Biofilm is one of the main causes of AMR and contributes to several chronic infectious diseases. A promising antibacterial strategy to deal with the development of AMR aims to interfere with bacterial quorum sensing (QS). The QS is the most effective cell-to-cell mechanism that bacteria use to communicate. One of the key bacterial enzymes that triggers and stokes the AI-2 mediated QS cascade is the kinase LsrK. LsrK catalyzes the phosphorylation of the AI-2 4,5-dihydroxypentane-2,3-dione (DPD), thus inducing QS. The exploitation of LsrK as a new target for the development of novel QS quenching drugs may represent a huge opportunity in fighting AMR. Because the LsrK pathway has been discovered quite recently, this target is still poorly explored from a pharmaceutical standpoint; thus, searching for new and developable synthetic LsrK inhibitors is challenging. In a previous work performed at LabMedChem, two natural compounds, namely HibA and HibB, were fished out by virtual screening from an in-house library of plant secondary metabolites. With the primary aim to evaluate the capability of the identified virtual hits to inhibit the LsrK kinase, my experimental work mainly focused on the preparation of all the chemicals required for setting up the in vitro enzymatic inhibitory assay. This required first of all the preparation of the LsrK substrate DPD. DPD is not commercially available. For this reason, I planned to develop a suitable synthetic strategy for the synthesis of the LsrK substrate. The proposed synthetic procedure for the synthesis of DPD allowed to prepare the DPD with a 10% overall yield. As a second part of my thesis work, I focused on the isolation and purification of HibA and Hib from Hibiscus sabdariffa, in suitable amounts to perform the in vitro enzymatic inhibitor assay, and of fumarprotocetaric acid from Cladonia foliacea to be used as positive control in the further LsrK inhibition assay. The third aim of my project was the identification of new synthetic hit compounds, potentially able to inhibit LsrK. At the present, the sole available crystallographic structures of LsrK are in the open conformation. For this reason, before proceeding with the virtual screening, the homology model of LsrK in close conformation was prepared. Both conformations of the kinase were exploited to perform a virtual screening on the in-house LabMedChem chemoteca. According to the results of the docking calculation, three virtual hits were proposed to bind at the LsrK active site in both conformations. MCL-0026 and MCL-022 were already available, whereas QS35 was re-synthesized. Lastly, I participated to the setup of the enzymatic inhibitor assay. However, at the moment, the assay is not still reliable to further investigate the inhibitory activity of the potential LsrK inhibitors identified and prepared. All the variables affecting the kinase activity will be investigated and the assay will be adapted accordingly. Moreover, besides the evaluation of the LsrK inhibitor activity, the binding of all the proposed compounds with the protein will be evaluated by measurement of the intrinsic tryptophan fluorescence. The results will be reported in due course.
La formazione di biofilm è una delle principali cause alla base dell’Antibiotico Resistenza in quanto fornisce ai batteri una minor suscettibilità all’azione degli antibiotici tradizionali. Il blocco della formazione del biofilm, mediante l’inibizione del quorum sensing (QS), può essere considerata una valida opportunità nella lotta all’AR. La proteina LsrK è un enzima chiave alla base di questo fenomeno in quanto attraverso la fosforilazione del 4,5-diidrossi pentan-2,3-dione (DPD), appartenente alla famiglia degli autoinduttori 2 (AI-2), induce l’innesco del QS. Poiché il pathway biochimico mediato da LsrK è stato scoperto di recente questo enzima è ancora poco studiato da un punto di vista farmaceutico. La sfida che il chimico farmaceutico si trova perciò ad affrontare è di individuare degli inibitori di questo enzima potenzialmente sviluppabili come innovativi farmaci antimicrobici. Il MedChemLab, ha individuato nei metaboliti secondari dell’H. sabdariffa, noti come HibA e HibB, due potenziali inibitori di LsrK. Il presente lavoro di tesi si inserisce in questo contesto ed è focalizzato in primis nella preparazione delle sostanze chimiche necessarie per la messa a punto di un saggio di inibizione enzimatico attraverso il quale poter valutare l’attività dei potenziali inibitori di LsrK. Il DPD, substrato di LsrK, non è commercialmente disponibile. Per questo motivo, il primo obiettivo di questo lavoro di tesi è stato la pianificazione di una strategia sintetica idonea per la preparazione del DPD. La procedura sintetica messa a punto ha consentito di ottenere il DPD con una resa complessiva del 10%. Il secondo aspetto del mio lavoro di tesi ha riguardato l'isolamento di HibA e