This thesis is inserted in the context of the project "More Medicines for Tubeculosis – MM4TB” (EC-VII Framework), whose main objective is the validation of at least 5 new targets and the identification of at least one new antitubercular drug candidate. Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, is responsible for about 1.5 million deaths/year, and the emergence and spread of M. tuberculosis multidrug-resistant strains has prompted the discovery for new drugs and new targets. In this context, previously in our laboratory the CTP synthetase PyrG was validated as the target of thiophene-carboxamide and carbamothioyl-propanamide prodrugs activated by EthA. Furthermore, by genetic and biochemical approaches, a secondary target of these three PyrG inhibitors was found: the panthotenate kinase PanK. These evidences suggested that PyrG and PanK enzymes could be successfully used for the screening of compound libraries, in order to identify potential multitargeting hits. For this reason, the publically available GSK TB-set library of 204 antitubercular compounds was firstly assayed against PyrG activity. From this first screen 15 compounds active against PyrG were found. In particular, the three more active compounds were three pyridine-thiazole. These compounds were confirmed to be effective competitive inhibitors of the ATP binding site of PyrG, with inhibition constants ranging from 3 to 20 μM. In order to verify if these compounds could be possible multitargeting hits, they have been assayed against recombinant PanK enzyme activity. One of them were effectively found to affect PanK activity. Biochemical analysis demonstrated that the compound inhibits PyrG and PanK with a similar competitive inhibition mechanism, and the molecular docking confirmed that it is able to interact with the ATP binding site of both proteins. In conclusion, the two targets PyrG and PanK have been demonstrated to be suitable tools for identification of new potential multitargeting antitubercular compounds.
Questa tesi si inserisce all’interno del progetto "More Medicines for Tubeculosis – MM4TB” (EC-VII Framework) i cui principali obiettivi sono: la validazione di almeno cinque nuovi bersagli farmacologici e l’identificazione di almeno una famiglia di potenziali farmaci antitubercolari. La tubercolosi, una malattia infettiva causata dal batterio patogeno Mycobacterium tuberculosis è responsabile di circa 1,5 milioni di morti all’anno, è infatti tutt’oggi un’emergenza globale, e l’insorgenza e la diffusione di ceppi multi resistenti di M. tuberculosis ha reso necessaria la scoperta di nuovi farmaci, e di nuovi bersagli terapeutici In questo ambito, nel nostro laboratorio in precedenza la CTP sintetasi PyrG è stata validata come bersaglio di due nuovi composti, un tiofene-carbossamide ed un carbamothioyl-propanammide, pro-farmaci attivati da EthA,. Inoltre, con approcci genetici e biochimici, è stato individuato un target secondario di questi inibitori: la pantotenato chinasi PanK. Questi risultati suggeriscono che i due enzimi PyrG e PanK potrebbero essere utilizzati con successo per lo screening di librerie di composti, al fine di identificare potenziali inibitori multitarget. Per questo motivo, la libreria GSK TB-set, costituita da 204 composti con attività antitubercolare è stata saggiata in primo luogo contro l’ attività enzimatica di PyrG, ottenuto in forma ricombinante. Da questo primo screening sono emersi 15 composti in grado di inibire l’attività di PyrG a diversi livelli. In particolare gli inibitori più efficaci sono risultati essere tre piridine-tiazolo. Questi composti sono stati dimostrati efficaci inibitori competitivi del sito di legame per l'ATP dell’enzima, con costanti di inibizione comprese fra 3 e 20 M. Al fine di verificare se questi 3 composti potessero risultare utili per lo sviluppo di potenziali farmaci multitarget, sono stati testati anche su l’enzima ricombinante PanK. Uno di essi è effettivamente risultato un buon inibitore di PanK, Le analisi biochimiche dimostrato che il composto inibisce PyrG e PanK con un simile meccanismo di inibizione competitiva, e il docking molecolare ha confermato che è in grado di interagire con il sito di legame ATP di entrambe le proteine. In conclusione, i due obiettivi PyrG e Pank hanno dimostrato di essere strumenti idonei per l'identificazione di nuovi potenziali composti multitargeting antitubercolari.
CTP sintetasi e pantotenato chinasi di Mycobacterium tuberculosis: due promettenti bersagli per lo sviluppo di farmaci antitubercolari
BUTTARI, NICOLETTA
2015/2016
Abstract
This thesis is inserted in the context of the project "More Medicines for Tubeculosis – MM4TB” (EC-VII Framework), whose main objective is the validation of at least 5 new targets and the identification of at least one new antitubercular drug candidate. Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, is responsible for about 1.5 million deaths/year, and the emergence and spread of M. tuberculosis multidrug-resistant strains has prompted the discovery for new drugs and new targets. In this context, previously in our laboratory the CTP synthetase PyrG was validated as the target of thiophene-carboxamide and carbamothioyl-propanamide prodrugs activated by EthA. Furthermore, by genetic and biochemical approaches, a secondary target of these three PyrG inhibitors was found: the panthotenate kinase PanK. These evidences suggested that PyrG and PanK enzymes could be successfully used for the screening of compound libraries, in order to identify potential multitargeting hits. For this reason, the publically available GSK TB-set library of 204 antitubercular compounds was firstly assayed against PyrG activity. From this first screen 15 compounds active against PyrG were found. In particular, the three more active compounds were three pyridine-thiazole. These compounds were confirmed to be effective competitive inhibitors of the ATP binding site of PyrG, with inhibition constants ranging from 3 to 20 μM. In order to verify if these compounds could be possible multitargeting hits, they have been assayed against recombinant PanK enzyme activity. One of them were effectively found to affect PanK activity. Biochemical analysis demonstrated that the compound inhibits PyrG and PanK with a similar competitive inhibition mechanism, and the molecular docking confirmed that it is able to interact with the ATP binding site of both proteins. In conclusion, the two targets PyrG and PanK have been demonstrated to be suitable tools for identification of new potential multitargeting antitubercular 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/20.500.14239/22955