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.

Chemoproteomics Reveals USP5 (Ubiquitin Carboxyl-Terminal Hydrolase 5) as Promising Target of the Marine Polyketide Gracilioether A.

Mass spectrometry-based chemical proteomic approaches using limited proteolysis have become a powerful tool for the identification and analysis of the interactions between a small molecule (SM) and its protein target(s). Gracilioether A (GeA) is a polyketide isolated from a marine sponge, for which we aimed to trace the interactome using this strategy. DARTS (Drug Affinity Responsive Target Stability) and t-LiP-MS (targeted-Limited Proteolysis-Mass Spectrometry) represented the main techniques used in this study. DARTS was applied on HeLa cell lysate for the identification of the GeA target proteins, and t-LiP-MS was employed to investigate the protein's regions involved in the binding with GeA. The results were complemented through the use of binding studies using Surface Plasmon Resonance (SPR) and in silico molecular docking experiments. Ubiquitin carboxyl-terminal hydrolase 5 (USP5) was identified as a promising target of GeA, and the interaction profile of the USP5-GeA complex was explained. USP5 is an enzyme involved in the pathway of protein metabolism through the disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. This activity is connected to different cellular functions concerning the maintenance of chromatin structure and receptors and the degradation of abnormal proteins and cancerogenic progression. On this basis, this structural information opens the way to following studies focused on the definition of the biological potential of Gracilioether A and the rational development of novel USP5 inhibitors based on a new structural skeleton.

Fatty Acid Synthase as Interacting Anticancer Target of the Terpenoid Myrianthic Acid Disclosed by MS-Based Proteomics Approaches.

This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of Myrianthus arboreus and from Oenothera maritima Nutt. (Onagraceae), using MS-based chemical proteomic techniques. Application of drug affinity responsive target stability (DARTS) and targeted-limited proteolysis coupled to mass spectrometry (t-LiP-MS) led to the identification of the enzyme fatty acid synthase (FAS) as an interesting macromolecular counterpart of myrianthic acid. This result, confirmed by comparison with the natural ursolic acid, was thoroughly investigated and validated in silico by molecular docking, which gave a precise picture of the interactions in the MA/FAS complex. Moreover, biological assays showcased the inhibitory activity of myrianthic acid against the FAS enzyme, most likely related to its antiproliferative activity towards tumor cells. Given the significance of FAS in specific pathologies, especially cancer, the myrianthic acid structural moieties could serve as a promising reference point to start the potential development of innovative approaches in therapy.

Target identification by structure-based computational approaches: Recent advances and perspectives.

The use of computational techniques in the early stages of drug discovery has recently experienced a boost, especially in the target identification step. Finding the biological partner(s) for new or existing synthetic and/or natural compounds by "wet" approaches may be challenging; therefore, preliminary in silico screening is even more recommended. After a brief overview of some of the most known target identification techniques, recent advances in structure-based computational approaches for target identification are reported in this digest, focusing on Inverse Virtual Screening and its recent applications. Moreover, future perspectives concerning the use of such methodologies, coupled or not with other approaches, are analyzed.

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.

A Classic Photochemical Approach Inducing an Unexpected Rearrangement: Exploring the Photoreactivity of Pentacyclic Triterpenic Acids.

The discovery of new bioactivities is closely related to the generation of novel scaffolds, and in the past few years different strategies have been proposed to obtain unknown architectures from the manipulation of known compounds. In the present study, we exploited a vintage photochemical approach for the discovery of an unexpected pathway of reactivity related to Δ1-3-oxo-pentacyclic triterpenic acids gaining access to a new class of natural-unnatural 5(10→1)abeo-pentacyclic triterpenic acids.

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.

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.

Structural Refinement of 2,4-Thiazolidinedione Derivatives as New Anticancer Agents Able to Modulate the BAG3 Protein.