HibB, in quantità adeguate per poter eseguire il saggio di inibizione enzimatica, e dell'acido fumarprotocetrarico dalla Cladonia foliacea da impiegare come controllo positivo. Il terzo obiettivo ha riguardato l'identificazione di piccole molecole di sintesi potenzialmente in grado di inibire LsrK sfruttando le informazioni strutturali recentemente disponibili sulla chinasi. Allo stato attuale, le sole strutture cristallografiche di LsrK disponibili sono in conformazione aperta. Per questo motivo, prima di procedere con il virtual screening, mi sono occupato della preparazione di un modello per omologia di LsrK in conformazione chiusa. Le strutture tridimensionali della proteina in entrambe le conformazioni sono state sfruttate per eseguire un virtual screening sulla chemoteca interna del MedChemLab. Sulla base dei risultati di docking, sono stati proposti tre virtual hits potenzialmente in grado di interagire con il sito attivo di LsrK in entrambe le conformazioni. MCL-0026 e MCL-022 erano già disponibili, mentre QS35 è stato re-sintetizzato. Infine, ho partecipato alla messa a punto del saggio di inibizione enzimatico. Tuttavia, al momento, il test non è ancora adeguatamente affidabile per la determinazione dell'attività inibitoria dei potenziali inibitori di LsrK identificati. Studi di binding alla proteina di tutti gli inibitori proposti mediante la misurazione della fluorescenza intrinseca del triptofano sono tuttora in corso.
Contrastare la resistenza agli antimicrobici mediante l'inibizione dell'enzima LsrK: virtual screening, sintesi e valutazione enzimatica di nuovi hits
MILLI, GIORGIO
2021/2022
Abstract
Biofilm is one of the main causes of AMR and contributes to several chronic infectious diseases. A promising antibacterial strategy to deal with the development of AMR aims to interfere with bacterial quorum sensing (QS). The QS is the most effective cell-to-cell mechanism that bacteria use to communicate. One of the key bacterial enzymes that triggers and stokes the AI-2 mediated QS cascade is the kinase LsrK. LsrK catalyzes the phosphorylation of the AI-2 4,5-dihydroxypentane-2,3-dione (DPD), thus inducing QS. The exploitation of LsrK as a new target for the development of novel QS quenching drugs may represent a huge opportunity in fighting AMR. Because the LsrK pathway has been discovered quite recently, this target is still poorly explored from a pharmaceutical standpoint; thus, searching for new and developable synthetic LsrK inhibitors is challenging. In a previous work performed at LabMedChem, two natural compounds, namely HibA and HibB, were fished out by virtual screening from an in-house library of plant secondary metabolites. With the primary aim to evaluate the capability of the identified virtual hits to inhibit the LsrK kinase, my experimental work mainly focused on the preparation of all the chemicals required for setting up the in vitro enzymatic inhibitory assay. This required first of all the preparation of the LsrK substrate DPD. DPD is not commercially available. For this reason, I planned to develop a suitable synthetic strategy for the synthesis of the LsrK substrate. The proposed synthetic procedure for the synthesis of DPD allowed to prepare the DPD with a 10% overall yield. As a second part of my thesis work, I focused on the isolation and purification of HibA and Hib from Hibiscus sabdariffa, in suitable amounts to perform the in vitro enzymatic inhibitor assay, and of fumarprotocetaric acid from Cladonia foliacea to be used as positive control in the further LsrK inhibition assay. The third aim of my project was the identification of new synthetic hit compounds, potentially able to inhibit LsrK. At the present, the sole available crystallographic structures of LsrK are in the open conformation. For this reason, before proceeding with the virtual screening, the homology model of LsrK in close conformation was prepared. Both conformations of the kinase were exploited to perform a virtual screening on the in-house LabMedChem chemoteca. According to the results of the docking calculation, three virtual hits were proposed to bind at the LsrK active site in both conformations. MCL-0026 and MCL-022 were already available, whereas QS35 was re-synthesized. Lastly, I participated to the setup of the enzymatic inhibitor assay. However, at the moment, the assay is not still reliable to further investigate the inhibitory activity of the potential LsrK inhibitors identified and prepared. All the variables affecting the kinase activity will be investigated and the assay will be adapted accordingly. Moreover, besides the evaluation of the LsrK inhibitor activity, the binding of all the proposed compounds with the protein will be evaluated by measurement of the intrinsic tryptophan fluorescence. The results will be reported in due course.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/20.500.14239/14570