Dottorato in BIOCHIMICA

Titolo della tesi: Anti-trypanosomatidal drug discovery: a challenge for structural biology

PhD thesis: “Anti-trypanosomatidal drug discovery: a challenge for structural biology” PhD candidate: Theo Battista, Biochemistry XXXIII Cycle Supervisor: Dr. Gianni Colotti PhD Coordinator: Prof. Stefano Gianni, Prof. Francesco Malatesta ABSTRACT Drug discovery and development is a lifelong challenge; the modern approach in the drug discovery process relies on the identification, validation and targeting of specific, unique and druggable molecular targets. So far, huge efforts have been made in order to maximize the number of active compounds identified in the lowest time possible, as in the case of large scale high-throughput screenings (HTS) or fragment-based screenings (FBS); at the same time computational tools together with biophysical methods, as X-ray crystallography, evolved so fast to make structure-based drug design (SBDD) a primary approach for drug development. These colossal knowledge and technology are now fundamental for the advancement in medicinal chemistry. The line of research on which this work is focused regards some of the most life-threatening and neglected diseases, globally named Neglected tropical diseases, namely Leishmaniasis, Human Africa trypanosomiasis (HAT) and American trypanosomiasis. The causative agents are all belonging to the parasitic family of Trypanosomatids and even if phenotypical characteristics are different from species to species, they all share unique and specific molecular features which can be targeted for the identification and development of new broad-spectrum drugs, in order to supersede current therapies, endowed with high toxicity and inefficacy profiles. With this aim, the trypanothione reductase (TR), a NADPH-dependent flavoenzyme, has been targeted as it fulfills all the requirements to be considered a good drug target: it is unique, as is present only in trypanosomatidal species and absent in the host, essential, playing a central role in the redox equilibrium of parasites and it is druggable, as can be efficiently addressed, and thus inhibited, by specific compounds. Employing different approaches, new active hit compounds against TR have been identified; a spiro-containing derivative, Compound 1, has been identified via HTS and fully characterized (Turcano et al., 2020). Its competitive mechanism of inhibition has been assessed and confirmed by SPR and X-ray crystallography respectively, and it was shown to inhibit the same enzyme from different sources, with high selectivity over the human homologue glutathione reductase (hGR). The most intriguing feature is represented by its chemotype, which is known to be able to cross the brain-blood barrier, particularly important in the treatment of the central nervous system phase of HAT. Simultaneously, screening of the 192 compounds contained in the LeishBox, a set of best antileishmanial compounds identified by a cell-based HTS performed by GlaxoSmithKline (GSK) over 1.8 million compounds (a HATBox and a ChagasBox were additionally built) (Peña et al., 2015), led to a list of seven compounds active against TR from L. infantum (Ilari et al., 2018); SPR experiments and docking predictions, together with a preliminary model of the crystal structure of TR in complex with compound A1/7, assessed the competitive fashion of the inhibition. A1/7 is among the most potent and selective inhibitors identified in the series and is contained also in the HATBox and ChagasBox, setting the basis for a broad-spectrum drug development. For this reason, it has been used as a scaffold for the synthesis of twelve derivatives (in collaboration with the medicinal chemistry group of Prof. Giuseppe Campiani, University of Siena, Italy), which are being tested at this time; three out of twelve display a better and/or comparable affinity with respect to A1/7, providing important information regarding the chemical modifications to be applied to boost affinity, supported by a preliminary crystallographic model of the TR-A1/7 complex. Finally, preliminary crystallographic studies have been performed, in order to fulfil pre-requirements needed for an FBS, to be performed in the near future at the Xchem-Screening platform, at Diamond Light Source (Didcot, UK). Reproducible high-diffracting, DMSO-resistant and ligand accessible crystals have been obtained by setting up an automated protocol. Future steps are those typical of the hit optimization round; the information so far collected will be used for intense SAR studies in order to obtain more potent and specific inhibitors. Efforts will be carried out in order to acquire structural information via X-ray crystallography, to proceed with a structure-based campaign. FBS results will help in designing new scaffolds and pharmacophores, facilitating the discovery of a single inhibitor for this family-specific and highly conserved target. References Ilari, A., Genovese, I., Fiorillo, F., Battista, T., De Ionna, I., Fiorillo, A., & Colotti, G. (2018). Toward a Drug Against All Kinetoplastids: From LeishBox to Specific and Potent Trypanothione Reductase Inhibitors. Molecular Pharmaceutics, 15(8), 3069–3078. Peña, I., Pilar Manzano, M., Cantizani, J., Kessler, A., Alonso-Padilla, J., Bardera, A. I., Alvarez, E., Colmenarejo, G., Cotillo, I., Roquero, I., De Dios-Anton, F., Barroso, V., Rodriguez, A., Gray, D. W., Navarro, M., Kumar, V., Sherstnev, A., Drewry, D. H., Brown, J. R., … Julio Martin, J. (2015). New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: An open resource. Scientific Reports, 5. Turcano, L., Battista, T., De Haro, E. T., Missineo, A., Alli, C., Paonessa, G., Colotti, G., Harper, S., Fiorillo, A., Ilari, A., & Bresciani, A. (2020). Spiro-containing derivatives show antiparasitic activity against trypanosoma brucei through inhibition of the trypanothione reductase enzyme. PLoS Neglected Tropical Diseases, 14(5), 1–17.