Using a technique known as drug repositioning, researchers at the University of São Paulo’s Institute of Biomedical Sciences (ICB-USP) have discovered seven possible drugs to inhibit replication of the novel coronavirus. The products are already approved by the Food and Drug Administration (FDA, the US regulatory agency), which would facilitate the transition to clinical trials if their efficacy were demonstrated in vitro, in tests carried out with cell lines.
The results of the research ?
1; supported by FAPESP through three projects (19 / 00195-2, 20 / 04680-0 and 16 / 09047-8) – were published in the Journal of Biomolecular Structure and Dynamics.
The investigation focused on the 3CLpro enzyme from Sars-CoV-2, considered essential for the replication of the virus. Using machine learning techniques, the scientists tested more than 11,000 molecules and selected those that showed greater affinity to the target molecule, as well as greater stability within the active site, the region of the protein where the chemical reaction occurs.
“With these computational predictions we have selected seven molecules that could hold promise for testing in cells. If they work in vitro, we can see them tested on humans. The advantage of testing existing drugs on the market is that the effects of toxicity and side effects are already widely known ”, underlines Cristiane Guzzo, coordinator of the study. “Therefore, after validation in in vitro assays, it would be possible to carry out clinical tests on patients with Covid-19”.
The discovery may also be important in establishing the criteria and properties the drug must have to inhibit the 3CLpro enzyme. “We have seen that the best compounds are those that interact favorably with the five specific amino acid residues of the enzyme. Therefore, these residues can be used to discover other inhibitors, ”explains postdoctoral fellow Anacleto Silva de Souza, first author of the article.
Enzymatic activation
Coronavirus 3CLpro is a protease responsible for breaking a chain of viral proteins (polyprotein) into its functional subunits, thus allowing RNA replication and the assembly of new viral particles that will infect other cells. The hypothesis is that, by inhibiting 3CLpro, it cannot perform its function and the virus stops replicating and proliferating, reducing the viral load and the severity of the disease.
This is a different goal from other studies, many of which have focused on the spike protein, which sits on the surface of the virus and is responsible for interacting with the host cell. “We chose 3CLpro because there was already a considerable amount of information about it, thanks to over 15 years of research with SARS-CoV’s 3CLpro [coronavírus que causou a epidemia de síndrome respiratória aguda grave entre 2002 e 2003]”Says Guzzo.
Methodology
To simulate the interaction of drugs with the protease, the researchers developed three mathematical models, using artificial neural networks and two regression models. These quantitative models, known as QSARs, were based on information from the literature on molecules already known to have inhibitory properties against the 3CLpro enzyme.
An affinity prediction of 11,000 molecules was made with the QSAR models, using the Drugbank database, a repository that combines detailed chemical and pharmacological data on approximately 4,300 drugs. Based on the computational results, 2,500 molecules were discarded for having low affinity for the enzyme. Of the 8,500 subsequent compounds, 14 molecules with different pharmacological properties (drugs for the treatment of migraine, respiratory diseases, antimicrobial action and natural products) were selected for the computational simulation between these drugs and the enzyme.
“To inhibit the enzyme it is not enough to have the expected high affinity, it is necessary that the molecule can remain attached to the target. Otherwise, the enzymatic function is reactivated. We then simulated the behavior of these 14 molecules within the 3CLpro active site. We make a docking [acoplamento entre moléculas] followed by molecular dynamics to check if the compound is stable within the enzyme, and this leaves seven candidates, “explain the researchers.
Scientists now intend to confirm these predictions in biochemical experiments, through the cloning of 3CLpro. If the finding is validated, the expectation is to establish partnerships with other ICB laboratories to test the drugs in vitro.
* With information from the ICB-USP Communication Department.