Extracellular HSP90α Induces MyD88-IRAK Complex-Associated IKKα/ß-NF-κB/IRF3 and JAK2/TYK2-STAT-3 Signaling in Macrophages for Tumor-Promoting M2-Polarization.

M2-polarization and the tumoricidal to tumor-promoting transition are commonly observed with tumor-infiltrating macrophages after interplay with cancer cells or/and other stroma cells. Our previous study indicated that macrophage M2-polarization can be induced by extracellular HSP90α (eHSP90α) secreted from endothelial-to-mesenchymal transition-derived cancer-associated fibroblasts. To extend the finding, we herein validated that eHSP90α-induced M2-polarized macrophages exhibited a tumor-promoting activity and the promoted tumor tissues had significant increases in microvascular density but decreases in CD4+ T-cell level. We further investigated the signaling pathways occurring in eHSP90α-stimulated macrophages. When macrophages were exposed to eHSP90α, CD91 and toll-like receptor 4 (TLR4) functioned as the receptor/co-receptor for eHSP90α binding to recruit interleukin (IL)-1 receptor-associated kinases (IRAKs) and myeloid differentiation factor 88 (MyD88), and next elicited a canonical CD91/MyD88-IRAK1/4-IκB kinase α/ß (IKKα/ß)-nuclear factor-κB (NF-κB)/interferon regulatory factor 3 (IRF3) signaling pathway. Despite TLR4-MyD88 complex-associated activations of IKKα/ß, NF-κB and IRF3 being well-known as involved in macrophage M1-activation, our results demonstrated that the CD91-TLR4-MyD88 complex-associated IRAK1/4-IKKα/ß-NF-κB/IRF3 pathway was not only directly involved in M2-associated CD163, CD204, and IL-10 gene expressions but also required for downregulation of M1 inflammatory cytokines. Additionally, Janus kinase 2 (JAK2) and tyrosine kinase 2 (TYK2) were recruited onto MyD88 to induce the phosphorylation and activation of the transcription factor signal transducer and activator of transcription-3 (STAT-3). The JAK2/TYK2-STAT-3 signaling is known to associate with tumor promotion. In this study, the MyD88-JAK2/TYK2-STAT-3 pathway was demonstrated to contribute to eHSP90α-induced macrophage M2-polarization by regulating the expressions of M1- and M2-related genes, proangiogenic protein vascular endothelial growth factor, and phagocytosis-interfering factor Sec22b.

Drug-like property optimization: Discovery of orally bioavailable quinazoline-based multi-targeted kinase inhibitors.

In an effort to develop new cancer therapeutics, we have reported clinical candidate BPR1K871 (1) as a potentanticancercompound in MOLM-13 and MV4-11 leukemia models, as well as in colorectal and pancreatic animal models. As BPR1K871 lacks oral bioavailability, we continued searching for orally bioavailable analogs through drug-like property optimization. We optimized both the physicochemical properties (PCP) as well as in vitro rat liver microsomal stability of 1, with concomitant monitoring of aurora kinase enzyme inhibition as well as cellular anti-proliferative activity in HCT-116 cell line. Structural modification at the 6- and 7-position of quinazoline core of 1 led to the identification of 34 as an orally bioavailable (F% = 54) multi-kinase inhibitor, which exhibits potent anti-proliferative activity against various cancer cell lines. Quinazoline 34 is selected as a promising oral lead candidate for further preclinical evaluation.

Multi-atlas tool for automated segmentation of brain gray matter nuclei and quantification of their magnetic susceptibility.