The multidomain BAG3 protein is a member of the BAG (Bcl-2-associated athanogene) family of co-chaperones, involved in a wide range of protein-protein interactions crucial for many key cellular pathways, including autophagy, cytoskeletal dynamics, and apoptosis. Basal expression of BAG3 is elevated in several tumor cell lines, where it promotes cell survival signaling and apoptosis resistance through the interaction with many protein partners. In addition, its role as a key player of several hallmarks of cancer, such as metastasis, angiogenesis, autophagy activation, and apoptosis inhibition, has been established. Due to its involvement in malignant transformation, BAG3 has emerged as a potential and effective biological target to control multiple cancer-related signaling pathways. Recently, by using a multidisciplinary approach we reported the first synthetic BAG3 modulator interfering with its BAG domain (BD), based on a 2,4-thiazolidinedione scaffold and endowed with significant anti-proliferative activity. Here, a further in silico-driven selection of a 2,4-thiazolidinedione-based compound was performed. Thanks to a straightforward synthesis, relevant binding affinity for the BAG3BD domain, and attractive biological activities, this novel generation of compounds is of great interest for the development of further BAG3 binders, as well as for the elucidation of the biological roles of this protein in tumors. Specifically, we found compound 6 as a new BAG3 modulator with a relevant antiproliferative effect on two different cancer cell lines (IC50: A375 = 19.36 µM; HeLa = 18.67 µM).

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.

Giffonins, Antioxidant Diarylheptanoids from Corylus avellana, and Their Ability to Prevent Oxidative Changes in Human Plasma Proteins.

With the aim to explore the ability of diarylheptanoids to reduce oxidative changes in human plasma proteins, a phytochemical investigation of the MeOH extract of Corylus avellana leaves was perfomed. Analysis by LC-ESI/LTQOrbitrap/MS/MSn guided the isolation of two new diarylheptanoid derivatives, giffonins W (1) and X (2). The structures 1 and 2 were assigned by analysis of NMR data combined with a QM (quantum mechanical)/NMR approach. The absolute configurations of 1 and 2 were established by analysis of electronic circular dichroism (ECD) spectra compared with the TDDFT-simulated curves. The antioxidant activity of the new and known giffonins was evaluated by inhibition of human plasma lipid peroxidation. Giffonins with the highest inhibitory activity were tested for their ability to reduce oxidation of thiol groups and carbonylation in plasma proteins, and some of them exhibited higher antioxidant activity than curcumin.

Inverse Virtual Screening for the rapid re-evaluation of the presumed biological safe profile of natural products. The case of steviol from Stevia rebaudiana glycosides on farnesoid X receptor (FXR).

Nonnutritive sweeteners (NNSs) are widely employed as dietary substitutes for classical sugars thanks to their safety profile and low toxicity. In this study, a re-evaluation of the biological effects of steviol (1), the main metabolite from Stevia rebaudiana glycosides, was performed using the Inverse Virtual Screening (IVS) target fishing computational approach. Starting from well-known pharmacological properties of Stevia rebaudiana glycosides, this computational tool was employed for predicting the putative interacting targets of 1 and, afterwards, of its five synthetic ester derivatives 2-6, accounting a large panel of proteins involved in cancer and inflammation events. Applying this methodology, the farnesoid X receptor (FXR) was identified as the putative target partner of 1-6. The predicted ligand-protein interactions were corroborated by transactivation assays, specifically disclosing the agonistic activity of 1 and the antagonistic activities of 2-6 on FXR. The reported results highlight the feasibility of IVS as a fast and potent tool for predicting the interacting targets of query compounds, addressing the re-evaluation of their bioactivity. In light of the obtained results, the presumably safe profile of known compounds, such as the case of steviol (1), is critically discussed.

Neurally adjusted ventilatory assist preserves cerebral blood flow velocity in patients recovering from acute brain injury.

