Martin Toul: Stroke treatment can be considerably more effective

In a detailed analysis of thrombolytic proteins, Ph.D. student Martin Toul discovered previously overlooked properties of the staphylokinase protein. Protein engineering methods can improve its efficiency by up to ten thousand times, which would make the protein a perfect candidate for stroke treatment. This discovery represents a breakthrough and could lead to a far more effective drug than those currently used clinically.

18 Mar 2022 Marie Hošťálková

Martin, why stroke treatment?

At Loschmidt labs, we work on proteins. We figure out their mechanisms of action and look for the most significant factors limiting them. Then, we attempt to remove those limiting factors to improve their properties. Specifically, we are looking for ways to make alteplase, a protein drug used in treating stroke, more effective or potentially replace it with a new effective protein. Alteplase has been around for over forty years. It works by breaking up and dissolving blood clots. While it is relatively effective, it is certainly not perfect. With tens of millions of people affected by stroke each year, it is time to develop something better.

Why did you decide to study at Masaryk University?

I am from Boskovice, a small village not far from Brno, so the close proximity was an obvious advantage. But more importantly, Masaryk University does high-quality science and research, which is on par with leading research institutions abroad.

And why Loschmidt Laboratories, RECETOX?

I studied Biochemistry at the Faculty of Science, but I always preferred chemistry. However, after an internship at an organic synthesis lab, I found that although I enjoy it all "on paper," in reality, classical synthetic chemistry was not really for me. Soon after, I was fortunate to attend a lecture on protein engineering by prof. Damborsky from Loschmidt labs. I was very drawn to the possibility of combining my knowledge in organic chemistry to study the mechanisms of biological processes. This led to the exciting research I am still involved in today. Apart from biochemistry, I have always enjoyed mathematics and physics, and my projects link all these together. Loschmidt Laboratories are open to high school students who can either work with us on their "SOČka" (student's professional activity) or as part of the Summer School of Protein Engineering, which we regularly organize for high-school and first-year college students.

You have made a groundbreaking discovery in your research work. You have uncovered new possibilities for developing a highly effective drug for stroke. What makes this finding so crucial?

We are studying staphylokinase as a potential alternative to alteplase. This protein is produced by the staphylococcus bacterium, which is present on human skin and mucous membranes.

Staphylokinase has several interesting properties overlooked because of its assumed lower efficacy compared to other proteins. However, in a detailed analysis of its entire mechanism, we have found that the situation is quite different. Activation of the drug involves two steps. In the first, the drug is activated by binding to a partner in our body. In the second, the actual dissolution of the blood clot occurs. Until now, it was thought that the second step was ineffective, i.e., the dissolution was too slow. However, our measurements have shown the exact opposite – dissolution is highly efficient and orders of magnitude faster than in the current drug alteplase.

How was this crucial fact missed?

The first step is problematic because only one in ten thousand molecules bind to their partners and become active. If only the average dissolution efficiency is determined, the results are obviously unremarkable. However, when looking closely, it becomes clear that the one molecule in ten thousand that participates in dissolution "works" extremely fast as opposed to the others who "do nothing". Thus, we now know that if we can perfect the first binding step and bind and activate all the molecules, the drug will be ten thousand times more effective in dissolving clots.

Illustation picture by Ella Maru Studio

What’s the next step?

An array of advanced protein engineering methods and tools are available for improving protein binding properties. Since we now precisely know what to target and that the limitations of staphylokinase are, we can make specific modifications. Using different strategies, we will prepare and test numerous protein variants to find the ones with the best properties. Our goal is to create a protein with improved binding properties. Ideally, all staphylokinase molecules will bind to partners in the first step leading to a significantly higher overall efficiency.

What preceded the actual discovery?

Ironically, it was unplanned. Initially, we just wanted to measure the properties of a group of proteins, including staphylokinase, to have data for comparing with other compounds under development. However, we found that our data, obtained by looking at staphylokinase from a slightly unique perspective, differed from previously published data. In fact, the prevailing analytical method led to a considerable underestimation of the data parameters. Our research is changing the paradigm of viewing staphylokinase, whose properties have been overlooked.

Who are you collaborating with on this research?

While I am the lead author of the newly published findings, many people have immensely contributed to the project. In addition to colleagues from Loschmidt laboratories of the Faculty of Science, we collaborate with groups from the Biophysical Institute at the Czech Academy of Sciences, the Faculty of Pharmacy and Medicine at Masaryk University, and the Masaryk Memorial Cancer Institute as part of the joint STROKE Brno platform. Thanks to the platform, we also work on clinically-relevant research, managed by experts from St. Anne's University Hospital in Brno, especially Prof. Robert Mikulík. Furthermore, without Prof. Jiří Damborský and Prof. Zbyněk Prokop, who supervised me and supported the entire project, these findings could have never been achieved.

What benefits does staphylokinase by itself offer?

Now that we know that the potential of improving staphylokinase binding by up to ten thousand times exists, we can use this knowledge for developing an effective drug. Also, staphylokinase is a smaller and simpler protein compared to alteplase. This makes it easier for designing and improving it to work the way we want it to. In addition, staphylokinase originates from bacteria and is thus easier to produce commercially and at a fraction of the cost of alteplase. This is especially crucial for making the drug available to stroke patients in developing countries.

At the same time, being of a bacterial origin is also a disadvantage - it is foreign to the human body (immunogenic), so our immune system develops antibodies against it. A modified non-immunogenic variant of staphylokinase (i.e., that does not cause an immune reaction) has already been created using protein engineering methods to counteract this response. Moreover, a recent clinical study has shown that it is as effective and safe as the original staphylokinase, paving the way for future research. We intend to modify the non-immunogenic variant to improve its binding and significantly increase its thrombolytic (clot dissolution) effect compared to the current drug. Clinical relevance is also a significant goal.

What motivates you in your work?

Since I was a child, I have enjoyed exploring, learning new things, and figuring out how the world around us works, so my work is a hobby for me. I love what I do - having the opportunity through research to develop innovative solutions that move us forward as humanity and make our lives better. That is probably the most significant driver for me.

And lastly, I wondered, having looked under the hood of your research, how do you manage to combine research with your Ph.D. studies?

My research is also the focus of my studies. Sometimes, combining science with leisure time, other hobbies, or family can be challenging. I need to set firm boundaries, as it is pretty easy to get caught up in exciting projects and sometimes almost get in over your head. But at the moment, I’m managing to balance my work and personal life well, so I'm very happy for all the projects I get to be involved in at Loschmidt Labs.

The scientific article is published in ACS Catalysis.

Martin, thank you for this interview. I´m wishing you the best in your career.




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