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Fragment binding validation by native mass spectrometry on the main protease from SARS-CoV-2
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Autores
Orientador(es)
Resumo(s)
COVID-19 emerged in the world to change it. This disease caused a lot of deaths, and the
continuous appearance of new viral variants created serious health problems, since they are resistant to
the action of current vaccines. Therefore, the discovery of new drugs to treat COVID-19 is essential.
Fragment-screen is based in the interaction between fragments with target proteins, initially with
low affinity but, when combined, enable the design of new molecules with far greater affinity, aiming
to inactivate the target. Native mass spectrometry is a new view to develop the fragment-screen
approach, allowing the fragment-protein complexes characterization, based on stoichiometry, binding
percentage and dissociation constant. The objective of this project is to validate the fragment screen, an
important approach for drug design, using native mass spectrometry.
Fragments used for screening are commonly prepared in DMSO and the unknown effect of this
solvent in the protein target, the SARS-CoV-2 main protease, led to the study of the protein’s
conformational behaviour in the presence of this co-solvent. Two model proteins, AHD and myoglobin,
as well as Mpro were incubated in the presence of different DMSO percentages to assess the effect on
protein’s charge state distribution and the solvent accessible surface area (SASA) were calculated for
each condition, revealing a higher protein compaction in the presence of this solvent in result of a
decrease in protein charge state distribution.
The last step in this project was the screening of fragments against the target. From the six
fragments tested, the two fragments with the strongest interaction were selected for a time-course
analysis, to assess the binding efficiency over time and select the best incubation time to perform the
titration necessary to quantify the interaction, calculating the dissociation constant and the binding
percentage associated to the protein-fragment complex.
Descrição
Tese de mestrado, Bioquímica e Biomedicina , 2024, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Q-TOF espetrometria de massa nativa fragment-screen SARS-CoV-2 Mpro Teses de mestrado - 2024