M. tuberculosis (Mtb) is the well-known causative agent of tuberculosis (TB) in humans, which today remains one of the top ten causes of death in the world, as well as the main cause of infection by a single infectious agent, above all HIV / AIDS (WHO Tb-report, 2020). This epidemiological incidence highlights the current need to find new treatments and drugs against tuberculosis. Considering the rich literature and studies carried out on Mtb in this regard, particular interest has been attributed to the iron uptake mechanisms, and in particular to the enzyme salicylate synthase MbtI, as a possible pharmacological target for tuberculosis. The MbtI enzyme represents an ideal target as it is essential in the course of infection and has no homologues in humans. For this reason, in collaboration with a group of pharmaceutical chemists, led by Prof. Stefania Villa and Fiorella Meneghetti of the DISFARM Department of Pharmaceutical Sciences of the University of Milan, a research has been launched for the development of new potential inhibitory molecules MbtI, which made it possible to obtain a series of benzo-furan derivatives, active against MbtI and with moderate anti-tuberculous activity (Chiarelli et al., 2018). Among the different potential targets of Mtb, PtpB tyrosine phosphatase has recently been shown to be inhibited by benzofuran salicylic derivatives, which have been shown to be good inhibitors (Zhou et al., 2010). As well as MbtI, PtpB is essential for the pathological development of TB and has no human orthologs, but unlike the latter, its substrate within the host macrophage has yet to be identified, while for MbtI it is known to be acid salicylic. Taking into account the fact that salicylate is a substrate of MbtI and that the best inhibitors obtained up to now are furan derivatives, it was natural to think about the possibility of developing dual inhibitors that target both enzymes in order to obtain multitarget compounds. The aim of my experimental thesis was therefore to identify and evaluate new dual inhibitors for MbtI and PtpB, through the screening of the MbtI inhibitor library, developed in our laboratory. The recombinant enzymes were then produced in E. coli, purified by chromatography, then subjected to enzyme inhibition assays to evaluate the effects of the different specially developed multitarget compounds.
M. tuberculosis (Mtb) è il noto agente eziologico della tubercolosi (TB) nell’uomo, che ad oggi rimane una fra le prime dieci cause di morte nel mondo, nonché la principale causa di infezione da singolo agente infettivo, sopra anche all’HIV/AIDS (WHO Tb-report, 2020). Tale incidenza epidemiologica evidenzia l’attuale necessità di trovare nuove cure e farmaci contro la tubercolosi. Considerando la ricca letteratura e gli studi effettuati su Mtb al riguardo, un interesse particolare è stato attribuito ai meccanismi di uptake del ferro, ed in particolare all’enzima salicilato sintasi MbtI, come possibile target farmacologico per la tubercolosi. L’enzima MbtI rappresenta un target ideale in quanto essenziale nel decorso dell’infezione e privo di omologhi nell’uomo. Per questo motivo in collaborazione con un gruppo di chimici farmaceutici, guidato dalle Prof.sse Stefania Villa e Fiorella Meneghetti del Dipartimento di Scienze Farmaceutiche DISFARM dell’Università degli Studi di Milano, è stata avviata una ricerca per lo sviluppo di nuove potenziali molecole inibitrici di MbtI, che ha permesso di ottenere una serie di derivati benzo-furanici, attive contro MbtI e con una moderata attività antitubercolare (Chiarelli et al., 2018). Fra i diversi potenziali target di Mtb, la tirosin fosfatasi PtpB è stata recentemente dimostrata essere inibita da derivati salicilici benzofuranici, i quali si sono dimostrati essere dei suoi buoni inibitori (Zhou et al., 2010). Così come MbtI, PtpB è essenziale per lo sviluppo patologico di TB e non presenta ortologhi umani, ma a differenza di quest’ultimo, il suo substrato all’interno del macrofago ospite deve ancora essere identificato, mentre per MbtI è noto essere l’acido salicilico. Tenendo conto del fatto che il salicilato è substrato di MbtI e che i migliori inibitori ottenuti fino ad ora sono dei derivati furanici, è risultato naturale pensare alla possibilità di sviluppare degli inibitori duali che colpissero entrambi gli enzimi al fine di ottenere dei composti multitarget. Scopo del mio lavoro di tesi sperimentale è stato quindi quello di identificare e valutare nuovi inibitori duali per MbtI e PtpB, attraverso lo screening della libreria inibitori di MbtI, sviluppata nel nostro laboratorio. Gli enzimi ricombinanti sono stati quindi prodotti in E. coli, purificati mediante cromatografia, quindi sottoposti a saggi enzimatici di inibizione per valutare gli effetti dei diversi composti multitarget appositamente sviluppati.
Screening di potenziali inibitori della salicilato sintasi MbtI e della fosfatasi PtpB di M. tuberculosis, per lo sviluppo di composti antitubercolari multitarget.
MONTI, JULIA DEL CARMEN
2019/2020
Abstract
M. tuberculosis (Mtb) is the well-known causative agent of tuberculosis (TB) in humans, which today remains one of the top ten causes of death in the world, as well as the main cause of infection by a single infectious agent, above all HIV / AIDS (WHO Tb-report, 2020). This epidemiological incidence highlights the current need to find new treatments and drugs against tuberculosis. Considering the rich literature and studies carried out on Mtb in this regard, particular interest has been attributed to the iron uptake mechanisms, and in particular to the enzyme salicylate synthase MbtI, as a possible pharmacological target for tuberculosis. The MbtI enzyme represents an ideal target as it is essential in the course of infection and has no homologues in humans. For this reason, in collaboration with a group of pharmaceutical chemists, led by Prof. Stefania Villa and Fiorella Meneghetti of the DISFARM Department of Pharmaceutical Sciences of the University of Milan, a research has been launched for the development of new potential inhibitory molecules MbtI, which made it possible to obtain a series of benzo-furan derivatives, active against MbtI and with moderate anti-tuberculous activity (Chiarelli et al., 2018). Among the different potential targets of Mtb, PtpB tyrosine phosphatase has recently been shown to be inhibited by benzofuran salicylic derivatives, which have been shown to be good inhibitors (Zhou et al., 2010). As well as MbtI, PtpB is essential for the pathological development of TB and has no human orthologs, but unlike the latter, its substrate within the host macrophage has yet to be identified, while for MbtI it is known to be acid salicylic. Taking into account the fact that salicylate is a substrate of MbtI and that the best inhibitors obtained up to now are furan derivatives, it was natural to think about the possibility of developing dual inhibitors that target both enzymes in order to obtain multitarget compounds. The aim of my experimental thesis was therefore to identify and evaluate new dual inhibitors for MbtI and PtpB, through the screening of the MbtI inhibitor library, developed in our laboratory. The recombinant enzymes were then produced in E. coli, purified by chromatography, then subjected to enzyme inhibition assays to evaluate the effects of the different specially developed multitarget compounds.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12453