Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), an infective disease which is one of the most death-causing from a single infectious agent worldwide, after COVID-19. To date, one of the best actions to decrease the risk of TB infection are the preventive treatments especially for who lives with HIV and groups subjected to clinical risks. Most of the knowledge about the therapies and prevention come from the complete genome of the M. tuberculosis H37Rv strain. Antimicrobial resistance (AMR) is one of the biggest problems in public health, which lead the common drugs to be ineffective against the increasing range of evolved infections. In particular what concern M. tuberculosis are the emerging challenges of the Multi-Drug Resistant (MDR) strains, which are resistant to two first-line drugs (isoniazid and rifampin) and eXtensively Drug Resistant (XDR) strains, MDR strains resistant also to two major second-line agents, such as aminoglycosides and fluoroquinolones. For this reason, it is mandatory the introduction of new drugs with novel molecular targets. Criteria for developing new TB drug candidates comprehend: More potent than the already available drug, in order to reduce the duration of therapy; Effective against MDR- and XDR-TB strains; Effective against Mtb in its different physiological states including latent TB. The tryptophan biosynthesis, an essential pathway for bacterial growth, but it is absent in humans, represents an interesting potential target for novel antitubercular drugs. Indeed, in recent years several compounds inhibiting enzymes of this pathway have been reported, including inhibitors of the tryptophan synthase. Mycobacterium tuberculosis tryptophan synthase TrpAB, encoded by trpA and trpB genes, is an heterotetrameric enzyme, that catalyses the two-step conversion indole-3-glycerol phosphate (IGP) and L-serine into L-tryptophan. In particular, the α-subunit hydrolyses IGP into glyceraldehyde 3-phosphate and indole, while the β-subunits catalyses the pyridoxal 5′-phosphate-dependent reaction of indole with L-Ser to produce L-Trp. In this work we report the cloning and production of the Mycobacterium tuberculosis TrpAB in recombinant form, and the assessment of the enzymatic activities assays. The achieved enzyme will represent a useful tool for the screening of potential inhibitors, to develop novel antitubercular molecules.
Produzione e caratterizzazione molecolare della triptofano sintasi di Mycobacterium tuberculosis e messa a punto del saggio di attività. Mycobacterium tuberculosis è l'agente eziologico della tubercolosi (TB), una malattia infettiva tra le più mortali al mondo, causata da un singolo agente infettivo. Ad oggi, una delle migliori azioni per diminuire il rischio di infezione da TB sono i trattamenti preventivi soprattutto per chi convive con l'HIV e gruppi soggetti a rischi clinici. La maggior parte delle conoscenze sulle terapie e sulla prevenzione provengono dal genoma completo del ceppo H37Rv di M. tuberculosis. La resistenza antimicrobica (AMR) è uno dei maggiori problemi riguardanti la sanità pubblica, che porta i comuni farmaci ad essere inefficaci contro la crescente gamma di infezioni che si sono evolute col passare del tempo. In particolare, per quanto riguarda M. tuberculosis i maggior problemi riguardano i ceppi Multi-Drug Resistant (MDR), resistenti a due farmaci di prima linea (isoniazide e rifampicina), ed eXtensively Drug Resistant (XDR) ceppi MDR resistenti anche a due principali agenti di seconda linea, come amminoglicosidi e fluorochinoloni. Per questo motivo l’introduzione di nuovi farmaci che colpiscono nuovi bersagli è di fondamentale importanza. I criteri per lo sviluppo di nuovi farmaci contro la tubercolosi comprendono: Famaci più potenti rispetto a quelli già disponibili, al fine di ridurre la durata della terapia; Efficaci contro i ceppi MDR e XDR-TB; Efficaci contro Mtb nei suoi diversi stati fisiologici inclusa la tubercolosi in fase latente. La biosintesi del triptofano rappresenta un interessante bersaglio farmacologico, in quanto è una via metabolica essenziale per la crescita del microorganismo, e assente nell’uomo. Infatti sono già state identificate alcune molecole in grado di inibire enzimi di questa via, compresi inibitori della triptofano sintasi. La triptofano sintasi di M. tuberculosis, TrpAB, è un enzima eterotetramerico che catalizza la conversione dell’indolo-glicero-fosfato (IGP) e L-serina in L-triptofano. In particolare la subunità α catalizza l’idrolisi dell’IGP in indolo e gliceraldeide-3-fosfato, mentre la subunità β catalizza la reazione PLP dipendente della L-ser con l’indolo a dare L-Trp. In questo lavoro di tesi TrpAB è stato clonato e prodotto in forma ricombinante, e sono stati messi a punti i saggi di attività per le due reazioni. Questo enzima sarà uno utile strumento per lo screening di potenziali inibitori, per lo sviluppo di nuove molecole antitubercolari.
Production and molecular characterization of the Mycobacterium tuberculosis’s tryptophan synthase and set up of the activity assay.
COCORULLO, MARIO
2021/2022
Abstract
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), an infective disease which is one of the most death-causing from a single infectious agent worldwide, after COVID-19. To date, one of the best actions to decrease the risk of TB infection are the preventive treatments especially for who lives with HIV and groups subjected to clinical risks. Most of the knowledge about the therapies and prevention come from the complete genome of the M. tuberculosis H37Rv strain. Antimicrobial resistance (AMR) is one of the biggest problems in public health, which lead the common drugs to be ineffective against the increasing range of evolved infections. In particular what concern M. tuberculosis are the emerging challenges of the Multi-Drug Resistant (MDR) strains, which are resistant to two first-line drugs (isoniazid and rifampin) and eXtensively Drug Resistant (XDR) strains, MDR strains resistant also to two major second-line agents, such as aminoglycosides and fluoroquinolones. For this reason, it is mandatory the introduction of new drugs with novel molecular targets. Criteria for developing new TB drug candidates comprehend: More potent than the already available drug, in order to reduce the duration of therapy; Effective against MDR- and XDR-TB strains; Effective against Mtb in its different physiological states including latent TB. The tryptophan biosynthesis, an essential pathway for bacterial growth, but it is absent in humans, represents an interesting potential target for novel antitubercular drugs. Indeed, in recent years several compounds inhibiting enzymes of this pathway have been reported, including inhibitors of the tryptophan synthase. Mycobacterium tuberculosis tryptophan synthase TrpAB, encoded by trpA and trpB genes, is an heterotetrameric enzyme, that catalyses the two-step conversion indole-3-glycerol phosphate (IGP) and L-serine into L-tryptophan. In particular, the α-subunit hydrolyses IGP into glyceraldehyde 3-phosphate and indole, while the β-subunits catalyses the pyridoxal 5′-phosphate-dependent reaction of indole with L-Ser to produce L-Trp. In this work we report the cloning and production of the Mycobacterium tuberculosis TrpAB in recombinant form, and the assessment of the enzymatic activities assays. The achieved enzyme will represent a useful tool for the screening of potential inhibitors, to develop novel antitubercular molecules.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/20.500.14239/14966