Design, synthesis, and biological evaluation of substituted hydrazone and pyrazole derivatives as selective COX-2 inhibitors: Molecular docking study

New arylhydrazone derivatives and a series of 1,5-diphenyl pyrazoles were designed and synthesized from 1-(4-chlorophenyl)-4,4,4-trifuorobutane-1,3-dione 1. The newly synthesized compounds were investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw oedema model. Moreover, they were tested for their inhibitory activity against ovine COX-1 and COX-2 using an in vitro cyclooxygenase (COX) inhibition assay. Some of the new compounds (2f, 6a and 6d) showed a reasonable in vitro COX-2 inhibitory activity, with IC?? value of 0.45 μM and selectivity index of 111.1. A virtual screening was carried out through docking the designed compounds into the COX-2 binding site to predict if these compounds have analogous binding mode to the COX-2 inhibitors. Docking study of the synthesized compounds 2f, 6a and 6d into the active site of COX-2 revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

Uncovering false positives on a virtual screening search for cruzain inhibitors

Some unexpected promiscuous inhibitors were observed in a virtual screening protocol applied to select cruzain inhibitors from the ZINC database. Physical-chemical and pharmacophore model filters were used to reduce the database size. The selected compounds were docked into the cruzain active site. Six hit compounds were tested as inhibitors. Although the compounds were designed to be nucleophilically attacked by the catalytic cysteine of cruzain, three of them showed typical promiscuous behavior, revealing that false positives are a prevalent concern in VS programs.     

Discovery of potential pancreatic cholesterol esterase inhibitors using pharmacophore modelling, virtual screening, and optimization studies

Pancreatic cholesterol esterase (CEase) is a serine hydrolase involved in the hydrolysis of variety of lipids and transport of free cholesterol. In this study, pharmacophore hypotheses based on known inhibitors were generated using common feature pharmacophore generation protocol available in Discovery Studio program. The best pharmacophore model containing two hydrogen bond acceptor and three hydrophobic features was selected and validated. It was further used in screening three diverse chemical databases. Hit compounds were subjected to drug-likeness and molecular docking studies. Four hits, namely SEW00846, NCI0040784, GK03167, and CD10645, were selected based on the GOLD fitness score and interaction with active site amino acids. All hit compounds were further optimized to improve their binding in the active site. The optimized compounds were found to have improved binding at the active site. Strongly binding optimized hits at the active site can act as virtual leads in potent CEase inhibitor designing.     

Lead compounds and key residues of ribosomal protein S1 in drug-resistant Mycobacterium tuberculosis

Ribosomal protein S1 (RpsA) has been identified as a novel target of pyrazinoic acid (POA), which is the active form of pyrazinamide (PZA), in vivo. RpsA plays a crucial role in trans-translation, which is widespread in microbes. In our investigation, we first described the discovery of promising RpsA antagonists for drug-resistant mycobacterium (MtRpsAd438A) and M. smegmatis, as well as wild-type M. tuberculosis. These antagonists were discovered via structure/ligand-based virtual screening approaches. A total of 21 targeted compounds were selected by virtual screening, combined scores, affinity, similarities and rules for potential as drugs. Next, the affinities of these compounds for three targeted proteins were tested in vitro by applying various technologies, including fluorescence quenching titration (FQT), saturation transfer difference (STD), and chemical shift perturbation (CSP) assays. The results showed that seven compounds had a high affinity for the targeted proteins. Our discovery set the stage for discovering new chemical entities (NCEs) for PZA-resistant tuberculosis and providing key residues for rational drug design to target RpsA.     

Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor

The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.     

Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies

The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ～90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC(50) values for each drug in both tests were similar, were lowest for posaconazole (<5 μM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.     

Novel GSK-3beta inhibitors from sequential virtual screening

Glycogen synthase kinase-3 (GSK-3beta) has been emerging as a key therapeutic target for type-2 diabetics, Alzheimer's disease, cancer, and chronic inflammation. For the purpose of finding biologically active and novel compounds and providing new idea for drug-design, we performed virtual screening using commercially available database. Three-dimensional common feature pharmacophore model was developed by using HipHop program provided in Catalyst software and it was used as a query for screening database. Recursive partitioning (RP) model was developed as a filtering system, which was able to classify active and inactive compounds. Eventually, a sequential virtual screening procedure (SQSP) was conducted by applying the common feature pharmacophore and RP model in succession to discover novel potent GSK-3beta inhibitors. The final 56 hit compounds were carefully selected considering predicted docking mode in crystal structures. Subsequent enzyme assay for human GSK-3beta protein confirmed that three compounds of these hit compounds exhibit micromolar inhibitory activity. Here, we report novel hit compounds and their binding mode in the active site of GSK-3beta crystal structure.     

