Discovery and development of novel salicylate synthase (MbtI) furanic inhibitors as antitubercular agents

We report on the virtual screening, synthesis, and biological evaluation of new furan derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based virtual screening procedure was applied to screen the Enamine database, identifying two compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active compound I as starting point for the development of novel MbtI inhibitors, we obtained new derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a emerged as the most potent MbtI inhibitor reported to date (Ki = 5.3 μM). Moreover, compound 1a showed a promising antimycobacterial activity (MIC99 = 156 μM), which is conceivably related to mycobactin biosynthesis inhibition

DESIGN E SINTESI DI NUOVI LIGANDI SELETTIVI PER IL RECETTORE BETA DEGLI ESTROGENI

Gli estrogeni sono ormoni sessuali femminili appartenenti alla classe degli ormoni steroidei. Questi sono implicati in molti processi fisiologici quali la riproduzione e lo sviluppo/mantenimento dei caratteri secondari femminili. Gli estrogeni inoltre hanno effetti positivi sull’omeostasi delle ossa, sul sistema cardiovascolare, sul fegato, sull’accumulo di tessuto adiposo, sul sistema nervoso e su molti altri organi. Sono coinvolti in diversi stati patologici quali alcune forme di tumore come il carcinoma mammario, il cancro alla prostrata, al colon e al polmone, ma anche in infiammazioni come l’artrite reumatoide o l’endometriosi. L'attività degli estrogeni è mediata da due recettori, ERalfa e ERbeta, che agiscono come fattori di trascrizione regolando l'espressione genica. I due sottotipi alfa e beta differiscono sia nella struttura a livello del binding pocket che nella distribuzione tissutale. I livelli di ERalfa ed ERbeta inoltre variano tra tessuto sano e tessuto malato. In questo progetto sono stati effettuati studi computazionali e sintetici volti alla ricerca di nuovi ligandi selttivi per il recettore beta degli estrogeni

Innovative strategies for the drug lead identification and optimization

This PhD thesis was focused on the exploration of several computational strategies that are employed in computer-aided drug design and in the study of small molecules interacting with different targets. The aim of the thesis was to validate the reliability and the effectiveness of these procedures and to combine them in order to develop efficient virtual screening (VS) protocols or target prediction platforms and to structurally optimize promising lead compounds. In the first part, the capability of a pharmacophore-enhanced consensus docking approach to improve VS results and reduce the normally required computing time was tested and validated. Chapter 1 reports the development of a VS protocol combining the pharmacophore filtering of a commercial database with a consensus docking approach that allowed the identification of the first STARD3 inhibitor. Herein, consensus docking was employed for both a qualitative and quantitative analysis. In Chapter 2, the same methodology was applied to a different target and led to the discovery of new potent MbtI inhibitors. Differently, Chapter 3 reports the extensive evaluation of docking-based reverse screening approaches in identifing the proper target of a query ligand. This analysis involved the use of several docking procedures in target prediction studies performed on a benchmark dataset including different targets and a set of known-active compounds. Finally, Chapter 4 describes the structural optimization of a class of potent MAGL inhibitors. Several in vitro analysis and preliminary in vivo studies were performed on the best representatives of this class of compounds

The energy transfer model of nonphotochemical quenching: Lessons from the minor CP29 antenna complex of plants

Antenna complexes in photosystems of plants and green algae are able to switch between a light-harvesting unquenched conformation and a quenched conformation so to avoid photodamage. When the switch is activated, nonphotochemical quenching (NPQ) mechanisms take place for an efficient deactivation of excess excitation energy. The molecular details of these mechanisms have not been fully clarified but different hypotheses have been proposed. Among them, a popular one involves excitation energy transfer (EET) from the singlet excited Chls to the lowest singlet state (S1) of carotenoids. In this work, we combine such model with μs-long molecular dynamics simulations of the CP29 minor antenna complex to investigate how conformational fluctuations affect the electronic couplings and the final EET quenching. The computational framework is applied to both CP29 embedding violaxanthin and zeaxantin in its L2 site. Our results demonstrate that the EET model is rather insensitive to physically reasonable variations in single chlorophyll-carotenoid couplings, and that very large conformational changes would be needed to see the large variation of the complex lifetime expected in the switch from light-harvesting to quenched state. We show, however, that a major role in regulating the EET quenching is played by the S1 energy of the carotenoid, in line with very recent spectroscopy experiments

