PharmaCore: The Automatic Generation of 3D Structure-Based Pharmacophore Models from Protein/Ligand Complexes.

In this work, we present PharmaCore: a new, completely automatic workflow aimed at generating three-dimensional (3D) structure-based pharmacophore models toward any target of interest. The proposed approach relies on using cocrystallized ligands to create the input files for generating the pharmacophore hypotheses, integrating not only the three-dimensional structural information on the ligand but also data concerning the binding mode of these molecules put in the protein cavity. We developed a Python library that, starting from the specific UniProt ID of the protein under investigation as the only element that requires user intervention, subsequently collects and aligns the corresponding structures bearing a known ligand in a fully automated fashion, bringing them all into the same coordinate system. The protocol includes a final phase in which the aligned small molecules are used to produce the pharmacophore hypotheses directly onto the protein structure using a specific software, e.g., Phase (Schrödinger LLC). To validate the entire procedure and highlight the possible applications in the field of drug discovery and repositioning, we first generated pharmacophores for soluble epoxide hydrolase (sEH) and compared with already-published ones. Then, we reproduced the binding profile of a reported selective binder of ATAD2 bromodomain (AM879), testing it against a panel of 1741 pharmacophores related to 16 epigenetic proteins and automatically generated with PharmaCore, finally disclosing putative unprecedented off-targets. The computational predictions were successfully validated with AlphaScreen assays, highlighting the applicability of the proposed workflow in drug discovery and repositioning. Finally, the process was also validated on tankyrase 2 and SARS-CoV-2 MPro, confirming the robustness of PharmaCore.

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 binding, 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 interfere with sEH activity, leading to inhibition percentages 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.

Identification and Development of BRD9 Chemical Probes.

The development of BRD9 inhibitors involves the design and synthesis of molecules that can specifically bind the BRD9 protein, interfering with the function of the chromatin-remodeling complex ncBAF, with the main advantage of modulating gene expression and controlling cellular processes. Here, we summarize the work conducted over the past 10 years to find new BRD9 binders, with an emphasis on their structure-activity relationships, efficacies, and selectivities in preliminary studies. BRD9 is expressed in a variety of cancer forms, hence, its inhibition holds particular significance in cancer research. However, it is crucial to note that the expanding research in the field, particularly in the development of new degraders, may uncover new therapeutic potentials.

2-Substituted 1,5-benzothiazepine-based HDAC inhibitors exert anticancer activities on human solid and acute myeloid leukemia cell lines.

Herein, we report the development of a new series of histone deacetylase inhibitors (HDACi) containing a 2-substituted 1,5-benzothiazepine scaffold. First, a virtual combinatorial library (∼1.6 × 103 items) was built according to a convenient synthetic route, and then it was submitted to molecular docking experiments on seven HDACs isoforms belonging to classes I and II. Integrated computational filters were used to select the most promising ones that were synthesized through an optimized approach, also amenable to generating both racemic and enantioenriched benzothiazepine-based derivatives. The obtained compounds showed potent HDAC inhibitory activity, especially those containing the sulphone moiety, endowed with IC50 in the nanomolar range. In addition, in vitro outcomes of our synthesized compounds demonstrated a cytotoxic effect on U937 and HCT116 cell lines and an arrest in the G2/M phase (13 ≤ IC50 ≤ 18 µM). Finally, Western blot analyses outlined the modulation of the histone acetyl markers such as H3K9/14, acetyl-tubulin, and the apoptotic indicator p21 in both cancer cell lines, disclosing a good HDAC inhibitor activity exerted by the designed items. Given the key role of HDACs in many cellular pathways, which makes these enzymes appealing and "hot" drug targets, our findings highlighted the importance of these 2-substituted 1,5-benzothiazepine-based compounds (both in the reduced and oxidized version) for the development of novel epidrugs.

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.

Targeting mPGES-1 by a Combinatorial Approach: Identification of the Aminobenzothiazole Scaffold to Suppress PGE2 Levels.

Microsomal prostaglandin E2 synthase-1 (mPGES-1), the terminal enzyme responsible for the production of inducible prostaglandin E2, has become an attractive target for the treatment of inflammation and cancer pathologies. Starting from an aminobenzothiazole scaffold, used as an unprecedented chemical core for mPGES-1 inhibition, a Combinatorial Virtual Screening campaign was conducted, using the X-ray crystal structure of human mPGES-1. Two combinatorial libraries (6 × 104) were obtained by decorating the aminobenzothiazole scaffold with all acyl chlorides and boronates available at the Merck database. The scientific multidisciplinary approach included virtual screening workflow, synthesis, and biological evaluation and led to the identification of three novel aminobenzothiazoles 1, 3, and 13 acting as mPGES-1 inhibitors. The three disclosed hits are able to inhibit mPGES-1 in a cell-free system (IC50 = 1.4 ± 0.2, 0.7 ± 0.1, and 1.7 ± 0.2 µM, respectively), and all are endowed with antitumoral properties against A549 human cancer cell lines at micromolar concentrations (28.5 ± 1.1, 18.1 ± 0.8, and 19.2 ± 1.3 µM, respectively).