A Teach-Discover-Treat Application of ZincPharmer: An Online Interactive Pharmacophore Modeling and Virtual Screening Tool

The 2012 Teach-Discover-Treat (TDT) community-wide experiment provided a unique opportunity to test prospective virtual screening protocols targeting the anti-malarial target dihydroorotate dehydrogenase (DHODH). Facilitated by ZincPharmer, an open access online interactive pharmacophore search of the ZINC database, the experience resulted in the development of a novel classification scheme that successfully predicted the bound structure of a non-triazolopyrimidine inhibitor, as well as an overall hit rate of 27% of tested active compounds from multiple novel chemical scaffolds. The general approach entailed exhaustively building and screening sparse pharmacophore models comprising of a minimum of three features for each bound ligand in all available DHODH co-crystals and iteratively adding features that increased the number of known binders returned by the query. Collectively, the TDT experiment provided a unique opportunity to teach computational methods of drug discovery, develop innovative methodologies and prospectively discover new compounds active against DHODH.     

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).     

The identification of novel PLC-γ inhibitors using virtual high throughput screening

Phospholipase C-γ (PLC-γ) has been identified as a possible biological target for anticancer drug therapy but suitable inhibitors are lacking. Therefore, in order to identify active compounds (hits) virtual high throughput screening was performed. The crystal structure of the PLC-δ isoform was used as a model docking scaffold since no crystallographic data are available on its γ counterpart. A pilot screen was performed using ～9.2 × 10⁴ compounds, where the robustness of the methodology was tested. This was followed by the main screening effort where ～4.4 × 10⁵ compounds were used. In both cases, plausible compounds were identified (virtual hits) and a selection of these was experimentally tested. The most potent compounds were in the single digit micro-molar range as determined from the biochemical (Flashplate) assay. This translated into ～15 μM in a functional assay in cells. About 30% of the virtual hits showed activity against PLC-γ (IC₅₀ < 50 μM).     

Screening of inhibitors for S130G inhibitor resistant mutants of TEM type beta-lactamase

Bacteria are remarkably adaptable organisms that acquire an almost limitless competence to survive under unpleasant conditions. The drastic emergence of antibiotic resistance among β-Lactamases is the most serious threat to hospitals and nosocomial settings. β-lactam inhibitors came into existence in order to overcome the problem of antibibiotic resistance in bacteria. The emergence of inhibitor resistant mutants has raised the alarms. In this study we have used structured based virtual screening approach and have screened out some inhibitors against S130G TEM mutant. All the compounds were tested in presence and absence of conserved active site water molecules. These compounds were found be showing much higher efficacy than known β-lactamase inhibitors. Amino acids G130, S70, N132, G130, Y105 and V216 were found crucial for the interaction of inhibitors within the active site.     

Identification of potent bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach

Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally distributed. The use of antivirals could complement vaccination as a tool of control and reduce economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus is essential for its genome replication and constitutes an attractive target for the identification of antivirals. With the aim of obtaining selective BVDV inhibitors, the crystal structure of BVDV RdRp was used to perform a virtual screening. Approximately 15,000 small molecules from commercial and in-house databases were evaluated and several structurally different compounds were tested in vitro for antiviral activity. Interestingly, of twelve evaluated compounds, five were active and displayed EC₅₀ values in the sub and low-micromolar range. Time of drug addition experiment and measured intracellular BVDV RNA showed that compound 7 act during RNA synthesis. Molecular Dynamics and MM/PBSA calculation were done to characterize the interaction of the most active compounds with RdRp, which will allow future ligand optimization. These studies highlight the use of in silico screening to identify a new class of BVDV inhibitors.     

Antibacterial activity of chalcones, hydrazones and oxadiazoles against methicillin-resistant Staphylococcus aureus

The increase in antibiotic resistance due to multiple factors has encouraged the search for new compounds which are active against multidrug-resistant pathogens. In this context, chalcones, dihydrochalcones, hydrazones and oxadiazoles were tested against Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus (MRSA) isolates, which were obtained from clinical laboratories and were characterized as MRSA using traditional and molecular methods. Among 65 tested compounds, two chalcones, one dihydrochalcone and two hydrazones were active against MRSA. Based on the minimal inhibitory concentration and cytotoxicity, hydrazones provided a better selectivity index than chalcones. Active hydrazones are promising antibiotic-like substances and they should be the subject of further microbiological studies.     

