On the development of B-Raf inhibitors acting through innovative mechanisms

B-Raf is a protein kinase participating to the regulation of many biological processes in cells. Several studies have demonstrated that this protein is frequently upregulated in human cancers, especially when it bears activating mutations. In the last years, few ATP-competitive inhibitors of B-Raf have been marketed for the treatment of melanoma and are currently under clinical evaluation on a variety of other types of cancer. Although the introduction of drugs targeting B-Raf has provided significant advances in cancer treatment, responses to ATP-competitive inhibitors remain limited, mainly due to selectivity issues, side effects, narrow therapeutic windows, and the insurgence of drug resistance. Impressive research efforts have been made so far towards the identification of novel ATP-competitive modulators with improved efficacy against cancers driven by mutant Raf monomers and dimers, some of them showing good promises. However, several limitations could still be envisioned for these compounds, according to literature data. Besides, increased attentions have arisen around approaches based on the design of allosteric modulators, polypharmacology, proteolysis targeting chimeras (PROTACs) and drug repurposing for the targeting of B-Raf proteins. The design of compounds acting through such innovative mechanisms is rather challenging. However, valuable therapeutic opportunities can be envisioned on these drugs, as they act through innovative mechanisms in which limitations typically observed for approved ATP-competitive B-Raf inhibitors are less prone to emerge. In this article, current approaches adopted for the design of non-ATP competitive inhibitors targeting B-Raf are described, discussing also on the possibilities, ligands acting through such innovative mechanisms could provide for the obtainment of more effective therapies

Refinement and rescoring of virtual screening results

High-throughput docking is an established computational screening approach in drug design. This methodology enables a rapid identification of biologically active hit compounds, providing an efficient and cost-effective complement or alternative to experimental high-throughput screenings. However, limitations inherent to the methodology make docking results inevitably approximate. Two major Achille’s heels include the use of approximated scoring functions and the limited sampling of the ligand-target complexes. Therefore, docking results require careful evaluation and further post-docking analyses. In this article, we will overview our approach to post-docking analysis in virtual screenings. BEAR (Binding Estimation After Refinement) was developed as a post-docking processing tool that refines docking poses by means of molecular dynamics (MD) and then rescores the ligands based on more accurate scoring functions (MM-PB(GB)SA). The tool has been validated and used prospectively in drug discovery applications. Future directions regarding refinement and rescoring in virtual screening are discussed

Molecular docking: Shifting paradigms in drug discovery

Molecular docking is an established in silico structure-based method widely used in drug discovery. Docking enables the identification of novel compounds of therapeutic interest, predicting ligand-target interactions at a molecular level, or delineating structure-activity relationships (SAR), without knowing a priori the chemical structure of other target modulators. Although it was originally developed to help understanding the mechanisms of molecular recognition between small and large molecules, uses and applications of docking in drug discovery have heavily changed over the last years. In this review, we describe how molecular docking was firstly applied to assist in drug discovery tasks. Then, we illustrate newer and emergent uses and applications of docking, including prediction of adverse effects, polypharmacology, drug repurposing, and target fishing and profiling, discussing also future applications and further potential of this technique when combined with emergent techniques, such as artificial intelligence

Design of dual inhibitors of histone deacetylase 6 and heat shock protein 90

Histone deacetylase 6 (HDAC6) and heat shock protein 90 (Hsp90) are widely investigated anticancer drug targets. Importantly, several lines of evidence indicate that their regulation and activity are intimately linked, and that their combined inhibition may lead to impressive therapeutic benefits. In this study, we developed and applied an integrated computational strategy to design dual inhibitors of HDAC6 and Hsp90. Although the two targets share very little homology, an integrated ligand-based and structure-based virtual screening approach indicated a subset of compounds possessing the key structural requirements for binding at both targets. In vitro tests demonstrated that some of the selected candidates are able to selectively inhibit HDAC6 over HDAC1, to increase the acetylation levels of tubulin on cell assays and to reduce cell proliferation. The discovered compounds represent valuable starting points for further hit optimization

