Repositioning of Small Molecules through the Inverse Virtual Screening in silico Tool: Case of Benzothiazole-Based Inhibitors of Soluble Epoxide Hydrolase (sEH).

Computational techniques accelerate drug discovery by identifying bioactive compounds for specific targets, optimizing molecules with moderate activity, or facilitating the repositioning of inactive items onto new targets. Among them, the Inverse Virtual Screening (IVS) approach is aimed at the evaluation of one or a small set of molecules against a panel of targets for addressing target identification. In this work, a focused library of benzothiazole-based compounds was re-investigated by IVS. Four items, originally synthesized and tested on bromodomain-containing protein 9 (BRD9) but yielding poor results, were critically re-analyzed, disclosing only a partial fit with 3D structure-based pharmacophore models, which, in the meanwhile, were developed for this target. Afterwards, these compounds were re-evaluated through IVS on a panel of proteins involved in inflammation and cancer, identifying soluble epoxide hydrolase (sEH) as a putative interacting target. Three items were subsequently confirmed as able to inhibit sEH activity, spanning from 70% up to 30% when tested at 10 µM. Finally, one benzothiazole-based compound emerged as the most promising inhibitor featuring an IC50 in the low micromolar range (IC50 = 6.62 ± 0.13 µM). Our data confirm IVS as a predictive tool for accelerating the target identification and repositioning processes.

Prodrug Approach to Exploit (S) Alanine as Arginine Mimic Moiety in the Development of Protein Arginine Methyltransferase 4 Inhibitors.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as an emerging target class in oncology and other diseases. A successful strategy to identify PRMT substrate-competitive inhibitors has been to exploit chemical scaffolds able to mimic the arginine substrate. (S)-Alanine amide moiety is a valuable arginine mimic for the development of potent and selective PRMT4 inhibitors; however, its high hydrophilicity led to derivatives with poor cellular outcomes. Here, we describe the development of PRMT4 inhibitors featuring a central pyrrole core and an alanine amide moiety. Rounds of optimization, aimed to increase lipophilicity and simultaneously preserve the inhibitory activity, produced derivatives that, despite good potency and physicochemical properties, did not achieve on-target effects in cells. On the other hand, masking the amino group with a NAD(P)H:quinone oxidoreductase 1 (NQO1)-responsive trigger group, led to prodrugs able to reduce arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155). These results indicate that prodrug strategies can be successfully applied to alanine-amide containing PRMT4 inhibitors and provide an option to enable such compounds to achieve sufficiently high exposures in vivo.

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents.

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi-preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1-5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1-5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues.

Exploring the chemical space of functionalized [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the bromodomain of BRD9.

Here we report a detailed structure-activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1-4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported "pharm-druglike2" 3D structure-based pharmacophore model, introducing it as "pharm-druglike2.1". Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

Identification of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based small molecules as selective BRD9 binders.

Targeting bromodomain-containing protein 9 (BRD9) represents a promising strategy for the development of new agents endowed with anticancer properties. With this aim, a set of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based compounds was investigated following a combined approach that relied on in silico studies, chemical synthesis, biophysical and biological evaluation of the most promising items. The protocol was initially based on molecular docking experiments, accounting a library of 1896 potentially synthesizable items tested in silico against the bromodomain of BRD9. A first set of 21 compounds (1-21) was selected and the binding on BDR9 was assessed through AlphaScreen assays. The obtained results disclosed compounds 17 and 20 able to bind BRD9 in the submicromolar range (IC50 = 0.35 ± 0.18 µM and IC50 = 0.14 ± 0.03 µM, respectively) showing a promising selectivity profile when tested against further nine bromodomains. Taking advantage of 3D structure-based pharmacophore models, additional 10 derivatives were selected in silico for the synthetic step and binding assessment, highlighting seven compounds (22, 23, 25, 26, 28, 29, 31) able to selectively bind BRD9 among different bromodomains. The ability of the identified BRD9 binders to cross artificial membranes in vitro was also assessed, revealing a very good passive permeability profile. Preliminary studies were carried out on a panel of healthy and cancer human cell lines to explore the biological behavior of the selected compounds, disclosing a moderate activity and significant selectivity profile towards leukaemia cells. These results highlighted the applicability of the reported multidisciplinary approach for accelerating the selection of promising items and for driving the chemical synthesis of novel selective BRD9 binders. Moreover, the low molecular weight of the reported 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based BRD9 binders suggests the possibility for further exploring the chemical space in order to obtain new analogues with improved potency.

Repositioning of Quinazolinedione-Based Compounds on Soluble Epoxide Hydrolase (sEH) through 3D Structure-Based Pharmacophore Model-Driven Investigation.

The development of new bioactive compounds represents one of the main purposes of the drug discovery process. Various tools can be employed to identify new drug candidates against pharmacologically relevant biological targets, and the search for new approaches and methodologies often represents a critical issue. In this context, in silico drug repositioning procedures are required even more in order to re-evaluate compounds that already showed poor biological results against a specific biological target. 3D structure-based pharmacophoric models, usually built for specific targets to accelerate the identification of new promising compounds, can be employed for drug repositioning campaigns as well. In this work, an in-house library of 190 synthesized compounds was re-evaluated using a 3D structure-based pharmacophoric model developed on soluble epoxide hydrolase (sEH). Among the analyzed compounds, a small set of quinazolinedione-based molecules, originally selected from a virtual combinatorial library and showing poor results when preliminarily investigated against heat shock protein 90 (Hsp90), was successfully repositioned against sEH, accounting the related built 3D structure-based pharmacophoric model. The promising results here obtained highlight the reliability of this computational workflow for accelerating the drug discovery/repositioning processes.

Introducing structure-based three-dimensional pharmacophore models for accelerating the discovery of selective BRD9 binders.

A well-structured in silico workflow is here reported for disclosing structure-based pharmacophore models against bromodomain-containing protein 9 (BRD9), accelerating virtual screening campaigns and facilitating the identification of novel binders. Specifically, starting from 23 known ligands co-crystallized with BRD9, three-dimensional pharmacophore models, namely placed in a reference protein structure, were developed. Specifically, we here introduce a fragment-related pharmacophore model, useful for the identification of new promising small chemical probes targeting the protein region responsible of the acetyllysine recognition, and two further pharmacophore models useful for the selection of compounds featuring drug-like properties. A pharmacophore-driven virtual screening campaign was then performed to facilitate the selection of new selective BRD9 ligands, starting from a large library of commercially available molecules. The identification of a promising BRD9 binder (7) prompted us to re-iterate this computational workflow on a second focused in-house built library of synthesizable compounds and, eventually, three further novel BRD9 binders were disclosed (8-10). Moreover, all these compounds were tested among a panel comprising other nine bromodomains, showing a high selectivity for BRD9. Preclinical bioscreens for potential anticancer activity highlighted compound 7 as that showing the most promising biological effects, proving the reliability of this in silico pipeline and confirming the applicability of the here introduced structure-based three-dimensional (3D) pharmacophore models as straightforward tools for the selection of new BRD9 ligands.