Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery

Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel.; Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD; +; /NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis.; For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline.; The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified

In silico protein modelling applied to the identification of new therapeutic agents

This thesis collects the results of three different research projects carried out in collaboration with the University of Parma. In chapter 2 the identification and characterization of antimicrobial compounds active as protein-protein interaction inhibitors of bacterial RNA polymerase subunit β' and transcription initiation factor σ is described. This peculiar protein-protein interaction has been identified as a promising target for antibacterial research and some small molecules were identified as disruptors of this interaction. In this project, a virtual screening campaign, aimed to the identification of novel antimicrobial molecules acting disrupting β'-σ interaction, was carried out. The newly identified compounds were experimentally tested in different assays, confirming their activity as PPI disruptors and highlighting their antimicrobial potential. A pharmacophore model was built for the most promising compounds to identify common patterns of interactions which were necessary for the activity and a binding hypothesis was proposed for each active compound that could be exploited as starting point for further optimization and for the discovery of new molecules. In the third chapter modelling studies were applied to NAPE-PLD, an enzyme responsible for the synthesis of bioactive lipids involved in the regulation of several physiological and pathological conditions. Modulation of NAPE-PLD activity could represent a promising therapeutic strategy for a wide range of diseases but, despite the important role played by this enzyme, no new active molecules have been reported so far. This work describes the identification and characterization of the first small molecule inhibitor of NAPE-PLD. Docking studies were performed, thanks to the availability of NAPE-PLD crystal structure, highlighting the binding pose of the compound and they were confirmed by SAR studies and mutagenesis experiments. Despite its low potency, this molecule represents a molecular probe which can help in better characterize and investigate NAPE-PLD mechanisms and its role in pathologic and physiological processes. NAPE-PLD together with other lipases, is characterized by a mechanism called “interfacial activation” which affects enzyme conformation and its catalytic machinery. The understanding of this phenomenon could be crucial to gain insight into enzyme’s behavior highlighting aspects of the catalytic mechanism that couldn’t be revealed by the X-ray structure alone. Molecular dynamics simulations were thus performed on the apo form of NAPE-PLD to better understand the conformational changes of the enzyme in both aqueous and membrane environment. Results highlights that the membrane environment stabilizes the open conformation, which is compatible with substrate recruitment, while water stabilizes a closed that block the access of the substrate to the active site, confirming the interfacial activation phenomenon. The last chapter of this thesis describes the statistical analysis that was performed on a large database of activity data collected by testing 400 compounds against Schistosoma mansoni, a parasite responsible for schistosomiasis, a tropical neglected disease. Three organizations, the University of California San Diego (UCSD), the Swiss Tropical and Public Health Institute (STPH) and Fiocruz (Fundação Oswaldo Cruz) Foundation in Brazil have experimentally tested the Pathogen Box, a collection of molecules active against different pathogens, monitoring the effects on the helminth. Different assays were performed and the results were collected using different numerical ranges, thus a mathematical transformation of the data was applied to the dataset to obtain activity values in the same scale. This was necessary to perform a statistical analysis to evaluate coherence of the collected data and to identify the most promising compounds.Questa tesi di dottorato raccoglie i risultati di tre diversi progetti di ricerca che ho svolto in collaborazione con l’Università di Parma. In particolare, il capitolo 2 descrive l’identificazione e la caratterizzazione di composti con attività antimicrobica che agiscono come inibitori dell’interazione proteina-proteina tra subunitá β' e fattore di trascrizione σ nell’RNA polimerasi batterica. Evidenze sperimentali hanno dimostrato che l’interfaccia tra le due proteine rappresenta un sito di legame per piccole molecole e composti che legano questa regione sono in grado di inibire l’interazione proteina-proteina arrestando la trascrizione e mostrando un’attività antibatterica. L’obiettivo di questo lavoro è stato identificare, tramite un protocollo di virtual-screening, all’interno di una libreria di composti, un sottoinsieme di molecole attive come inibitori dell’interazione β'-σ. Test sperimentali sono poi stati eseguiti sui composti più promettenti confermando l’attività per alcuni e provando la loro efficacia come composti antibatterici. Un modello farmacoforico è stato poi costruito per razionalizzare la relazione tra struttura e attività e si è inoltre ipotizzata una modalità di legame per i composti attivi con la subunità target β' che potrà essere utilizzata come punto di partenza per lo screening di nuove librerie o per la progettazione di nuovi composti. Nel capitolo 3 vengono invece descritti studi di modellistica molecolare effettuati sull’enzima NAPE-PLD. Questa lipasi di membrana è responsabile della produzione di lipidi bioattivi coinvolti in diversi processi fisiologici e patologici e la sua modulazione risulta essere particolarmente importante nel trattamento di diverse patologie. La disponibilità della struttura cristallografica dell’enzima umano ha reso possibile effettuare studi di docking per ipotizzare le modalità di legame della prima molecola capace di inibire NAPE-PLD, identificata mediante HTS. La posa di docking ottenuta risulta compatibile con i dati SAR in nostro possesso ed è inoltre confermata da studi di mutagenesi. Nonostante l’attività inibitoria limitata del composto esso può essere considerato il punto di partenza per sviluppare nuovi inibitori. Sono poi state effettuate simulazioni di dinamica molecolare per valutare i cambiamenti conformazionali dell’enzima in due ambienti differenti: in presenza di solvente acquoso oppure in presenza di membrana cellulare per testare l’ipotesi di “attivazione interfacciale”, un fenomeno caratteristico delle lipasi. Questo meccanismo è caratterizzato dall’equilibrio conformazionale tra uno stato definito “aperto”, compatibile con l’accesso del substrato e uno stato “chiuso” in cui l’accesso del substrato è bloccato. Durante il tempo di una simulazione di dinamica molecolare si sono potuti evidenziare cambiamenti conformazionali della proteina compatibili con questa teoria permettendoci di ipotizzare un meccanismo di “recruitment” del substrato quando l’enzima si trova in presenza della membrana cellulare. L’ultimo capitolo descrive il progetto di ricerca che ho seguito presso la University of California San Diego: qui ho svolto un lavoro di analisi di dati di attività biologica ottenuti testando 400 composti di una libreria chiamata Pathogen Box sul platelminta Schistosoma mansoni per identificare composti attivi contro la schistosomiasi, una parassitosi comune nei paesi sottosviluppati. Tre organizzazioni, la University of California San Diego (UCSD), il Swiss Tropical and Public Health Institute di Basilea (STPH) e la fondazione brasiliana Fiocruz (Fundação Oswaldo Cruz) hanno eseguito diversi saggi collezionando una grande quantità di dati di attività che ho successivamente analizzato per verificare diversi aspetti tra cui l’identificazione dei composti più potenti e la coerenza dei dati raccolti tra le diverse organizzazioni