Unified QSAR approach to antimicrobials. Part 3: first multi-tasking QSAR model for input-coded prediction, structural back-projection, and complex networks clustering of antiprotozoal compounds

Several pathogen parasite species show different susceptibilities to different antiparasite drugs. Unfortunately, almost all structure-based methods are one-task or one-target Quantitative Structure-Activity Relationships (ot-QSAR) that predict the biological activity of drugs against only one parasite species. Consequently, multi-tasking learning to predict drugs activity against different species by a single model (mt-QSAR) is vitally important. In the two previous works of the present series we reported two single mt-QSAR models in order to predict the antimicrobial activity against different fungal (Bioorg. Med. Chem.2006, 14, 5973-5980) or bacterial species (Bioorg. Med. Chem.2007, 15, 897-902). These mt-QSARs offer a good opportunity (unpractical with ot-QSAR) to construct drug-drug similarity Complex Networks and to map the contribution of sub-structures to function for multiple species. These possibilities were unattended in our previous works. In the present work, we continue this series toward other important direction of chemotherapy (antiparasite drugs) with the development of an mt-QSAR for more than 500 drugs tested in the literature against different parasites. The data were processed by Linear Discriminant Analysis (LDA) classifying drugs as active or non-active against the different tested parasite species. The model correctly classifies 212 out of 244 (87.0%) cases in training series and 207 out of 243 compounds (85.4%) in external validation series. In order to illustrate the performance of the QSAR for the selection of active drugs we carried out an additional virtual screening of antiparasite compounds not used in training or predicting series; the model recognized 97 out of 114 (85.1%) of them. We also give the procedures to construct back-projection maps and to calculate sub-structures contribution to the biological activity. Finally, we used the outputs of the QSAR to construct, by the first time, a multi-species Complex Networks of antiparasite drugs. The network predicted has 380 nodes (compounds), 634 edges (pairs of compounds with similar activity). This network allows us to cluster different compounds and identify on average three known compounds similar to a new query compound according to their profile of biological activity. This is the first attempt to calculate probabilities of antiparasitic action of drugs against different parasites.     

Synthesis,molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC₅₀ values of 0.22–2.3 μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30 μM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.     

Design, synthesis and evaluation of 2,4-diaminoquinazolines as inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper describes the design, synthesis and evaluation of a series of 2,4-diaminoquinazolines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were designed by a generating virtual library of compounds and docking them into the enzyme active site. Following their synthesis, they were found to be potent and selective inhibitors of leishmanial dihydrofolate reductase. The compounds were also found to have potent activity against Trypanosoma brucei rhodesiense, a causative organism of African trypanosomiasis and also against Trypanosoma cruzi, the causative organism of Chagas disease. There was significantly lower activity against Leishmania donovani, one of the causative organisms of leishmaniasis.     

Novel 1,3,4-oxadiazole/oxime hybrids: Synthesis,docking studies and investigation of anti-inflammatory,ulcerogenic liability and analgesic activities

A novel group of 1,3,4-oxadaiazoles, a group known for their anti-inflammatory activity, is hybridized with nitric oxide (NO) releasing group, oxime, for its gastro-protective action and potential synergistic effect. The synthesized hybrids were evaluated for their anti-inflammatory, analgesic, antioxidant and ulcerogenic activities. Most of the tested compounds showed excellent anti-inflammatory activity with compound 8e being more active than indomethacin. They also showed moderate analgesic activity but no antioxidant one. The ability of the synthesized compounds to inhibit COX-1 and COX-2 is studied and the prepared compounds were able to inhibit both COXs non-selectively with IC₅₀s of 0.75–70.50 μM. Docking studies revealed the mode of interaction of the tested compounds into the empty pocket of the isozymes. All of the synthesized compounds interact with COXs active site with energy scores comparable to that of ibuprofen. All compounds showed a safer profile on the stomach tissue integrity compared to conventional NSAIDs. The designed strategy was applied to ibuprofen to introduce ibuprofen/oxadiazole/NO hybrid. The synthesized ibuprofen hybrid is a promising alternative to ibuprofen having similar anti-inflammatory activity but with safer GIT profile.     

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17)--part II: Core rigidification and influence of substituents at the methylene bridge

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.     

CCLab--a multi-objective genetic algorithm based combinatorial library design software and an application for histone deacetylase inhibitor design

The introduction of the multi-objective optimization has dramatically changed the virtual combinatorial library design, which can consider many objectives simultaneously, such as synthesis cost and drug-likeness, thus may increase positive rates of biological active compounds. Here we described a software called CCLab (Combinatorial Chemistry Laboratory) for combinatorial library design based on the multi-objective genetic algorithm. Tests of the convergence ability and the ratio to re-take the building blocks in the reference library were conducted to assess the software in silico, and then it was applied to a real case of designing a 5×6 HDAC inhibitor library. Sixteen compounds in the resulted library were synthesized, and the histone deactetylase (HDAC) enzymatic assays proved that 14 compounds showed inhibitory ratios more than 50% against tested 3 HDAC enzymes at concentration of 20 μg/mL, with IC(50) values of 3 compounds comparable to SAHA. These results demonstrated that the CCLab software could enhance the hit rates of the designed library and would be beneficial for medicinal chemists to design focused library in drug development (the software can be downloaded at: http://202.127.30.184:8080/drugdesign.html).