Leishmaniasis is a parasitic disease, which afflicts more than 1.3 million people throughout the world. It is more common in tropical and subtropical areas, where recurs by two main forms, Visceral and Cutaneous leishmaniasis, both caused by protozoan parasite of genus Leishmania and transmitted by the bite of the female sandfly. The current treatments for leishmaniasis are based on drugs, such as pentavalent antimonials, that are characterized by a high toxicity. In addition, cases of drug resistance have recently arisen in endemic countries and every year an increasing number of resistant strains is recorded. Consequently, there is a urgent need to project new, more affordable and less toxic drugs against this disease. Unlike the mammals, Leishmania has a unique thiol-based metabolism, in which the trypanothione (N1,N8-bis(glutathionyl)spermidine), T(SH)2, trypanothione reductase (TR), tryparedoxin (TXN) and tryparedoxin peroxidase (TXNPx) replace the redox systems present in the human host. In fact, they are part of an electron transport chain that transfers electrons from NADPH to H2O2 or organic peroxides produced by the host macrophages during the infection. Thus, the knowledge of the structures and mechanisms of action of these enzymes is very important for the development of new lead compounds since they are fundamental for the parasite survival and are absent in the mammalian host. The work reported in this thesis focuses on the functional and structural study of TR and TXNPx. In particular, the kinetic studies performed on trypanothione reductase from Leishmania infantum allowed the discovery of the promiscuous behavior by this protein, which can still oxidize NADPH in the absence of trypanothione using as electron acceptor the molecular oxygen. Furthermore, the inhibitory assays performed using an in-house library allowed the identification of a new compound, RDS777, (6-sec- Butoxy-2-[(3-chlorophenyl)sulfanyl]-4-pyrimidinamine) able to inhibit L.infantum TR with high efficiency (KI = 5.2 ± 3.8 μM). Moreover, I solved the X-ray structure of TR in complex with RDS777 which disclosed the mechanism of TR inhibition by that compound and allowed the identification of the key residues necessary for the binding. In addition, in this thesis is also reported the structural analysis of TXNPx from Leishmania major in Fully Folded (FF) conformation which has been solved during my PhD thesis at a resolution higher than that of TXNPx in Locally Unfolded (LU) conformation (2.34 Å). Through this structure, I was able to visualize all the residues involved in the catalysis not visible in the already solved LU TXNPx. The structural information was used for high throughput docking (HTD) studies in order to identify new potential inhibitors of this enzyme. The compounds selected by HTD were tested in vitro for the binding using the SPR technique and for its enzymatic activity using the HRP spectrophotometric assay. These studies allowed the identification of 9 compounds able to inhibit TXNPx with a KD in a range between 39 and 290 μM.
La leishmaniosi è una malattia parassitaria che ogni anno affligge più di 1.3 milioni di persone in tutto il mondo. È' principalmente diffusa nelle zone tropicali e subtropicali ed è caratterizzata da due forme principali: Viscerale e Cutanea. Entrambe sono causate da un protozoo del genere Leishmania e trasmesse per mezzo di pappataci del genere Phlebotomus e Lutzomya. Le terapie attualmente disponibili per la leishmaniosi sono basate su farmaci antimoniali, i quali sono caratterizzati sia da costi poco accessibili che da un'elevata tossicità. Inoltre, casi di farmaco-resistenza a questi trattamenti stanno via via aumentando, in particolare nei paesi dove la malattia è endemica. Quindi, c'è un urgente bisogno di nuovi farmaci che siano allo stesso tempo più economici e meno tossici. A differenza dei mammiferi, il metabolismo di Leishmania è basato su un ditiolo a basso peso molecolare, nel quale il tripanotione (N1,N8-bis(glutathionyl)spermidine), T(SH)2, tripanotione reduttasi (TR), triparedossina (TXN) e triparedossina perossidasi (TXNPx) sostituiscono i sistemi redox presenti nell'ospite mammifero. Infatti, questi fanno parte di un catena di trasporto elettronico che trasferisce equivalenti riducenti dall'NADPH all'H2O2 o altri perossidi organici prodotti dal macrofago durante l'infezione. Di conseguenza, la conoscenza della struttura e del meccanismo di azione di questi enzimi gioca un ruolo fondamentale nello sviluppo di nuovi inibitori, dal momento che questi risultano fondamentali alla sopravvivenza del parassita e assenti nell'ospite mammifero. Il lavoro riportato in questa tesi si focalizza sullo studio strutturale e funzionale di due di questi enzimi: TR e TXNPx. In particolare, esperimenti cinetici effettuati sulla tripanotione reduttasi da Leishmania infantum hanno