Extensive Reliability Evaluation of Docking-Based Target-Fishing Strategies

The development of target-fishing approaches, aimed at identifying the possible protein targets of a small molecule, represents a hot topic in medicinal chemistry. A successful target-fishing approach would allow for the elucidation of the mechanism of action of all therapeutically interesting compounds for which the actual target is still unknown. Moreover, target-fishing would be essential for preventing adverse effects of drug candidates, by predicting their potential off-targets, and it would speed up drug repurposing campaigns. However, due to the huge number of possible protein targets that a small-molecule might interact with, experimental target-fishing approaches are out of reach. In silico target-fishing represents a valuable alternative, and examples of receptor-based approaches, exploiting the large number of crystallographic protein structures determined to date, have been reported in the literature. To the best of our knowledge, no proper evaluation of such approaches is, however, reported yet. In the present work, we extensively assessed the reliability of docking-based target-fishing strategies. For this purpose, a set of X-ray structures belonging to different targets was selected, and a dataset of compounds, including 10 experimentally active ligands for each target, was created. A target-fishing benchmark database was then obtained, and used to assess the performance of 13 different docking procedures, in identifying the correct target of the dataset ligands. Moreover, a consensus docking-based target-fishing strategy was developed and evaluated. The analysis highlighted that specific features of the target proteins could affect the reliability of the protocol, which however, proved to represent a valuable tool in the proper applicability domain. Our study represents the first extensive performance assessment of docking-based target-fishing approaches, paving the way for the development of novel efficient receptor-based target fishing strategies

A Virtual Screening Study for Lactate Dehydrogenase 5 Inhibitors by using a Pharmacophore-Based Approach

Inhibitors of human lactate dehydrogenase 5 (hLDH5) are promising therapeutic agents against cancer. This enzyme is generally found to be overexpressed in most invasive cancer cells and is linked to their vitality especially under hypoxic conditions. In this study, with the aim of identifying new hLDH5 inhibitors, a receptor-based pharmacophore modeling approach has been tested and, in order to verify the reliability of the reported approach, the Gold and Platinum database from Asinex were filtered. The top-ranked compounds were experimentally tested for their hLDH5 inhibition activity and enzymatic assays revealed that, among the ten selected compounds, two proved to inhibit the enzyme activity with Ki values in the micromolar range (Ki=33.1-76.7μM)

Cyclic Ketoximes as Estrogen Receptorβ Selective Agonists

The development of estrogen receptorβ (ERβ)-selective agonists represents a therapeutic strategy against several kinds of cancers, but the high homology between the two receptor subtypes, ERα and ERβ, makes the achievement of this goal very challenging. In the past, we developed salicylaldoxime- and salicylketoxime-based molecules that proved to bind well to ERβ. In this paper, further structural evolution of the salicylketoximes is presented: two of the newly synthesized five-membered cyclic ketoximes bind with nanomolar affinities to ERβ, and they show selectivity for this subtype over ERα. Their agonist character was confirmed by cell-free coactivator recruitment assays, in which we demonstrated the ability of these compounds to form an active complex with ERβ capable of recruiting coactivator proteins; this indicated their efficacy as agonists. Finally, their potency and selectivity for ERβ binding were rationalized by molecular-modeling studies

Computationally driven discovery of phenyl(piperazin-1-yl)methanone derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors

Monoacylglycerol lipase (MAGL) is an attractive therapeutic target for many pathologies, including neurodegenerative diseases, cancer as well as chronic pain and inflammatory pathologies. The identification of reversible MAGL inhibitors, devoid of the side effects associated to prolonged MAGL inactivation, is a hot topic in medicinal chemistry. In this study, a novel phenyl(piperazin-1-yl)methanone inhibitor of MAGL was identified through a virtual screening protocol based on a fingerprint-driven consensus docking (CD) approach. Molecular modeling and preliminary structure-based hit optimization studies allowed the discovery of derivative 4, which showed an efficient reversible MAGL inhibition (IC 50 = 6.1 µM) and a promising antiproliferative activity on breast and ovarian cancer cell lines (IC 50 of 31–72 µM), thus representing a lead for the development of new and more potent reversible MAGL inhibitors. Moreover, the obtained results confirmed the reliability of the fingerprint-driven CD approach herein developed

Binding investigation and preliminary optimisation of the 3-amino-1,2,4-triazin-5(2H)-one core for the development of new Fyn inhibitors

Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer’s and Parkinson’s diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (VS6), a hit compound that showed a micromolar inhibition of Fyn (IC50= 4.8 μM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimisation. This analysis led us to confirm the hypothesised binding mode of VS6 and to identify a new derivative that is about 6-fold more active than VS6 (compound 3, IC50= 0.76 μM)