New Antimicrobials Targeting Bacterial RNA Polymerase Holoenzyme Assembly Identified with an in Vivo BRET-Based Discovery Platform

Bacterial resistance represents a major health threat worldwide, and the development of new therapeutics, including innovative antibiotics, is urgently needed. We describe a discovery platform, centered on in silico screening and in vivo bioluminescence resonance energy transfer in yeast cells, for the identification of new antimicrobials that, by targeting the protein-protein interaction between the β'-subunit and the initiation factor σ70 of bacterial RNA polymerase, inhibit holoenzyme assembly and promoter-specific transcription. Out of 34 000 candidate compounds, we identified seven hits capable of interfering with this interaction. Two derivatives of one of these hits proved to be effective in inhibiting transcription in vitro and growth of the Gram-positive pathogens Staphylococcus aureus and Listeria monocytogenes. Upon supplementation of a permeability adjuvant, one derivative also effectively inhibited Escherichia coli growth. On the basis of the chemical structures of these inhibitors, we generated a ligand-based pharmacophore model that will guide the rational discovery of increasingly effective antibacterial agents

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery.

BACKGROUND:Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. METHODS:Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. RESULTS:For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. CONCLUSIONS:The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified

Synthesis and characterization of the first inhibitor of: N -acylphosphatidylethanolamine phospholipase D (NAPE-PLD)

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is a membrane-associated zinc enzyme that catalyzes the hydrolysis of N-acylphosphatidylethanolamines (NAPEs) into fatty acid ethanolamides (FAEs). Here, we describe the identification of the first small-molecule NAPE-PLD inhibitor, the quinazoline sulfonamide derivative 2,4-dioxo-N-[4-(4-pyridyl)phenyl]-1H-quinazoline-6-sulfonamide, ARN19874