permesso di studiare l'attività O2-ossidoreduttasica di tale enzima, che può ancora ossidare l'NADPH in assenza di tripanotione usando come accettare di elettroni ossigeno molecolare. Inoltre, saggi di inibizione hanno permesso l'identificazione di un nuovo composto, RDS777, (6-sec- Butoxy-2-[(3-chlorophenyl)sulfanyl]-4-pyrimidinamine), capace di inibire TR con alta efficienza (KI = 5.2 ± 3.8 μM). Per di più, la struttura cristallografica di TR in complesso con RDS777 è stata risolta e questa ha permesso l'identificazione dei residui chiave necessari all'interazione enzima-inibitore. In questa tesi è anche riportata la struttura cristallografica di TXNPx nella conformazione Fully Folded (FF), ottenuta ad una risoluzione maggiore (2.34 Å) rispetto alla precedente nella conformazione Locally Unfolded (LU). Attraverso questa nuova struttura, è stato possibile visualizzare tutti i residui che prendono parte al processo catalitico, non visibili nella conformazione LU. Queste informazioni strutturali sono state usate in studi di high throughput docking (HTD), allo scopo di identificare nuovi potenziali inibitori per questo enzima. I composti ottenuti tramite HTD sono stati testati in vitro attraverso esperimenti SPR, mentre la loro attività enzimatica è stata studiata attraverso saggi spettrofotometrici. Entrambi hanno permesso l'identificazione di 9 composti capaci di inibire TXNPx con una KD compresa tra 39 e 290 μM.
Trypanothione Reductase And Tryparedoxin Peroxidase: Structure And Action Mechanism Of Two Essential Leishmania Enzymes / Angiulli, Gabriella. - ELETTRONICO. - (2015).
Trypanothione Reductase And Tryparedoxin Peroxidase: Structure And Action Mechanism Of Two Essential Leishmania Enzymes
ANGIULLI, GABRIELLA
01/01/2015
Abstract
Leishmaniasis is a parasitic disease, which afflicts more than 1.3 million people throughout the world. It is more common in tropical and subtropical areas, where recurs by two main forms, Visceral and Cutaneous leishmaniasis, both caused by protozoan parasite of genus Leishmania and transmitted by the bite of the female sandfly. The current treatments for leishmaniasis are based on drugs, such as pentavalent antimonials, that are characterized by a high toxicity. In addition, cases of drug resistance have recently arisen in endemic countries and every year an increasing number of resistant strains is recorded. Consequently, there is a urgent need to project new, more affordable and less toxic drugs against this disease. Unlike the mammals, Leishmania has a unique thiol-based metabolism, in which the trypanothione (N1,N8-bis(glutathionyl)spermidine), T(SH)2, trypanothione reductase (TR), tryparedoxin (TXN) and tryparedoxin peroxidase (TXNPx) replace the redox systems present in the human host. In fact, they are part of an electron transport chain that transfers electrons from NADPH to H2O2 or organic peroxides produced by the host macrophages during the infection. Thus, the knowledge of the structures and mechanisms of action of these enzymes is very important for the development of new lead compounds since they are fundamental for the parasite survival and are absent in the mammalian host. The work reported in this thesis focuses on the functional and structural study of TR and TXNPx. In particular, the kinetic studies performed on trypanothione reductase from Leishmania infantum allowed the discovery of the promiscuous behavior by this protein, which can still oxidize NADPH in the absence of trypanothione using as electron acceptor the molecular oxygen. Furthermore, the inhibitory assays performed using an in-house library allowed the identification of a new compound, RDS777, (6-sec- Butoxy-2-[(3-chlorophenyl)sulfanyl]-4-pyrimidinamine) able to inhibit L.infantum TR with high efficiency (KI = 5.2 ± 3.8 μM). Moreover, I solved the X-ray structure of TR in complex with RDS777 which disclosed the mechanism of TR inhibition by that compound and allowed the identification of the key residues necessary for the binding. In addition, in this thesis is also reported the structural analysis of TXNPx from Leishmania major in Fully Folded (FF) conformation which has been solved during my PhD thesis at a resolution higher than that of TXNPx in Locally Unfolded (LU) conformation (2.34 Å). Through this structure, I was able to visualize all the residues involved in the catalysis not visible in the already solved LU TXNPx. The structural information was used for high throughput docking (HTD) studies in order to identify new potential inhibitors of this enzyme. The compounds selected by HTD were tested in vitro for the binding using the SPR technique and for its enzymatic activity using the HRP spectrophotometric assay. These studies allowed the identification of 9 compounds able to inhibit TXNPx with a KD in a range between 39 and 290 μM.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