LigAdvisor: A versatile and user-friendly web-platform for drug design

Although several tools facilitating in silico drug design are available, their results are usually difficult to integrate with publicly available information or require further processing to be fully exploited. The rational design of multi-target ligands (polypharmacology) and the repositioning of known drugs towards unmet therapeutic needs (drug repurposing) have raised increasing attention in drug discovery, although they usually require careful planning of tailored drug design strategies. Computational tools and data-driven approaches can help to reveal novel valuable opportunities in these contexts, as they enable to efficiently mine publicly available chemical, biological, clinical, and disease-related data. Based on these premises, we developed LigAdvisor, a data-driven webserver which integrates information reported in DrugBank, Protein Data Bank, UniProt, Clinical Trials and Therapeutic Target Database into an intuitive platform, to facilitate drug discovery tasks as drug repurposing, polypharmacology, target fishing and profiling. As designed, LigAdvisor enables easy integration of similarity estimation results with clinical data, thereby allowing a more efficient exploitation of information in different drug discovery contexts. Users can also develop customizable drug design tasks on their own molecules, by means of ligand- and target-based search modes, and download their results. LigAdvisor is publicly available at https://ligadvisor.unimore.it/

Design and Synthesis of Hsp90 Inhibitors with B-Raf and PDHK1 Multi-Target Activity

5noopenThe design of multi-target ligands has become an innovative approach for the identification of effective therapeutic treatments against complex diseases, such as cancer. Recent studies have demonstrated that the combined inhibition of Hsp90 and B-Raf provides synergistic effects against several types of cancers. Moreover, it has been reported that PDHK1, which presents an ATP-binding pocket similar to that of Hsp90, plays an important role in tumor initiation, maintenance and progression, participating also to the senescence process induced by B-Raf oncogenic proteins. Based on these premises, the simultaneous inhibition of these targets may provide several benefits for the treatment of cancer. In this work, we set up a design strategy including the assembly and integration of molecular fragments known to be important for binding to the Hsp90, PDHK1 and B-Raf targets, aided by molecular docking for the selection of a set of compounds potentially able to exert Hsp90-B-Raf-PDHK1 multi-target activities. The designed compounds were synthesized and experimentally validated in vitro. According to the in vitro assays, compounds 4 a, 4 d and 4 e potently inhibited Hsp90 and moderately inhibited the PDHK1 kinase. Finally, molecular dynamics simulations were performed to provide further insights into the structural basis of their multi-target activity.openPinzi L.; Foschi F.; Christodoulou M.S.; Passarella D.; Rastelli G.Pinzi, L.; Foschi, F.; Christodoulou, M. S.; Passarella, D.; Rastelli, G

2-Phenyloxazole-4-carboxamide as a Scaffold for Selective Inhibition of Human Monoamine Oxidase B

A series of 2-phenyloxazoles bearing an amide group at position 4 were designed and synthesized for evaluation as potential inhibitors of human recombinant monoamine oxidases (hrMAOs). Results of kinetics experiments demonstrated that all compounds behave as competitive MAO inhibitors, with good selectivity toward the MAO-B isoform. The most potent and selective derivatives are characterized by inhibition constant (Ki ) values in the sub-micromolar range and a good selectivity index (Ki\u2009MAO-A /Ki\u2009MAO-B >50). Some derivatives were also found to be able to inhibit MAO activity in nerve growth factor (NGF)-differentiated PC12 cells, taken as a model of neuronal cells. In particular, 2-(2-hydroxyphenyl)-N-phenyloxazole-4-carboxamide (compound 4\u2009a) may be a promising new scaffold, exerting the highest selectivity and inhibitory effect toward MAOs in NGF-differentiated PC12 cell lysates, without compromising cell viability. Molecular docking analysis allowed a rationalization of the experimentally observed binding affinity and selectivity

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections

Promising Non-cytotoxic Monosubstituted Chalcones to Target Monoamine Oxidase-B

A library of monosubstituted chalcones (1-17) bearing electron-donating and electron-withdrawing groups on both aromatic rings were selected. The cell viability on human tumor cell lines was evaluated first. The compounds unable to induce detectable cytotoxicity (1, 13, and 14) were tested using the monoamine oxidase (MAO) activity assay. Interestingly, they inhibit MAO-B, acting as competitive inhibitors, with 13 and 14 showing the best profiles. In particular, 13 exhibited a potency higher than that of safinamide, taken as a reference. Docking studies and crystallographic analysis showed that in human MAO-B 13 binds with the halogen-substituted aromatic ring in the entrance cavity, similar to safinamide, whereas 14 is accommodated in the opposite way. The main conclusion of this cell biology, biochemistry, and structural study is to highlights 13 as a chalcone derivative that is worth consideration for the development of novel MAO-B-selective inhibitors for the treatment of neurodegenerative diseases