Quantification of tissue magnetic susceptibility using MRI offers a non-invasive measure of important tissue components in the brain, such as iron and myelin, potentially providing valuable information about normal and pathological conditions during aging. Despite many advances made in recent years on imaging techniques of quantitative susceptibility mapping (QSM), accurate and robust automated segmentation tools for QSM images that can help generate universal and sharable susceptibility measures in a biologically meaningful set of structures are still not widely available. In the present study, we developed an automated process to segment brain nuclei and quantify tissue susceptibility in these regions based on a susceptibility multi-atlas library, consisting of 10 atlases with T1-weighted images, gradient echo (GRE) magnitude images and QSM images of brains with different anatomic patterns. For each atlas in this library, 10 regions of interest in iron-rich deep gray matter structures that are better defined by QSM contrast were manually labeled, including caudate, putamen, globus pallidus internal/external, thalamus, pulvinar, subthalamic nucleus, substantia nigra, red nucleus and dentate nucleus in both left and right hemispheres. We then tested different pipelines using different combinations of contrast channels to bring the set of labels from the multi-atlases to each target brain and compared them with the gold standard manual delineation. The results showed that the segmentation accuracy using dual contrasts QSM/T1 pipeline outperformed other dual-contrast or single-contrast pipelines. The dice values of 0.77 ±â€¯0.09 using the QSM/T1 multi-atlas pipeline rivaled with the segmentation reliability obtained from multiple evaluators with dice values of 0.79 ±â€¯0.07 and gave comparable or superior performance in segmenting subcortical nuclei in comparison with standard FSL FIRST or recent multi-atlas package of volBrain. The segmentation performance of the QSM/T1 multi-atlas was further tested on QSM images acquired using different acquisition protocols and platforms and showed good reliability and reproducibility with average dice of 0.79 ±â€¯0.08 to manual labels and 0.89 ±â€¯0.04 in an inter-protocol manner. The extracted quantitative magnetic susceptibility values in the deep gray matter nuclei also correlated well between different protocols with inter-protocol correlation constants all larger than 0.97. Such reliability and performance was ultimately validated in an external dataset acquired at another study site with consistent susceptibility measures obtained using the QSM/T1 multi-atlas approach in comparison to those using manual delineation. In summary, we designed a susceptibility multi-atlas tool for automated and reliable segmentation of QSM images and for quantification of magnetic susceptibilities. It is publicly available through our cloud-based platform (www.mricloud.org). Further improvement on the performance of this multi-atlas tool is expected by increasing the number of atlases in the future.

Identification of Novel Cdc7 Kinase Inhibitors as Anti-Cancer Agents that Target the Interaction with Dbf4 by the Fragment Complementation and Drug Repositioning Approach.

BACKGROUND: Cdc7-Dbf4 is a conserved serine/threonine kinase that plays an important role in initiation of DNA replication and DNA damage tolerance in eukaryotic cells. Cdc7 has been found overexpressed in human cancer cell lines and tumor tissues, and the knockdown of Cdc7 expression causes an p53-independent apoptosis, suggesting that Cdc7 is a target for cancer therapy. Only a handful Cdc7 kinase inhibitors have been reported. All Cdc7 kinase inhibitors, including PHA-767491, were identified and characterized as ATP-competitive inhibitors. Unfortunately, these ATP-competitive Cdc7 inhibitors have no good effect on clinical trial. METHODS: Here, we have developed a novel drug-screening platform to interrupt the interaction between Cdc7 and Dbf4 based on Renilla reniformis luciferase (Rluc)-linked protein-fragment complementation assay (Rluc-PCA). Using drug repositioning approach, we found several promising Cdc7 inhibitors for cancer therapy from a FDA-approved drug library. FINDINGS: Our data showed that dequalinium chloride and clofoctol we screened inhibit S phase progression, accumulation in G2/M phase, and Cdc7 kinase activity. In addition, in vivo mice animal study suggests that dequalinium chloride has a promising anti-tumor activity in oral cancer. Interestingly, we also found that dequalinium chloride and clofoctol sensitize the effect of platinum compounds and radiation due to synergistic effect. In conclusion, we identified non-ATP-competitive Cdc7 kinase inhibitors that not only blocks DNA synthesis at the beginning but also sensitizes cancer cells to DNA damage agents. INTERPRETATION: The inhibitors will be a promising anti-cancer agent and enhance the therapeutic effect of chemotherapy and radiation for current cancer therapy. FUND: This work was supported by grants from the Ministry of Science and Technology, Ministry of Health and Welfare, and National Health Research Institutes, Taiwan.

Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity.

BACKGROUND: Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and cytochrome P450 reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. METHODS: We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. RESULTS: Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. CONCLUSIONS: These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.

Identification of neuraminidase inhibitors against dual H274Y/I222R mutant strains.

Influenza is an annual seasonal epidemic that has continually drawn public attentions, due to the potential death toll and drug resistance. Neuraminidase, which is essential for the spread of influenza virus, has been regarded as a valid target for the treatment of influenza infection. Although neuraminidase drugs have been developed, they are susceptible to drug-resistant mutations in the sialic-binding site. In this study, we established computational models (site-moiety maps) of H1N1 and H5N1 to determine properties of the 150-cavity, which is adjacent to the drug-binding site. The models reveal that hydrogen-bonding interactions with residues R118, D151, and R156 and van der Waals interactions with residues Q136, D151, and T439 are important for identifying 150-cavitiy inhibitors. Based on the models, we discovered three new inhibitors with IC50 values <10 µM that occupies both the 150-cavity and sialic sites. The experimental results identified inhibitors with similar activities against both wild-type and dual H274Y/I222R mutant neuraminidases and showed little cytotoxic effects. Furthermore, we identified three new inhibitors situated at the sialic-binding site with inhibitory effects for normal neuraminidase, but lowered effects for mutant strains. The results suggest that the new inhibitors can be used as a starting point to combat drug-resistant strains.

Glucocorticoids may compromise the effect of gefitinib in non-small cell lung cancer.

The epidermal growth factor receptor (EGFR)-targeting tyrosine kinase inhibitors (TKIs) have shown remarkable benefits in non-small cell lung cancer (NSCLC) patients with drug-sensitive mutations in the EGFR gene. Responsive patients are usually continuously prescribed with TKIs until disease progression. Glucocorticoids (GCs) are potent homeostasis maintaining drugs and are frequently used in cancer patients to alleviate discomforts caused by anti-cancer therapies. Several previous studies reported that concomitant use of GCs may compromise the efficacy of chemo-therapeutics in patients with solid tumors. Little is known in the concomitant use of target therapy with GCs in treating NSCLC. In this study, we hypothesized that concomitant use of GCs in EGFR-TKI therapy may be detrimental and addressed this issue using cell cultures and xenograft studies followed by a retrospective population study based on data from the Taiwan national health insurance system. In cell cultures and xenograft studies, GCs were shown to unequally compromise the anti-cancer efficacy of TKIs in both PC9 and NCI-H1975 NSCLC cells models. In the retrospective population study, patients with similar disease status that were co-medicated with GCs had a significantly higher risk of disease progression.

Discovery of BPR1K871, a quinazoline based, multi-kinase inhibitor for the treatment of AML and solid tumors: Rational design, synthesis, in vitro and in vivo evaluation.

The design and synthesis of a quinazoline-based, multi-kinase inhibitor for the treatment of acute myeloid leukemia (AML) and other malignancies is reported. Based on the previously reported furanopyrimidine 3, quinazoline core containing lead 4 was synthesized and found to impart dual FLT3/AURKA inhibition (IC50 = 127/5 nM), as well as improved physicochemical properties. A detailed structure-activity relationship study of the lead 4 allowed FLT3 and AURKA inhibition to be finely tuned, resulting in AURKA selective (5 and 7; 100-fold selective over FLT3), FLT3 selective (13; 30-fold selective over AURKA) and dual FLT3/AURKA selective (BPR1K871; IC50 = 19/22 nM) agents. BPR1K871 showed potent anti-proliferative activities in MOLM-13 and MV4-11 AML cells (EC50 ~ 5 nM). Moreover, kinase profiling and cell-line profiling revealed BPR1K871 to be a potential multi-kinase inhibitor. Functional studies using western blot and DNA content analysis in MV4-11 and HCT-116 cell lines revealed FLT3 and AURKA/B target modulation inside the cells. In vivo efficacy in AML xenograft models (MOLM-13 and MV4-11), as well as in solid tumor models (COLO205 and Mia-PaCa2), led to the selection of BPR1K871 as a preclinical development candidate for anti-cancer therapy. Further detailed studies could help to investigate the full potential of BPR1K871 as a multi-kinase inhibitor.

