Translation. Region: Russian Federation –
Source: Novosibirsk State University –
An important disclaimer is at the bottom of this article.
Russian scientists have developed a new combined approach aimed at identifying the binding sites of drugs used in photodynamic oncotherapy with the protein responsible for drug transport in the human body. This approach will accelerate the search for the most effective cancer drugs and minimize side effects on patients. This study was conducted by a team of scientists from Novosibirsk State University, the International Tomography Center of the Siberian Branch of the Russian Academy of Sciences, and staff from the Russian Technological University MIREA.
The results of the study were published in Journal of the American Chemical SocietyThe mere fact of publication in such a prestigious and highly cited journal is already considered a success for young researchers, and this article was accepted for publication in the "Editor's Choice" section, demonstrating the recognition of the research by the international professional community. The publication's lead author, Mikhail Kolokolov, a second-year graduate student in the Department of Chemical and Biological Physics at the NSU Physics Faculty and a junior researcher at the Electron Paramagnetic Resonance Laboratory of the International Tomography Center, received the prestigious youth award from the International Society for EPR Spectroscopy for best scientific paper. The young scientist conducted his research with fourth-year graduate student Natalia Sannikova from the same department, under the supervision of Olesya Krumkacheva, Doctor of Physical and Mathematical Sciences.
When medications enter the human body, they primarily bind to proteins in the blood. The effectiveness of a particular drug depends on its binding to serum albumin, a protein found in blood plasma responsible for transporting substances within the body. The degree of binding to this protein significantly influences the drug's action. If binding is too strong, the drug's concentration in the blood will be reduced, while if binding is weak, the drug may be unevenly distributed throughout the body or even destroyed without achieving its intended effect.
“To create an effective drug and control its binding to the transport protein, it is important to know where on the protein its molecules will attach. Identifying such sites will lead to understanding the mechanism of action of drugs, predicting side effects and identifying the causes of drug resistance in some patients. However, traditional methods of structural biology are not effective enough if there are several binding sites or the interaction of the drug with the protein surface is unstable. Then researchers use the molecular modeling method, but its results are not enough, since drugs often bind to the protein in several places. Thus, several small drug molecules can be attached to one protein simultaneously and at different sites. Thus, many variants of the structure of such complexes are obtained, which becomes difficult to take into account by molecular modeling. We proposed our own combined approach that allows us to measure the distance between various elements of the complex and use them to obtain its structure. Previously used methods produce average values, but in our case it is possible to achieve atomic precision in measuring the distribution of distances between binding sites, “see” all possible conformations (that is, the spatial arrangements of atoms in a molecule of a certain configuration) and find places where small molecules of a substance bind to a protein. This is the most important element of our work. In our approach, we measure distances within the complex using spin labels. A special small molecule containing an unpaired spin is selectively introduced into a region of the protein that we know. After binding the protein to the drug, we can measure the spin-spin distances between the spin label and the drug molecules on the protein,” explained Mikhail Kolokolov.
In their approach, the scientists combined molecular modeling methods with experimental data obtained using electron paramagnetic resonance, which allows the structure of compounds to be determined based on their microwave absorption. They first identified potential drug-protein binding sites using calculations, then conducted EPR spectroscopy studies, and then applied the experimental results and computer calculations to refine the configuration of these sites. This work was carried out by Mikhail Kolokolov and Natalia Sannikova, graduate students from the NSU Physics Department and junior research fellows at the EPR Laboratory of the International Tomography Center. It was discovered that binding for various types of photosensitizers can occur at non-standard sites on albumin and at several sites simultaneously.
"In theory, you can even determine where a molecule binds to a protein without any experiments, simply using computational methods. However, in practice, it turns out that these methods lead to significant inaccuracies and even errors because the calculation algorithms are relatively simplified. For this reason, scientists are often unsure of their results. Furthermore, computational methods can yield several possible binding sites and their locations. And often, from a calculation standpoint, these options are equally likely. The question is which one is correct. For this reason, the computational method is not precise enough and should not be relied upon entirely. However, it is still useful because it provides direction for experimental research, allowing us to narrow the range of possible binding sites. Thanks to this, we can use our experimental distances, which we are confident in, along with the computational methods, to determine the presence of a molecule on a protein with sufficient accuracy," explained Mikhail Kolokolov.
The scientists tested their combined approach by studying the binding of albumin to photosensitizers.
Photosensitizers are natural or synthetic substances that are used in medicine, for example in photodynamic therapy (PDT), where they accumulate in pathological cells and are activated when irradiated with light, causing their death.
Photodynamic oncotherapy is considered a very promising method because, unlike traditional chemotherapy, it targets only the tumors that are exposed to light. However, this cancer treatment method is currently not widely used due to the imperfections of photosensitizers. Scientists are faced with the challenge of improving their light absorption, diffusion throughout the body, and accumulation in tumors. This study of the albumin-binding sites of photosensitizers is important for further improving their diffusion throughout the body and increasing their concentration in tumors, which will contribute to increased therapeutic efficacy. Therefore, work in this area has significant clinical significance.
Scientists have identified the locations of binding sites for seven compounds whose structural interactions with albumin were previously unclear. The new approach demonstrated that binding can occur at non-standard sites on albumin and at multiple sites simultaneously for different types of photosensitizers.
The scientists tested the effectiveness of their combined approach using several photosensitizers. To demonstrate different binding mechanisms, they used compounds whose molecules had different electrical charges—negative, positive, and neutral. It turned out that, depending on this charge, they bind differently to the protein, which in this case was negatively charged. Molecules with a positive or neutral charge "sit" on the negatively charged surface of the protein and form an unstable bond—they can temporarily detach and reattach.
Negatively charged molecules behave differently—they penetrate pockets on the protein surface and remain there stably. However, in this case, their size plays a key role. Relatively small molecules fit completely into these pockets and formed very effective binding, while larger molecules behave differently.
Experiments have shown that the smaller the molecule and the more completely it fits into these pockets, the higher the site population. Experiments with larger molecules that fit less freely into these pockets yield lower populations and less effective binding. The researchers observed these processes directly in experiments. This molecular behavior is logical, but computational methods don't account for it. While they can determine how a molecule binds to a protein, they don't determine how this affects the protein itself. If small molecules fit freely into the pocket, no significant changes occur. However, large molecules can alter the protein structure. Computational methods often don't capture this, but the researchers corrected these errors and inaccuracies through experiments.
"Throughout all our experiments in this study, we demonstrated with atomic precision where the molecules of these compounds bind to albumin, which is undoubtedly a novelty in terms of photostabilizer development. The combined approach we developed will make the analysis of anticancer compounds significantly more accurate, and the development of new oncotherapy drugs simpler and faster. By combining computer analysis and electron paramagnetic resonance data, we were able to significantly reduce the number of labor-intensive calculations and experiments, simplifying the determination of interactions between albumin and photosensitizers. We believe our work will enable us to predict the most promising compounds for photodynamic anticancer therapy. We now plan to apply our approach to study how photosensitizers bind to DNA molecules," explained Mikhail Kolokolov.
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