Computational investigation of the Plasmodium falciparum fatty acid biosynthetic pathway toward the discovery of novel antimalarials

The structural peculiarities of a protein are related to its biological function. In the fatty acid elongation cycle, one small carrier protein shuttles and delivers the acyl intermediates from one enzyme to the other. The carrier has to recognize several enzymatic counterparts, specifically interact with each of them, and finally transiently deliver the carried substrate to the active site. Carry out such a complex game requires the players to be flexible and efficiently adapt their structure to the interacting protein or substrate. In a drug discovery effort, the structure-function relationships of a target system should be taken into account to optimistically interfere with its biological function. In this doctoral work, the essential role of structural plasticity in key steps of fatty acid biosynthesis in Plasmodium falciparum is investigated by means of molecular simulations. The key steps considered include the delivery of acyl substrates and the structural rearrangements of catalytic pockets upon ligand binding. The ground-level bases for carrier/enzyme recognition and interaction are also put forward. The structural features of the target have driven the selection of proper drug discovery tools, which captured the dynamics of biological processes and could allow the rational design of novel inhibitors. The model may be perspectively used for the identification of novel pathway-based antimalarial compounds.

E-cadherin and Choline Kinase: two challenging drug discovery targets

The data presented in this thesis was generated using molecular biology, protein chemistry and X-ray crystallography techniques. However, while the methodologies employed are essentially the same, the research work presented here refers to two different proteins, which are part of different research projects in the laboratory. For this reason, the content of this thesis is divided in two independent parts, each provided with an introduction and a general overview of the research topic and state-ofthe- art, a materials and methods section discussing the techniques used and the protocols followed, and a section where the results are presented and discussed in detail. The first half of the thesis deals with the structural characterization of the complex between human E-cadherin and three different small molecule potential inhibitors identified via a fragment-based drug discovery (FBDD) screening campaign that was conducted using a library of commercially available small fluorinated chemical fragments. For this screening phase, we used 19F-NMR as readout. The NMR experiments were done by our collaborator Dr. Marina Veronesi at the D3 PharmaChemistry division of the Italian Institute of Technology (IIT) in Genova (Italy). Functional cell adhesion assays to validate the inhibitory effects of the fragments thus identified were carried out in collaboration with Prof. Frédéric André at the University of Marseille (France). The second half of the thesis describes the structural characterization of Plasmodium falciparum Choline Kinase (PfChoK), an important pharmaceutical target in the fight against malaria, as well as the biochemical characterization of a library of potential inhibitors of PfChoK. These inhibitors were synthetized in the group of Prof. Luisa Carlota López-Cara at the Department of Pharmaceutical and Organic Chemistry of the University of Granada (Spain) in the framework of an ongoing collaboration between the two groups.