Discovery of novel inhibitors of Aurora kinases with indazole scaffold: In silico fragment-based and knowledge-based drug design.

Aurora kinases have emerged as important anticancer targets so that there are several inhibitors have advanced into clinical study. Herein, we identified novel indazole derivatives as potent Aurora kinases inhibitors by utilizing in silico fragment-based approach and knowledge-based drug design. After intensive hit-to-lead optimization, compounds 17 (dual Aurora A and B), 21 (Aurora B selective) and 30 (Aurora A selective) possessed indazole privileged scaffold with different substituents, which provide sub-type kinase selectivity. Computational modeling helps in understanding that the isoform selectivity could be targeted specific residue in the Aurora kinase binding pocket in particular targeting residues Arg220, Thr217 or Glu177.

Mycotoxin Patulin Suppresses Innate Immune Responses by Mitochondrial Dysfunction and p62/Sequestosome-1-dependent Mitophagy.

Innate immune responses are important for pathogen elimination and adaptive immune response activation. However, excess inflammation may contribute to immunopathology and disease progression (e.g. inflammation-associated hepatocellular carcinoma). Immune modulation resulting from pattern recognition receptor-induced responses is a potential strategy for controlling immunopathology and related diseases. This study demonstrates that the mycotoxin patulin suppresses Toll-like receptor- and RIG-I/MAVS-dependent cytokine production through GSH depletion, mitochondrial dysfunction, the activation of p62-associated mitophagy, and p62-TRAF6 interaction. Blockade of autophagy restored the immunosuppressive activity of patulin, and pharmacological activation of p62-dependent mitophagy directly reduced RIG-I-like receptor-dependent inflammatory cytokine production. These results demonstrated that p62-dependent mitophagy has an immunosuppressive role to innate immune response and might serve as a potential immunomodulatory target for inflammation-associated diseases.

Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC) patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs. RESULTS: Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits cell adhesion-related response and greatly enhances the cell-killing effects of EGFR TKI (gefitinib for the PC9 cells; afatinib for the H1975 cells) in NSCLC cells, which would otherwise escape the TKI-induced apoptosis. CONCLUSION: Results from this study indicate that NSCLC cells can employ the adhesion response as a survival pathway to survive under EGFR-targeted therapy. Simultaneous targeting of EGFR signaling and adhesion pathways would further boost the efficacy of EGFR-targeted therapy in NSCLC.

Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.

Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP's RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.

Identification of Substituted Naphthotriazolediones as Novel Tryptophan 2,3-Dioxygenase (TDO) Inhibitors through Structure-Based Virtual Screening.

A structure-based virtual screening strategy, comprising homology modeling, ligand-support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

Homology modeling of DFG-in FMS-like tyrosine kinase 3 (FLT3) and structure-based virtual screening for inhibitor identification.

