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.

Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors.

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

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.

Structure-based screening for the discovery of 1,2,4-oxadiazoles as promising hits for the development of new anti-inflammatory agents interfering with eicosanoid biosynthesis pathways.

The multiple inhibition of biological targets involved in pro-inflammatory eicosanoid biosynthesis represents an innovative strategy for treating inflammatory disorders in light of higher efficacy and safety. Herein, following a multidisciplinary protocol involving virtual combinatorial screening, chemical synthesis, and in vitro and in vivo validation of the biological activities, we report the identification of 1,2,4-oxadiazole-based eicosanoid biosynthesis multi-target inhibitors. The multidisciplinary scientific approach led to the identification of three 1,2,4-oxadiazole hits (compounds 1, 2 and 5), all endowed with IC50 values in the low micromolar range, acting as 5-lipoxygenase-activating protein (FLAP) antagonists (compounds 1 and 2), and as a multi-target inhibitor (compound 5) of arachidonic acid cascade enzymes, namely cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1). Moreover, our in vivo results demonstrate that compound 5 is able to attenuate leukocyte migration in a model of zymosan-induced peritonitis and to modulate the production of IL-1ß and TNF-α. These results are of interest for further expanding the chemical diversity around the 1,2,4-oxadiazole central core, enabling the identification of novel anti-inflammatory agents characterized by a favorable pharmacological profile and considering that moderate interference with multiple targets might have advantages in re-adjusting homeostasis.

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).

Identification of the 2-Benzoxazol-2-yl-phenol Scaffold as New Hit for JMJD3 Inhibition.

JMJD3 is a member of the KDM6 subfamily and catalyzes the demethylation of lysine 27 on histone H3 (H3K27). This protein was identified as a useful tool in understanding the role of epigenetics in inflammatory conditions and in cancer as well. Guided by a virtual fragment screening approach, we identified the benzoxazole scaffold as a new hit suitable for the development of tighter JMJD3 inhibitors. Compounds were synthesized by a microwave-assisted one-pot reaction under catalyst and solvent-free conditions. Among these, compound 8 presented the highest inhibitory activity (IC50 = 1.22 ± 0.22 µM) in accordance with molecular modeling calculations. Moreover, 8 induced the cycle arrest in S-phase on A375 melanoma cells.

Discovery of new erbB4 inhibitors: Repositioning an orphan chemical library by inverse virtual screening.

Inverse Virtual Screening (IVS) is a docking based approach aimed to the evaluation of the virtual ability of a single compound to interact with a library of proteins. For the first time, we applied this methodology to a library of synthetic compounds, which proved to be inactive towards the target they were initially designed for. Trifluoromethyl-benzenesulfonamides 3-21 were repositioned by means of IVS identifying new lead compounds (14-16, 19 and 20) for the inhibition of erbB4 in the low micromolar range. Among these, compound 20 exhibited an interesting value of IC50 on MCF7 cell lines, thus validating IVS in lead repurposing.

Virtual Fragment Screening Identification of a Quinoline-5,8-dicarboxylic Acid Derivative as a Selective JMJD3 Inhibitor.

The quinoline-5,8 dicarboxylic acid scaffold has been identified by a fragment-based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3-(2,4-dimethoxypyrimidin-5-yl)quinoline-5,8-dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain-containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.

Fulvitalea axinellae gen. nov., sp. nov., a member of the family Flammeovirgaceae isolated from the Mediterranean sponge Axinella verrucosa.

The yellow-pigmented, non-motile, Gram-negative, strictly aerobic, rod-shaped bacterial strain VI.18(T) was isolated from the Mediterranean sponge Axinella verrucosa collected off the coast near Sdot Yam, Israel. Results from 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flammeovirgaceae. The highest nucleotide similarity (91.4 %) occurred with Aureibacter tunicatorum A5Q-118(T). The predominant cellular fatty acids of strain VI.18(T) were iso-C15 : 0 (56.0 %), iso-C17 : 1ω9c (22.8 %) and C16 : 0 (7.4 %) and its major respiratory quinone was MK-7. The DNA G+C content was 47.5 mol%. The strain could readily be distinguished from its phylogenetically closest relatives by phenotypic, physiological and chemotaxonomic properties. On the basis of the data from the present polyphasic study, we propose a novel genus and species within the family Flammeovirgaceae, with the name Fulvitalea axinellae gen. nov., sp. nov. Strain VI.18(T) ( = ATCC BAA-2395(T)  = LMG 26722(T)) is the type strain of Fulvitalea axinellae.

