Structure, conformation and inhibition of kinases from Leishmania

Author(s)

Plínio Salmazo Vieira

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-05-08

Examining board members

Mário Tyago Murakami; Richard John Ward; Gustavo Costa Bressan; Marcos Antonio de Oliveira; Juliana Helena Costa Smetana

• Advisor: Priscila Oliveira de Giuseppe; Mário Tyago Murakami

Abstract

Kinases are key enzymes in many aspects of cellular metabolism, gene regulation and signal transmission in the cell. Unicellular intracellular parasites can make use of these proteins in various functions such as division, signaling and its interaction with the host. In particular, the Leishmania parasite that causes leishmaniasis in different parts of the world, is of great interest. In this respect, the understanding of proteins that participate in their metabolic pathways, many of which kinases are involved, can bring benefits for the control and eradication of the disease, where the number of new cases is about a million and a half people, endangering other three hundred and ten million. In this work, we studied two classes of kinases: the NEK kinases and nucleoside diphosphate kinases. The NEK kinases are Ser/Thr kinases with functions related primarily to the cilia and flagella with large expansion in these organisms, as in the case of Leishmania. Phylogenetic analysis showed that many NEKs are unique to the parasite, while others are close to the human host. A construction NEK LbrM.21.1750 among the initial 21 to 210 residues were tested under conditions of expression, purified efficiently and in sufficient quantities for structural and biochemical assays. Despite extensive efforts employed in an attempt to crystallization, the protein proved recalcitrant to formation of crystals. However, the low resolution three dimensional structure was obtained from the SAXS data, indicating a good spatial correlation with the atomic coordinates generated by molecular modeling by homology. Extrinsic fluorescence assays revealed three analog compounds that bind to the ATP pocket of this kinase, of which NU6140 compound was used in kinetic experiments, being a competitive inhibitor. The three molecules were tested in vitro, being active on the promastigote and amastigote forms of the parasite, except PD153035 molecule that possibly did not penetrate the membrane of the macrophage. Thus, new molecules were identified through studies of the NEK kinases that can be used as starting templates for the development of new drugs. The other protein studied, NDK, is important in the maintenance of intracellular levels of NTPs and NDPs, among other functions including some extracellular, as it was found to be secreted by Leishmania species. The L. braziliensis NDK and mutant forms P95S, 'delta'5Cterm and P100S-'delta'5Cterm of Leishmania major NDK were purified and crystallized, and it was possible to resolve the three-dimensional structure. SAXS envelopes have shown that, with the exception of P100S-'delta'5Cterm mutant protein which is a dimer, all others are hexameric. Through a comparative structural study of the native proteins of L. braziliensis, L. major and mutant forms of the latter, it was possible to notice the important influence of the C-terminal region of this enzyme, affecting their correct oligomerization and therefore in its enzymatic activity. Thus, it was demonstrated that this region has potential as a target for enzyme inhibition since it alters not only its quaternary structure, but as well as their function.Collectively, the studies of both proteins provide new biochemical and structural insights about the kinase's repertoire of these parasites and bring potential strategies for inhibition and the consequent development of new therapies (AU)

• FAPESP's process: 11/24178-8 - Structural studies of NEK kinases from Leishmania braziliensis.
• Grantee: Plínio Salmazo Vieira
• Support Opportunities: Scholarships in Brazil - Doctorate