Neurally adjusted ventilatory assist (NAVA) has never been applied in patients recovering from acute brain injury (ABI) because neural respiratory drive could be affected by intracranial disease with detrimental effects on cerebral blood flow (CBF) velocity. Our primary aim was to assess the impact of NAVA and pressure support ventilation (PSV) on CBF velocity. In fifteen adult patients recovering from ABI and undergoing invasive assisted ventilation, PSV and NAVA were applied over 30-min-lasting trials, in the following sequence: PSV1, NAVA, and PSV2. While PSV was set to deliver a tidal volume ranging between 6 and 8 ml kg-1 of predicted body weight, in NAVA the level of assistance was chosen to achieve the same inspiratory peak airway pressure as PSV. At the end of each trial, a sonographic evaluation of CBF mean velocity was bilaterally obtained on the middle cerebral artery and an arterial blood gas sample was taken for analysis. CBF mean velocity was 51.8 [41.9,75.2] cm  s-1 at baseline, 51.9 [43.4,71.0] cm s-1 in PSV1, 53.6 [40.7,67.7] cm s-1 in NAVA, and 49.5 [42.1,70.8] cm s-1 in PSV2 (p = 0.0514) on the left and 50.2 [38.0,77.7] cm s-1 at baseline, 47.8 [41.7,68.2] cm s-1 in PSV1, 53.9 [40.1,78.5] cm s-1 in NAVA, and 55.6 [35.9,74.1] cm s-1 in PSV2 (p = 0.8240) on the right side. No differences were detected for pH (p = 0.0551), arterial carbon dioxide tension (p = 0.8142), and oxygenation (p = 0.0928) over the entire study duration. NAVA and PSV preserved CBF velocity in patients recovering from ABI.Trial registration: The present trial was prospectively registered at www.clinicatrials.gov (NCT03721354) on October 18th, 2018.

Insights into the Ligand Binding to Bromodomain-Containing Protein 9 (BRD9): A Guide to the Selection of Potential Binders by Computational Methods.

The estimation of the binding of a set of molecules against BRD9 protein was carried out through an in silico molecular dynamics-driven exhaustive analysis to guide the identification of potential novel ligands. Starting from eight crystal structures of this protein co-complexed with known binders and one apo form, we conducted an exhaustive molecular docking/molecular dynamics (MD) investigation. To balance accuracy and an affordable calculation time, the systems were simulated for 100 ns in explicit solvent. Moreover, one complex was simulated for 1 µs to assess the influence of simulation time on the results. A set of MD-derived parameters was computed and compared with molecular docking-derived and experimental data. MM-GBSA and the per-residue interaction energy emerged as the main indicators for the good interaction between the specific binder and the protein counterpart. To assess the performance of the proposed analysis workflow, we tested six molecules featuring different binding affinities for BRD9, obtaining promising outcomes. Further insights were reported to highlight the influence of the starting structure on the molecular dynamics simulations evolution. The data confirmed that a ranking of BRD9 binders using key parameters arising from molecular dynamics is advisable to discard poor ligands before moving on with the synthesis and the biological tests.

Mechanical Ventilation Guided by Uncalibrated Esophageal Pressure May Be Potentially Harmful.

BACKGROUND: Esophageal balloon calibration was proposed in acute respiratory failure patients to improve esophageal pressure assessment. In a clinical setting characterized by a high variability of abdominal load and intrathoracic pressure (i.e., pelvic robotic surgery), the authors hypothesized that esophageal balloon calibration could improve esophageal pressure measurements. Accordingly, the authors assessed the impact of esophageal balloon calibration compared to conventional uncalibrated approach during pelvic robotic surgery. METHODS: In 30 adult patients, scheduled for elective pelvic robotic surgery, calibrated end-expiratory and end-inspiratory esophageal pressure, and the associated respiratory variations were obtained at baseline, after pneumoperitoneum-Trendelenburg application, and with positive end-expiratory pressure (PEEP) administration and compared to uncalibrated values measured at 4-ml filling volume, as per manufacturer recommendation. Data are expressed as median and [25th, 75th percentile]. RESULTS: Ninety calibrations were successfully performed. Chest wall elastance worsened with pneumoperitoneum-Trendelenburg and PEEP (19.0 [15.5, 24.6] and 16.7 [11.4, 21.7] cm H2O/l) compared to baseline (8.8 [6.3, 9.8] cm H2O/l; P < 0.0001 for both comparisons). End-expiratory and end-inspiratory calibrated esophageal pressure progressively increased from baseline (3.7 [2.2, 6.0] and 7.7 [5.9, 10.2] cm H2O) to pneumoperitoneum-Trendelenburg (6.2 [3.8, 10.2] and 16.1 [13.1, 20.6] cm H2O; P = 0.014 and P < 0.001) and PEEP (8.8 [7.7, 15.6] and 18.9 [16.3, 22.0] cm H2O; P < 0.0001 vs. baseline for both comparison; P < 0.001 and P = 0.002 vs. pneumoperitoneum-Trendelenburg) and, at each study step, they were persistently lower than uncalibrated esophageal pressure (P < 0.0001 for all comparisons). Overall, difference among uncalibrated and calibrated esophageal pressure was 5.1 [3.8, 8.4] cm H2O at end-expiration and 3.8 [3.0, 6.3] cm H2O at end-inspiration. Uncalibrated esophageal pressure swing was always lower than calibrated one (P < 0.0001 for all comparisons) with a difference of -1.0 [-1.8, -0.4] cm H2O. CONCLUSIONS: In a clinical setting with variable chest wall mechanics, uncalibrated measurements substantially overestimated absolute values and underestimated respiratory variations of esophageal pressure. Calibration could substantially improve mechanical ventilation guided by esophageal pressure.

