Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA

• Autor(a) principal: Fernandes, Andreia Catarina Fernandes Pinto
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/88726
• topic: Tuberculosis; Mycobacterium tuberculosis; Protein Tyrosine Phosphatase A (PtpA); Chalcones and Thiosemicarbazones; Chalcones and Thiosemicarbazones; In silico studies; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Tuberculosis (TB) is one of the top causes of death remaining a major public health problem worldwide. Mycobacterium tuberculosis is the agent of TB, infecting the human respiratory tract. Its remarkable pathogenicity hinges upon the ability to challenge the immune system of the host by secreting phosphatases into macrophages. Among them, Protein Tyrosine Phosphatase A (PtpA) plays a key role on the infection process, preventing the phagosome-lysosome fusion and promoting the inhibition of phagosome acidification. Thus, PtpA becomes a promising target for the development of new anti-TB drugs. The aim of this work is to contribute to find new structure-based drug design approaches against TB, studying the inhibitory properties of three different families of compounds towards PtpA – chalcones, thiosemicarbazones and azaindoles. The protein was overexpressed in E. coli – final yield of 20 mg protein/ liter of culture – and successfully purified using affinity chromatography. To provide new insights into the binding mode of the studied compounds, molecular docking studies were performed suggesting thiosemicarbazones as non-competitive inhibitors and the chalcones and azaindoles with a preferential active site binding. The protein was also biophysically characterized. The oligomeric state was confirmed by SEC, proving that PtpA is a monomer in solution. The protein stability was assessed through TSA revealing that, with 10% glycerol, PtpA resists to the effects of 10% DMSO. TSA was also used to find a suitable protein storage condition (-80°C) and to confirm PEG400 as an alternative solvent for the inhibitors. In addition, distinct biophysical approaches – TSA, MST and urea-gel electrophoresis – were implemented to detect protein-ligand interactions but definitive evidence were not obtained. Ligand-free and co-crystallization assays were extensively explored and several crystals were tested at the ESRF, Diamond and MAX IV. Two crystal structures were obtained: a co-crystallization PtpA-Lap11 structure at 3.6 Å resolution and a soaking PtpA-C33 structure at 2.8 Å resolution. Despite the low/medium resolution obtained, both structures reveal the potential binding of the inhibitors with suspicious density blobs near His120B for Lap11 and at the active site for C33. The ligands were preliminarily modelled but further refinement cycles are required to elucidate the respective binding.