IRON ACQUISITION AS TARGET FOR NEW ANTITUBERCULAR DRUGS
Creators: name: MORI, MATTEO
Publisher: name: Universit� degli Studi di Milano
Year: 2020

description: Tuberculosis nowadays ranks among the leading causes of death worldwide. The growing emergence of resistant strains of Mycobacterium tuberculosis poses a serious threat to the public?s health. Moreover, the inadequacy of the available drugs and the difficult treatment regimens contribute to the spread of the disease. Therefore, the discovery of new antitubercular drugs, acting on new pharmacological targets, is assuming critical importance. The iron uptake pathways have been identified as a potential option, considering the essentiality of this metal ion for the survival and virulence of the microorganism. In particular, the inhibition of the mycobacterium-specific salicylate synthase MbtI has been recently validated as a new promising approach for the development of novel antitubercular agents. In this context, the current research project was focused on the identification of new, potent MbtI inhibitors for the development of innovative therapeutic agents. Our investigations started with a structure-based virtual screening, employing the available co-crystal structures of MbtI: we built a pharmacophore model and we used it to screen the Enamine database. Subsequent steps of consensus docking and molecular dynamics allowed us to identify a competitive furan-based inhibitor, endowed with a promising activity on the enzyme. With the aim of understanding the key structural features required for the inhibitory effect, we modified the hit compound to derive preliminary information. Then, we performed an in-depth exploration of the chemical space around this scaffold through a structure-activity relationship study; this investigation led to the identification of the most potent MbtI inhibitors available to date. All the compounds were submitted to a complete biological evaluation. Considering the difficulty in sourcing the natural substrate of the enzyme for the biological analyses, we decided to produce, purify and characterise it, using an in-house optimised process. Apart from an excellent inhibitory activity on the target enzyme, our candidate inhibitors also exhibited an interesting antimycobacterial action, related to the reduced production of siderophores by the pathogen. Notably, we managed to obtain, for the first time, Iron Acquisition as Target for New Antitubercular Drugs iii a solid correlation between an effective inhibition of MbtI and a promising antimycobacterial effect in whole-cell assays. Parallelly, we submitted the best compounds to co-crystallisation experiments to empirically define the binding mode of this class of inhibitors within the active site of MbtI. The obtainment of high-quality crystals of the protein proved particularly challenging; therefore, we dedicated our efforts to the optimisation of the whole protein production and crystallisation process. These studies allowed us to achieve valuable results, thanks to the obtainment of good crystal samples. The analysis of the structural data allowed to uncover the key protein-ligand interactions at a molecular level, paving the way for the optimisation of the computational model, and for the design and synthesis of improved candidates. Moreover, thanks to these crystallographic investigations, we managed to gather unprecedented experimental evidences regarding the catalytic activity of MbtI. Finally, we assessed the possibility to design liposome-based nanocarriers for antitubercular inhalation therapy. The promising results obtained from our preliminary experiments, performed on liposomes loaded with one or two first line drugs, laid the foundations for the development of novel delivery systems for the administration of combinations of new and established antitubercular agents. This multidisciplinary project was developed thanks to the cooperation of scientists from the University of Pavia (Italy), the University of Pisa (Italy), the University of Modena and Reggio Emilia (Italy) and the Institut Pasteur (Paris, France).