DFT/NMR Approach for the Configuration Assignment of Groups of Stereoisomers by the Combination and Comparison of Experimental and Predicted Sets of Data.

Quantum mechanical/nuclear magnetic resonance (NMR) approaches are widely used for the configuration assignment of organic compounds generally comparing one cluster of experimentally determined data (e.g., 13C NMR chemical shifts) with those predicted for all possible theoretical stereoisomers. More than one set of experimental data, each related to a specific stereoisomer, may occur in some cases, and the accurate stereoassignments can be obtained by combining the experimental and computed data. We introduce here a straightforward methodology based on the simultaneous analysis, combination, and comparison of all sets of experimental/calculated 13C chemical shifts for aiding the correct configuration assignment of groups of stereoisomers. The comparison of the differences between the calculated/experimental chemical shifts instead of the shifts themselves led to the advantage of avoiding errors arising from calibration procedures, reducing systematic errors, and highlighting the most diagnostic differences between calculated and experimental data. This methodology was applied on a tetrad of synthesized cladosporin stereoisomers (cladologs) and further corroborated on a tetrad of pochonicine stereoisomers, obtaining the correct correspondences between experimental and calculated sets of data. The new MAEΔΔδ parameter, useful for indicating the best fit between sets of experimental and calculated data, is here introduced for facilitating the stereochemical assignment of groups of stereoisomers.

Discovery of noscapine derivatives as potential ß-tubulin inhibitors.

Twenty novel 1,2,3-triazole noscapine derivatives were synthesized starting from noscapine by consecutive N-demethylation, reduction of lactone ring, N-propargylation and Huisgen 1,3-dipolar cycloaddition reaction. In order to select the most promising molecules to subject to further biophysical and biological evaluation, a molecular docking analysis round was performed using noscapine as reference compound. The molecules featuring docking predicted binding affinity better than that of noscapine were then subjected to MTT assay against MCF7 cell line. The obtained results disclosed that all the selected triazole derivatives exhibited a remarkably lower cell viability compared to noscapine in the range of 20 µM in 48 h. In an attempt to correlate the biological activity with the ability to bind tubulin, the surface plasmon resonance (SPR) assay was employed. Compounds 8a, 8h, 9c, 9f and 9j were able to bind tubulin with affinity constant values in the nanomolar range and higher if compared to noscapine. Integrating computational predictions and experimental evaluation, two promising compounds (8h and 9c) were identified, whose relevant cytotoxicity was supposed to be correlated with tubulin binding affinity. These findings shed lights onto structural modifications of noscapine toward the identification of more potent cytotoxic agents targeting tubulin.

Oesophageal balloon calibration during pressure support ventilation: a proof of concept study.

