University and pharmaceutical company create new drug discovery tools | AGÊNCIA FAPESP

Researchers use computer simulations to investigate how kinase AAK1 interacts with small molecules capable of inhibiting its activity

University and pharmaceutical company create new drug discovery tools

March 15, 2017

By Karina Toledo  |  Agência FAPESP – The open platform bioRxiv recently published one of the first fruits of a partnership between the Brazilian pharmaceutical company Aché Laboratórios Farmacêuticos and the Protein Kinase Chemical Biology Center at the University of Campinas (SGC-UNICAMP) in São Paulo State, Brazil, supported by FAPESP.

The article describes a chemical compound that could be used in future research as a selective inhibitor of the enzyme AAK1 (AP2-associated kinase 1; also known as adaptor-associated protein kinase 1), which is considered a potential target for drug development. 

SGC-UNICAMP is part of the Structural Genomics Consortium (SGC), a public-private partnership that supports drug discovery through open-access research by more than 400 scientists at universities, pharmaceutical companies and nonprofits in several countries. Led by researchers Jonathan Elkins, Katlin Massirer, Opher Gileadi and Paulo Arruda, SGC-UNICAMP investigated the functions of protein kinases, which regulate important processes such as cell division, proliferation and differentiation. Although protein kinases are considered priority targets for drug development, it is estimated that only 40 of over 500 protein kinases identified in the human genome have been studied in depth.

According to Rafael Couñago, a researcher at SGC-UNICAMP and first author of the article, AAK1 is a kinase that is involved in endocytosis, the process by which various types of particles from the extracellular medium, such as neurotransmitters, micronutrients and even some viruses (for example, hepatitis, dengue and Zika), are transported into the cell.

“Evidence from the scientific literature shows that endocytosis occurs less frequently when AAK1 is inhibited,” Couñago said. “But to understand its exact function in the cell and find out whether it’s a promising therapeutic target, we need a tool called a chemical probe – a small molecule that can selectively bind to this enzyme and inhibit its function in a biological model.”

AAK1 is known to be responsible for phosphorylating AP2 (adaptor protein 2). In other words, it catalyzes the transfer of high-energy phosphate molecules, such as ATP (adenosine triphosphate) to AP2, and this modifies the activity of the target protein in the cell, making the process of endocytosis occur more frequently.

“We still need to understand how this regulation works in more detail. For example, exactly when during endocytosis is the action of AAK1 important?” Couñago asked.

High-throughput screening

In their search for a chemical probe with which to study AAK1, researchers affiliated with the SGC at the University of North Carolina at Chapel Hill, USA, analyzed a library of chemical molecules by high-throughput screening, a biochemical assay in which the enzyme was tested against hundreds of candidate inhibitors simultaneously.

The researchers at SGC-UNICAMP used X-ray crystallography to elucidate the three-dimensional structure of the complex formed between the molecule that proved most promising in the high-throughput screening tests and BIKE, the protein kinase that most closely resembles AAK1 in humans.

“The crystallographic structure shows how intimate contact between the small molecule and the protein takes place, enabling us to identify which part of the chemical probe is responsible for the strong affinity with AAK1 it displays,” Couñago explained.

With the collaboration of Aché’s radical innovation team, led by director Cristiano Guimarães, computational studies were conducted to calculate the energy cost during stabilization of the catalytic process.

“This analysis showed us that when we substituted an isopropyl group for a cyclopropyl group in the structure of the candidate chemical probe, its activity was dramatically reduced,” Couñago said. “The reason was the much higher energy cost to form the complex. That can’t be seen just by looking at the structure.”

This discovery is important, he explained, because it means that the alternative isopropyl-containing version of the small molecule can be used as a “negative control” in experiments. Although interaction with AAK1 is switched off, the candidate probe continues to interact normally with all other proteins in the cell.

“So we can do the same experiments with the chemical probe and the negative control, and then compare the results. This increases the degree of certainty that the biological effects observed with the inhibitor are due to inhibition of the function of AAK1,” Couñago said.

Open science

One of the key principles of the SGC’s partnership model is open science, meaning that all members of the consortium strive to make their research findings public as soon as possible. As Couñago explained, this is why the article was posted on the internet even before it had been peer reviewed.

“We believe this increases the number of people who can comment on the paper, so that we can make the necessary corrections and enhance the impact of the discoveries,” he said.

While the small molecule showed potential to become a chemical probe, he added, more studies will be necessary for quality-control purposes.

“We’re now at the stage of finding collaborators who are interested in studying the endocytosis pathway and the function of AAK1 in this process. The SGC doesn’t have the capacity to work with a variety of biological models, and for this reason, it tries to reach the scientific community as soon as possible in order to attract collaborators,” Couñago concluded.

The article “Development of narrow spectrum ATP-competitive kinase inhibitors as probes for BIKE and AAK1” can be read at




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