Luteivirga sdotyamensis gen. nov., sp. nov., a novel bacterium of the phylum Bacteroidetes isolated from the Mediterranean sponge Axinella polypoides.

A novel aerobic bacterium, designated strain PIII.02(T), was isolated from a Mediterranean sponge (Axinella polypoides) collected off the Israeli coast near Sdot Yam. The non-motile cells were Gram-staining-negative, oxidase-positive and catalase-positive. The orange pigment of colonies growing on marine agar was neither diffusible nor flexirubin-like. Strain PIII.02(T) grew at 15-35 °C, at pH 6.0-9.0, with 2.0-7.0 % (w/v) NaCl, and with 1.0-8.0 % (w/v) sea salts. The predominant fatty acids were iso-C15 : 0, iso-C16 : 1 H, iso-C16 : 0, C16 : 0, anteiso-C15 : 0 and C16 : 1ω7c. The major respiratory quinone was MK-7. The genomic DNA G+C content of the novel strain was 38.1 mol%. Results from 16S rRNA gene sequence analysis indicated that strain PIII.02(T) was distantly related to established members of the phylum Bacteroidetes. The established species found to be most closely related to the novel strain was Persicobacter diffluens NCIMB 1402(T) (87.6 % 16S rRNA gene sequence similarity). Based on the phenotypic and chemotaxonomic data and the results of the phylogenetic analyses, strain PIII.02(T) represents a novel species of a new genus, for which the name Luteivirga sdotyamensis gen. nov., sp. nov. is proposed. The type strain is PIII.02(T) ( = ATCC BAA-2393(T)  = LMG 26723(T)).

Aureivirga marina gen. nov., sp. nov., a marine bacterium isolated from the Mediterranean sponge Axinella verrucosa.

Two bacterial strains, VI.14 and VIII.04(T), were isolated from the Mediterranean sponge Axinella verrucosa collected off the Israeli coast near Sdot Yam. The non-motile, aerobic, Gram-negative isolates were oxidase-negative and catalase-positive, and formed golden-brown colonies on marine agar 2216. The pigment was neither diffusible nor flexirubin-like. Strain VIII.04(T) grew at 15-37 °C, at pH 6.0-9.0, in the presence of 20-50 g NaCl l(-1) and 20-80 g sea salts l(-1), The spectrum was narrower for strain VI.14, with growth at pH 7.0-8.0. and in the presence of 30-50 g NaCl l(-1) and 30-70 g sea salts l(-1). The predominant fatty acid (>50 %) in both strains was iso-C15 : 0, and the major respiratory quinone was MK-6. The DNA G+C content was 30.7 and 31.1 mol% for VIII.04(T) and VI.14, respectively. Results from 16S rRNA sequence similarity and phylogenetic analyses indicated that both strains are closely related to members of the family Flavobacteriaceae within the phylum Bacteroidetes, with as much as 91.7 % 16S rRNA sequence similarity. On the basis of data from the polyphasic analysis, we suggest that the strains represent a novel species in a new genus within the family Flavobacteriaceae, for which the name Aureivirga marina gen. nov., sp. nov. is proposed. Strain VIII.04(T) ( = ATCC BAA-2394(T) = LMG 26721(T)) is the type strain of Aureivirga marina.

Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation.

Phosphomannomutase 2 (PMM2) deficiency represents the most frequent type of congenital disorders of glycosylation. For this disease there is no cure at present. The complete loss of phosphomannomutase activity is probably not compatible with life and people affected carry at least one allele with residual activity. We characterized wild-type PMM2 and its most common hypomorphic mutant, p.F119L, which is associated with a severe phenotype of the disease. We demonstrated that active species is the dimeric enzyme and that the mutation weakens the quaternary structure and, at the same time, affects the activity and the stability of the enzyme. We demonstrated that ligand binding stabilizes both proteins, wild-type and F119L-PMM2, and promotes subunit association in vitro. The strongest effects are observed with glucose-1,6-bisphosphate (Glc-1,6-P2) or with monophosphate glucose in the presence of vanadate. This finding offers a new approach for the treatment of PMM2 deficiency. We propose to enhance Glc-1,6-P2 concentration either acting on the metabolic pathways that control its synthesis and degradation or exploiting prodrugs that are able to cross membranes.

