Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAF(V600E) mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAF(V600E) has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC(50) values in the 40–88 nM range. Selected compounds inhibited BRAF(V600E) signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior

A community effort in SARS-CoV-2 drug discovery.

peer reviewedThe COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against Covid-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments.R-AGR-3826 - COVID19-14715687-CovScreen (01/06/2020 - 31/01/2021) - GLAAB Enric

Inhibition of Patched chemotherapy resistance activity : biocomputational, chemical and cellular approaches

Malgré des efforts continus dans le développement de nouveaux médicaments, la résistance aux chimiothérapies reste un défi majeur dans le traitement du cancer. L'un des principaux mécanismes responsables de la résistance aux médicaments est l'efflux des agents thérapeutiques hors des cellules cancéreuses par les transporteurs multidrogues.Le récepteur Hedgehog Patched1 (PTCH1), qui fait partie de la voie de signalisation Hedgehog, est surexprimé dans de nombreux cancers. Outre son rôle physiologique de transporteur de cholestérol, PTCH1 est aussi capable de transporter des agents anticancéreux hors des cellules cancéreuses, contribuant ainsi à la résistance aux médicaments avec les transporteurs ABC (ATP binding cassette). Contrairement à ces derniers, qui sont exprimés de manière endogène dans les cellules normales et jouent un rôle crucial dans la survie cellulaire, l'activité d'efflux de PTCH1 n'a lieu que dans les cellules cancéreuses. Cela fait de PTCH1 une nouvelle cible prometteuse pour le traitement du cancer. Trois inhibiteurs de son activité d'efflux ont été identifiés à ce jour : l'astémizole, la méthiothépine et un composé naturel, la panicéine A hydroquinone (PAH). Ces composés augmentent l'efficacité des chimiothérapies contre les cellules de mélanome in vitro et in vivo, prouvant que l'inhibition de PTCH1 est une stratégie anticancéreuse pertinente. Cependant, ils présentent des limites intrinsèques et doivent être optimisés. Par exemple, la PAH a une très faible stabilité métabolique, ce qui empêche d'envisager son développement en tant que médicament.Dans ce contexte, le projet a pour objectif de développer un inhibiteur de l'efflux de PTCH1, en tant que médicament “first-in-class” pour lutter contre la résistance à la chimiothérapie et améliorer la survie des patients grâce à une optimisation rationnelle de la PAH.Nous avons donc étudié l'interaction entre la PAH et PTCH1 au moyen de méthodologies in silico et effectué une comparaison structure-ligand entre les trois inhibiteurs connus. En utilisant un protocole de docking couplé à des simulations de dynamiques moléculaires, nous avons extrait des informations importantes sur la conformation active d'un inhibiteur de PTCH1 et identifié un site de liaison supposé dans le canal hydrophobe de PTCH1.Nous avons mis au point une nouvelle synthèse totale de l'E-PAH qui répond à certaines limitations importantes de la précédente, telles que sa non-stéréosélectivité et sa grande spécificité de substrat. Après optimisation, nous avons finalement obtenu un nouveau protocole de synthèse stéréosélective et robuste pour les E et Z PAH. Ceci a permis de synthétiser plusieurs analogues de PAH dont l'activité a été évaluée sur des cellules de mélanome, en combinaison avec le vemurafenib, dans le but de réaliser une étude structure-activité pour le motif hydroquinone. Nous avons également évalué certaines propriétés pharmacocinétiques de ce motif, telles que la stabilité plasmatique et le profil de sécurité.La partie hydroquinone de la PAH est importante pour son activité biologique, mais c'est aussi l'un de ses principaux inconvénients en raison de son oxydation facile en quinone. En combinant les connaissances acquises grâce aux études in silico et SAR dans la dernière partie de ce projet, nous avons cherché à synthétiser des nouvelles molécules en appliquant un remplacement "bioisostérique", une approche souvent utilisée avec succès en chimie médicinale. Tout d'abord, le remplacement de la double liaison de la PAH par des groupes chimiques biologiquement équivalents a permis une stratégie de synthèse robuste, convergente et efficace, qui s'inscrit dans les tendances modernes des approches de chimie durable. Ensuite, il a été possible de synthétiser facilement un grand nombre de composés, en remplaçant le motif hydroxyquinone, et de cribler l'activité biologique in cellulo d'un espace chimique diversifié avec des résultats biologiques très prometteurs.Despite the continuous efforts in the design and development of new drugs with innovative mode of action, resistance to both chemo- and targeted therapy remains a major challenge in cancer treatment. One of the major mechanisms responsible for multidrug resistance is the efflux of therapeutics out of cancer cells by multidrug transporters.The Hedgehog receptor Patched1 (PTCH1), part of the Hedgehog signaling pathway, is over-expressed in many cancers. In addition to its physiological role as cholesterol transporter, PTCH1 is also able to transport anticancer agents out of cancer cells, thus contributing to multidrug resistance together with the ATP binding cassette (ABC) transporters. Unlike ABC transporters which are endogenously expressed in normal cells and play a crucial role in cell survival, PTCH1 efflux activity takes place only in cancer cells. This makes PTCH1 a new attractive target for cancer treatment. Three inhibitors of PTCH1 efflux activity have been identified to date, namely, astemizole, methiothepin and the natural compound panicein A hydroquinone (PAH). These compounds increased the efficacy of both conventional and targeted chemotherapies against melanoma cells in vitro and in vivo, showing the proof of concept of PTCH1 inhibition as a successful anticancer strategy. However, they have some intrinsic limitations and require optimization. For instance, PAH, has a very low metabolic stability which prevents its advance in a drug development pipeline towards the clinics.In that context, this PhD project is aimed at the ultimate goal of developing a PTCH1 drug efflux inhibitor as a first-in-class drug candidate to fight chemotherapy resistance and improve patient survival. In particular, it comprehends the medicinal chemistry efforts made in the direction of rational optimization of the natural compound PAH.To this aim we studied the interaction between PAH and PTCH1 by means of in silico methodologies and we performed structure- and ligand based comparison between the three known inhibitors. By using an ensemble docking protocol coupled to molecular dynamics simulations of the ligands we extracted important information about the active conformation of a PTCH1 inhibitor and we identified a putative binding site within the hydrophobic channel on PTCH1.We developed a new total synthesis for E-PAH which addresses some important limitations of the previous one, such as its non-stereoselectivity and high substrate specificity. We applied several strategies and eventually obtained a stereoselective and robust new synthesis protocol for E and Z PAH. This allowed for the synthesis of several PAH analogs whose activity was evaluated on melanoma cells, in combination with vemurafenib, with the aim to assess a structure-activity relationship for the hydroquinone scaffold. We further evaluated some PK properties of the hydroquinone scaffold such as plasma stability and safety profile.The hydroquinone moiety of PAH is important for its biological activity but it is also one of its major drawbacks due to its easy oxidation to quinone. By combining the knowledge acquired from the in silico and SAR studies on the hydroquinone, in the last part of this PhD project we aimed at the synthesis of innovative scaffolds by applying a “bioisosteric” replacement on the hydroquinone, an approach often successfully used in medicinal chemistry. First, the replacement of the double bond of PAH with biologically equivalent chemical groups allowed for a robust, convergent and efficient synthetic strategy, which is line with the modern trends of sustainable chemistry approaches. Next, it made possible to readily synthesize a large number of compounds, substituting the hydroxyquinone motif, and screen the in cellulo biological activity of a diversified chemical space with quite promising biological results

Chasing new targets to fight antimicrobial resistance: identification of small molecules affecting the interaction of human hemoglobin with the IsdB hemophore of Staphylococcus aureus

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad-hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the sub-micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns

A community effort to discover small molecule SARS-CoV-2 inhibitors

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of a community effort, the “Billion molecules against Covid-19 challenge”, to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 potentially active molecules, which were subsequently ranked to find ‘consensus compounds’. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (Nsp12 domain), and (alpha) spike protein S. Overall, 27 potential inhibitors were experimentally confirmed by binding-, cleavage-, and/or viral suppression assays and are presented here. All results are freely available and can be taken further downstream without IP restrictions. Overall, we show the effectiveness of computational techniques, community efforts, and communication across research fields (i.e., protein expression and crystallography, in silico modeling, synthesis and biological assays) to accelerate the early phases of drug discovery