Oesophageal balloon calibration improves the oesophageal pressure (Pes) assessment during invasive controlled mechanical ventilation. The primary aim of the present investigation was to ascertain the feasibility of oesophageal balloon calibration during pressure support ventilation (PSV). Secondarily, the calibrated Pes (Pescal) was compared to uncalibrated one acquired at 4 ml-filling volume (PesV4), as per manufacturer recommendation. After a naso-gastric tube equipped with oesophageal balloon was correctly positioned in 21 adult patients undergoing invasive volume-controlled ventilation (VCV) for acute hypoxemic respiratory failure, the balloon was progressively inflated, applying a series of end-inspiratory and end-expiratory holds at each filling volume during VCV and PSV. Upon optimal balloon filling volume (Vbest) was identified, Pescal was computed by correcting the Pes measured at Vbest for the oesophageal wall pressure elicited at same filling volume. Finally, end-expiratory and end-inspiratory PesV4 were recorded too. A total of 42 calibrations, 21 per ventilatory mode, were performed. Vbest was 1.9 ± 1.6 ml in VCV and 1.7 ± 1.6 ml in PSV (p = 0.5217). PesV4 was overestimated compared to Pescal at end-expiration and end-inspiration (p <0.0001 for all comparisons) in both VCV (13.4 ± 3.4 cmH2O and 15.4 ± 3 cmH2O vs. 8.5 ± 2.9 cmH2O and 11.4 ± 3 cmH2O) and PSV (14.7 ± 4.2 cmH2O and 17 ± 3.9 cmH2O vs. 8.9 ± 3.4 cmH2O and 12.4 ± 3.9 cmH2O). In PSV, oesophageal balloon calibration is feasible and allows to obtain a reliable Pes assessment compared to uncalibrated approach.

Protein Preparation Automatic Protocol for High-Throughput Inverse Virtual Screening: Accelerating the Target Identification by Computational Methods.

Structure-based virtual screening is highly used in the early stages of drug discovery to identify new putative lead compounds for a given target. However, when a small molecule elicits a biological effect, but its target is unknown, or the side effects it causes arise from its undesired interaction with unknown counterparts, the identification of its interacting targets represents an indispensable task. The computational procedure named inverse virtual screening, which relies on docking a molecule (or a small set of compounds) against panels of target proteins to select the most promising complexes, could be useful to overcome these issues. Panels can contain thousands of proteins, and they must be correctly prepared to assure the best docking performance. Therefore, the preparation of panels of proteins collected in the Protein Data Bank ( www.rcsb.org ), if manually performed, may be costly in terms of time and efforts, and this can limit the applicability of this approach in high-throughput virtual screening workflows. We here show an automated workflow to speed up panel preparation and development, and to test its performance, this protocol was initially applied to a panel of 628 viral proteins and, afterward, to a panel of transferase proteins (2789 entries) to perform a large inverse virtual screening study, testing a small set of compounds synthesized in our laboratory. Tankyrase 2 (PARP 5b) was selected as their preferred target of interaction, and the predicted binding was validated by means of surface plasmon resonance experiments. This protocol is useful for the rapid identification of the interacting target for a bioactive compound; accordingly, it facilitates the re-evaluation of the pharmacological activity of known active compounds, addressing the repurposing and the polypharmacology concepts.

Immunomodulatory Biscembranoids and Assignment of Their Relative and Absolute Configurations: Data Set Modulation in the Density Functional Theory/Nuclear Magnetic Resonance Approach.

Five new biscembranoids, bistrochelides A-E (3-7), were isolated together with glaucumolides A (1) and B (2) from the soft coral Sarcophyton trocheliophorum. Their structures and absolute configurations were determined by spectroscopic methods, X-ray crystal diffraction, and DFT/NMR (density functional theory/nuclear magnetic resonance) and TDDFT/ECD (time-dependent density functional theory/electronic circular dichroism) calculations. A new approach is introduced to determine the relative configuration of a stereocenter through the dynamic evaluation of the mean absolute errors (MAEs) between the investigated diastereoisomers, moving from an "extended" to a more diagnostic "restricted" set of atoms. This research leads to the structure revision of glaucumolides A and B. In in vitro immunomodulatory screening, compounds 1 and 4 significantly induced the proliferation of CD3+ T cells, while compounds 1 and 5 significantly increased the CD4+/CD8+ ratio at 3 µM.