Pseudomonas litoralis sp. nov., isolated from Mediterranean seawater.

Strains 2SM5(T) and 2SM6, two strictly aerobic chemo-organotrophic gammaproteobacteria, were isolated from Mediterranean seawater off the coast of Vinaroz, Castellón, Spain, in February, 1990. They were extensively characterized by a polyphasic study that placed them in the genus Pseudomonas. Phylogenetic analysis of 16S rRNA gene sequences showed that both strains shared 100 % sequence similarity and were closely related to members of the Pseudomonas pertucinogena clade, with less than 97.3 % similarity to strains of established species; Pseudomonas xiamenensis was the closest relative. Analysis of sequences of three housekeeping genes, rpoB, rpoD and gyrB, further confirmed the phylogenetic assignment of the Mediterranean isolates. Chemotaxonomic traits such as quinone and polar lipid composition also corroborated the placement of strains 2SM5(T) and 2SM6 in the gammaproteobacteria. Other phenotypic traits, including fatty acid composition, enabled clear differentiation of both isolates from other species of Pseudomonas. We therefore conclude that strains 2SM5(T) and 2SM6 represent a novel species of Pseudomonas, for which the name Pseudomonas litoralis is proposed; the type strain is 2SM5(T) ( = CECT 7670(T) = KCTC 23093(T)).

Salinivibrio sharmensis sp. nov., a novel haloalkaliphilic bacterium from a saline lake in Ras Mohammed Park (Egypt).

A novel haloalkaliphilic, facultative anaerobic and Gram-negative Salinivibrio-like microorganism (designated strain BAG(T)) was recovered from a saline lake in Ras Mohammed Park (Egypt). Cells were motile, curved rods, not spore-forming and occurred singly. Strain BAG(T) grew optimally at 35°C (temperature growth range 25-40°C) with 10.0% (w/v) NaCl [NaCl growth range 6.0-16.0% (w/v)] and at pH 9.0 (pH growth range 6.0-10.0). Strain BAG(T) had phosphatidylethanolamine (PEA) and phosphatidylglycerol (PG) as the main polar lipids, C16:0 (54.0%) and C16:1 (26.0%) as the predominant cellular fatty acids and Q-8 as the major respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BAG(T) was a member of Salinivibrio genus, with the highest sequence similarities of 99.1, 98.4 and 98.1% to Salinivibrio siamensis JCM 14472(T), Salinivibrio proteolyticus DSM 19052(T) and Salinivibrio costicola subsp. alcaliphilus DSM 16359(T), respectively. DNA-DNA hybridization values of strain BAG(T) with members of Salinivibrio genus were lower than 55.0%. DNA G + C content was 51.0 mol%. On the basis of the polyphasic taxonomic results revealed in this study, strain BAG(T) should be classified as a novel species of Salinivibrio genus, for which the name Salinivibrio sharmensis sp. nov. is proposed, with the type strain BAG(T) (=ATCC BAA-1319(T) = DSM 18182(T)).

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Euzebyella saccharophila gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae.

Strain 7SM30(T)(,) an aerobic marine, Gram-negative, heterotrophic and yellow- to orange-pigmented bacterium isolated from seawater from Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence showed that the isolate represented a novel lineage within the family Flavobacteriaceae. The most closely related genera were Pseudozobellia, Zobellia and Kriegella. Cells of strain 7SM30(T) were non-motile rods that required sea salts for growth, used a wide variety of carbohydrates as sole carbon and energy sources and, unlike species of the genera Pseudozobellia and Zobellia, did not possess flexirubin-type pigment or hydrolyse agar. Strain 7SM30(T) contained MK6 as the sole respiratory quinone. Phosphatidylethanolamine (PE) was the only identifiable polar lipid, although other lipids were also detected. The predominant cellular fatty acids were saturated C(15) and monounsaturated C(15). The DNA G+C content was around 40 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the new strain represents a novel genus and species, for which the name Euzebyella saccharophila gen. nov., sp. nov., is proposed. The type strain of the type species is 7SM30(T) (=CECT 7477(T)=KCTC 22655(T)).

Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

Identification of AglE, a second glycosyltransferase involved in N glycosylation of the Haloferax volcanii S-layer glycoprotein.

Archaea, like Eukarya and Bacteria, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea.