Discovery of new GSK-3β inhibitors through structure-based virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an attractive therapeutic target for human diseases, such as diabetes, cancer, neurodegenerative diseases, and inflammation. Thus, structure-based virtual screening was performed to identify novel scaffolds of GSK-3β inhibitors, and we observed that conserved water molecules of GSK-3β were suitable for virtual screening. We found 14 hits and D1 (IC₅₀ of 0.71?μM) were identified. Furthermore, the neuroprotection activity of D1–D3 was validated on a cellular level. 2D similarity searches were used to find derivatives of high inhibitory compounds and an enriched structure–activity relationship suggested that these skeletons were worthy of study as potent GSK-3β inhibitors.     

Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with K_i of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.     

Discovery of a natural PI3Kδ inhibitor through virtual screening and biological assay study

Phosphoinositide-3-kinase-δ (PI3Kδ) is a key regulator in the process of IgE mediated mast cell degranulation, which directly induces allergic diseases, such as asthma. This study is aimed at discovery of natural PI3Kδ inhibitors from Chinese medicine and evaluating their anti-mast cell degranulation activity. A combined virtual screening based on 3D pharmacophore model and molecular docking was used to screen for bioactive ingredients directly targeting PI3Kδ. Then, an in vitro kinase inhibition assay was conducted to evaluate the PI3Kδ inhibitory activity of the virtual screening hits. Subsequently, a β-hexosaminidase release assay was performed to verify the anti-mast cell degranulation activity of the active compounds. Finally, ginkgoneolic acid was identified as a PI3Kδ inhibitor (IC₅₀?=?2.49?μM) and exhibited anti-mast cell degranulation activity in vitro (IC₅₀?=?2.40?μM). Docking studies showed that Glu826, Val827 and Val828 were key amino acid residues for PI3Kδ inhibitory activity. Ginkgoneolic acid may be a potential lead compound for developing effective and safe PI3Kδ-inhibiting drugs.     

Silibinin: a novel inhibitor of Aβ aggregation

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β peptide (Aβ) into extracellular fibrillar deposits known as amyloid plaque. Inhibition of Aβ aggregation is therefore viewed as a potential method to halt or slow the progression of AD. It is reported that silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), attenuates cognitive deficits induced by Aβ25-35 peptide and methamphetamine. However, it remains unclear whether silibinin interacts with Aβ peptide directly and decreases Aβ peptide-induced neurotoxicity. In the present study, we identified, through employing a ThT assay and electron microscopic imaging that silibinin also appears to act as a novel inhibitor of Aβ aggregation and this effect showed dose-dependency. We also show that silibinin prevented SH-SY5Y cells from injuries caused by Aβ(1-42)-induced oxidative stress by decreasing H(2)O(2) production in Aβ(1-42)-stressed neurons. Taken together, these results indicate that silibinin may be a novel therapeutic agent for the treatment of AD.     

Structure–activity relationship of novel DAPK inhibitors identified by structure-based virtual screening

Death-associated protein kinase (DAPK) is a serine/threonine protein kinase implicated in diverse programmed cell death pathways. DAPK is a promising target protein for the treatment of ischemic diseases. We identified novel potent and selective DAPK inhibitors efficiently by structure-based virtual screening, then further developed the hit compounds. In this paper, we describe the development of the hit compounds and the structure–activity relationship studies of the DAPK inhibitors in detail, including calculation of the solvated interaction energy (SIE), and verification of selectivity using a kinase panel.     

Description and assessment of a model for GSK-3β database virtual screening

Molecular docking was used in order to prioritize organic syntheses or experimental evaluations. Different GSK-3β protein models were generated in silico from a known X-ray structure. A set of 42 known inhibitors were then flexibly docked into each rigid model and re-scored with various functions, which led to different rankings. The biological activities of the chemicals were then compared to each set of results and one of the rigid models emerged in combination with two scoring functions as giving the best correlation. This methodology constitutes an easy and accurate way to generate reliable models for virtual database screening.     

Discovery of atorvastatin as a tetramer stabilizer of nuclear receptor RXRα through structure-based virtual screening

Retinoid X receptor alpha (RXRα), a central member of the nuclear receptor superfamily and a key regulator of many signal transduction pathways, has been an attractive drug target. We previously discovered that an N-terminally truncated form of RXRα can be induced by specific ligands to form homotetramers, which, as a result of conformational selection, forms the basis for inhibiting the nongenomic activation of RXRα. Here, we report the identification and characterization of atorvastatin as a new RXRα tetramer stabilizer by using structure-based virtual screening and demonstrate that virtual library screening can be used to aid in identifying RXRα ligands that can induce its tetramerization. In this study, docking was applied to screen the FDA-approved small molecule drugs in the DrugBank 4.0 collection. Two compounds were selected and purchased for testing. We showed that the selected atorvastatin could bind to RXRα to promote RXRα-LBD tetramerization. We also showed that atorvastatin possessed RXRα-dependent apoptotic effects. In addition, we used a chemical approach to aid in the studies of the binding mode of atorvastatin.     

Discovery of novel 5α-reductase type II inhibitors by pharmacophore modelling,virtual screening,molecular docking and molecular dynamics simulations

Benign prostatic hyperplasia (BPH) is caused by augmented levels of androgen dihydrotestosterone (DHT) which is involved in the growth of the prostate in humans. 5α-Reductase type II (5αR2) is an intracellular enzyme that catalyses the formation of DHT from testosterone; hence, the inhibition of 5αR2 has emerged as one of the most promising strategies for the treatment of BPH. In this study, a computational approach that integrates ligand-based pharmacophore modelling, virtual screening, molecular docking and molecular dynamics (MD) simulations was adopted to discover novel 5αR2 inhibitors with less side effects. After validating by Fischer's randomisation and Güner–Henry test, the best quantitative pharmacophore model (Hypo1), consisting of two hydrogen-bond acceptors and three hydrophobic features, was subsequently used as a three-dimensional-query in virtual screening to identify potential hits from Maybridge and National Cancer Institute databases. These hits were further filtered by ADMET (absorption, distribution, metabolism, elimination and toxicology) and molecular docking experiments, and their binding stabilities were validated by 10-ns MD simulations. Finally, only one hit was identified as a potential lead based on higher predicted inhibitory activity to 5αR2 compared with the most active inhibitor (finasteride). Our results further suggest that this potential lead could easily be synthesised and has structural novelty, making it a promising candidate for treating BPH.     

Discovery of a novel hybrid from finasteride and epristeride as 5α-reductase inhibitor

Finasteride and epristeride both inhibit 5α-reductase with high potency via competitive and non-competitive mechanism, respectively. A new hybrid of finasteride and epristeride was designed as a new 5α-reductase inhibitor based on combination principles in medicinal chemistry. Human 5β-reductase was chosen as a plausible surrogate of 5α-reductase type II and the results indicate that although the hybrid compound possesses the main bulk of epristeride, its inhibitory mechanism is same as of finasteride. The hybrid turned out to be a potent 5α-reductase inhibitor in low IC₅₀ ranges.     

Identification of novel scaffold of benzothiazepinones as non-ATP competitive glycogen synthase kinase-3β inhibitors through virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that has been proved as a key target for neurodegenerative diseases and diabetes. Up to date, most of known inhibitors are bound to the ATP-binding pocket of GSK-3β, which might lead widespread effects due to the high homology between kinases. Recently, some of its non-ATP competitive inhibitors had been confirmed having therapeutical effects owing to their high selectivity. This finding opens a new pathway to study hopeful drugs for treatment of these diseases. However, it is still a challenge nowadays on how to efficiently find non-ATP competitors. Here, we successfully discovered a novel scaffold of benzothiazepinones (BTZs) as selective non-ATP competitive GSK-3β inhibitors through virtual screening approach. A 3D receptor model of substrate binding site of GSK-3β was constructed and applied to screen against drug-like Maybridge database through Autodock program. BTZ compounds were top ranked as efficient hits and were then synthesized for further screening. Among them, the representative compound 4j showed activity to GSK-3β (IC₅₀: 25 μM) in non-ATP competitive mechanism, and nearly no inhibitory effect on other 10 related protein kinases. Overall, the results point out that BTZ compounds might be useful in treatment of Alzheimer’s disease and diabetes mellitus as novel GSK-3β inhibitors. It also suggests, on the other hand, that virtual screening would provide a valuable tool in combination with in vitro assays for the identification of novel selective and potent inhibitors.     

Virtual ligand screening of α-glucosidase: Identification of a novel potent noncarbohydrate mimetic inhibitor

5-Thiazoleacetamide derivatives of AR122 and AR125 were screened as α-glucosidase inhibitors by in silico high-throughput screening from commercial drug-like small compound libraries. Inhibition of α-glucosidase with AR122 and AR125 is time dependent: with no preincubation, AR122 and AR125 are relatively moderate inhibitors, but interestingly, after a 120 min incubation, they were 50-fold more potent (AR122: IC(50)=2.47 μM and AR125: IC(50)=27.1 μM). Plots of ln [residual α-glucosidase activity %] versus preincubation time show a pseudo-first order kinetics for both inhibitors. Through dialysis of enzyme-inhibitor complexes, no activity recovery was shown. These results suggest that AR122 and AR125 constitute a new class of noncarbohydrate mimetic inhibitor with an irreversible mechanism.     

Discovery of a novel covalent non-β-lactam inhibitor of the metallo-β-lactamase NDM-1

The inhibition of metallo-β-lactamases (MBL) can prevent the hydrolysis of β-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-β-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (K_I¹) was 580 nM and the inhibition constant for the initial complex (K_I) was 76 μM. To our knowledge, this inhibitor is the first example for a reversible covalent non-β-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.     

Screening and identification of a novel class of TGF-β type 1 receptor kinase inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC(50) value of 0.7 μM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC(50) value of 0.8 μM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.     

Discovery of highly potent, nonsteroidal 17β-hydroxysteroid dehydrogenase type 1 inhibitors by virtual high-throughput screening

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the formation of the potent proliferation-stimulating hormone estradiol, and it is thus involved in the development of hormone-dependent breast cancer. Due to its high substrate specificity and the known relationships between its overexpression and disease incidence, 17β-HSD1 is considered an attractive target for drug development. Here, we have used structure-based virtual high-throughput screening to successfully identify potent nonsteroidal 17β-HSD1 inhibitors. Computational screening of a drug-like database containing 13 million compounds identified hits with a 2-benzylidenebenzofuran-3(2H)-one scaffold that we show to be highly potent 17β-HSD1 inhibitors. The most potent in the series, compound 1, showed an IC(50) of 45nM in our 17β-HSD1 inhibition assay, and also showed good selectivity for 17β-HSD1 over 17β-HSD2.     

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).     

Identification of small molecules that inhibit GSK-3β through virtual screening

Glycogen synthase kinase-3β (GSK-3β) is involved in glycogen metabolism, neuronal cell development, osteoblast differentiation. Small molecule inhibitors of GSK-3β have various therapeutic potential for the treatment of diabetes type II, bipolar disorders, stroke and chronic inflammatory disease.To identify GSK-3β inhibitors with novel scaffold from chemical library, we primarily screened out putative inhibitors through computer modeling and subsequently evaluated the inhibitory activity of selected compounds against GSK-3β by in vitro Z’-LYTE? assay. A series of compound KRMs strongly inhibited phosphorylation of its substrate with IC₅₀ value of approximately 0.5 μM. Also, we demonstrated that KRM-189 and KRM-191 competed with ATP for GSK-3β, leading to decreased V_max and constant K_m with increasing concentrations of ATP as determined from Lineweaver–Berk equation. Moreover, they showed the selectivity for GSK-3β over other kinases with IC₅₀ values of 2 to 10 μM or more Incubation of cells with KRM-191 with highly selective and potent inhibitory activity caused accumulation of β-catenin, downstream of GSK-3β signaling pathway, indicating that small molecule can prevent degradation of β-catenin via GSK-3β inhibition. Our results suggest that modeling in combination with in vitro assays can be used for the identification of selective and potent inhibitors.     

E-pharmacophore-based virtual screening to identify GSK-3β inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3β increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer’s disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3β to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3β. The 50?ns MD simulations run confirmed the stable nature of GSK-3β-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3β.