, including allergy, pain, hypertension, and diabetes, among others. The new biosensors are expected to be relevant to the discovery and development of new drugs for the treatment of even more diseases,” said Claudio Miguel da Costa-Neto, a professor in the Biochemistry and Immunology Department of the University of São Paulo's Ribeirão Preto Medical School (FMRP-USP) in Brazil and one of the authors of the article.
Costa-Neto explained that up to a few years ago, the in vitro tests used in new drug progress—performed before clinical experiments in animal models and humans—demonstrated only whether the compound triggered or blocked a particular cellular response.
To risk an analogy, until a few years ago these tests were performed as if there were a lamp that could be switched on or off. We've recently found it possible to analyze the different pathways that can be activated by a receptor and to what extent a given signaling pathway is activated. So it's no longer just a matter of an on or off switch. It's as if we had a room with several LED lights or a dimmer, and could say how many pathways were activated, which ones, and above all how the pathways were activated or blocked. Our biosensors, and others that are being developed by other groups, provide a more complete answer, a signaling profile, as it were.
Claudio Miguel da Costa-Neto, Study Co-Author and Professor, Department of Biochemistry and Immunology, Ribeirão Preto Medical School (FMRP-USP), University of São Paulo.
The research describes how the scientists created, validated, and used a set of bioluminescence resonance energy transfer (BRET)—based biosensors to compute the varied intracellular signaling pathways and detect the action of drugs in cultured cells. The signaling cascades ensuing from interactions between GPCRs and their ligands (including neurotransmitters and hormones) regulate a broad array of processes in cells, rendering them core targets for new drug discovery.
The international team of scientists used genetic engineering and molecular biology methods to incorporate fluorescent and luminescent enzymes (such as luciferase, the light-producing material found in glow-worms) to the GPCR ligands.
“When one of these receptors is activated by a drug and the protein inside the cell interacts with the receptor, light emitted by luciferase is transferred to the fluorescent protein and activates it. In this manner, we can accurately measure different levels of a drug's action,” Costa-Neto said.
The research resulted from a broad collaboration among researchers affiliated with FMRP-USP and Canada's University of Montreal and McGill University. It was aided by FAPESP via a Thematic Project, a project nominated in a call issued by São Paulo Researchers in International Collaboration (SPRINT), a research scholarship abroad awarded to postdoctoral researcher Larissa de Bortoli, and a doctoral scholarship awarded to Sarah Capelupe Simões, as well as the March 2018 São Paulo School of Advanced Science on Medicines: from Target to Market.
Validation
The 13 biosensors were examined on numerous drugs and on more than a few mutant receptors that simulated polymorphisms, some of which are related to genetic diseases.
To validate such important tools for the discovery of new drugs, the biosensors were tested on different ligands and mutant receptors. The goal was to show that it's possible to detect a distinct signaling pattern not only when a different drug is used but also when the receptor is changed.
Claudio Miguel da Costa-Neto, Study Co-Author and Professor, Department of Biochemistry and Immunology, Ribeirão Preto Medical School (FMRP-USP), University of São Paulo.
According to the scientists, the research embodies progress in the understanding of the refined signaling mechanism, because of the number of biosensors built and the method applied in the research.
“Mechanisms like these had already been described, but we significantly advanced our understanding of them. For this reason, we believe the new biosensors will have a major impact on new drug development,” Costa-Neto said.
“In addition to the importance of GPCRs as a target, another point is that the biosensors were extensively validated. We showed that they work, respond well and are highly reliable for the characterization of these signaling pathways.”
Besides the precision of the tools they built, Costa-Neto underlined creativity as an important feature of the research.
Developing proteins with genetic engineering involves a great deal of knowledge and creativity. Some of these biosensors are veritable ‘Frankensteins’, fabricated by splicing together various structural parts of different proteins. It was a highly creative study.
Claudio Miguel da Costa-Neto, Study Co-Author and Professor, Department of Biochemistry and Immunology, Ribeirão Preto Medical School (FMRP-USP), University of São Paulo.