The inhibition of FMS-like tyrosine kinase 3 (FLT3) activity using small-molecule inhibitors has emerged as a target-based alternative to traditional chemotherapy for the treatment of acute myeloid leukemia (AML). In this study, we report the use of structure-based virtual screening (SBVS), a computer-aided drug design technique for the identification of new chemotypes for FLT3 inhibition. For this purpose, homology modeling (HM) of the DFG-in FLT3 structure was carried using two template structures, including PDB ID: 1RJB (DFG-out FLT3 kinase domain) and PDB ID: 3LCD (DFG-in CSF-1 kinase domain). The modeled structure was able to correctly identify known DFG-in (SU11248, CEP-701, and PKC-412) and DFG-out (sorafenib, ABT-869 and AC220) FLT3 inhibitors, in docking studies. The modeled structure was then used to carry out SBVS of an HTS library of 125,000 compounds. The top scoring 97 compounds were tested for FLT3 kinase inhibition, and two hits (BPR056, IC50 = 2.3 and BPR080, IC50 = 10.7 µM) were identified. Molecular dynamics simulation and density functional theory calculation suggest that BPR056 (MW: 325.32; cLogP: 2.48) interacted with FLT3 in a stable manner and could be chemically optimized to realize a drug-like lead in the future.

Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with activity against drug resistance-conferring point mutations.

Numerous FLT3 inhibitors have been explored as a viable therapy for the treatment of acute myeloid leukemia (AML). However, clinical data have been underwhelming due to incomplete inhibition of FLT3 or the emergence of resistant mutations treated with these older agents. We previously developed a series of 3-phenyl-1H-5-pyrazolylamine derivatives as highly potent and selective FLT3 inhibitors with good in vivo efficacy using an intravenous (IV) route. However, the poor bioavailability of these pyrazole compounds limits the development of these promising antileukemic compounds for clinical use. Herein, we describe a novel class of 5-phenyl-thiazol-2-ylamine compounds that are multi-targeted FLT3 inhibitors. From this class of compounds, compound 7h was very potent against AML cell lines and exhibited excellent oral efficacy in AML xenograft models. In addition, further studies demonstrated that compound 7h exhibited potent in vitro and in vivo activities against clinically relevant AC220 (3)-resistant kinase domain mutants of FLT3-ITD.

Anchor-based classification and type-C inhibitors for tyrosine kinases.

Tyrosine kinases regulate various biological processes and are drug targets for cancers. At present, the design of selective and anti-resistant inhibitors of kinases is an emergent task. Here, we inferred specific site-moiety maps containing two specific anchors to uncover a new binding pocket in the C-terminal hinge region by docking 4,680 kinase inhibitors into 51 protein kinases, and this finding provides an opportunity for the development of kinase inhibitors with high selectivity and anti-drug resistance. We present an anchor-based classification for tyrosine kinases and discover two type-C inhibitors, namely rosmarinic acid (RA) and EGCG, which occupy two and one specific anchors, respectively, by screening 118,759 natural compounds. Our profiling reveals that RA and EGCG selectively inhibit 3% (EGFR and SYK) and 14% of 64 kinases, respectively. According to the guide of our anchor model, we synthesized three RA derivatives with better potency. These type-C inhibitors are able to maintain activities for drug-resistant EGFR and decrease the invasion ability of breast cancer cells. Our results show that the type-C inhibitors occupying a new pocket are promising for cancer treatments due to their kinase selectivity and anti-drug resistance.

Comparing fractional anisotropy in patients with childhood-onset schizophrenia, their healthy siblings, and normal volunteers through DTI.

BACKGROUND: Diffusion tensor imaging is a neuroimaging method that quantifies white matter (WM) integrity and brain connectivity based on the diffusion of water in the brain. White matter has been hypothesized to be of great importance in the development of schizophrenia as part of the dysconnectivity model. Childhood-onset schizophrenia (COS), is a rare, severe form of the illness that resembles poor outcome adult-onset schizophrenia. We hypothesized that COS would be associated with WM abnormalities relative to a sample of controls. METHODS: To evaluate WM integrity in this population 39 patients diagnosed with COS, 39 of their healthy (nonpsychotic) siblings, and 50 unrelated healthy volunteers were scanned using a diffusion tensor imaging (DTI) sequence during a 1.5 T MRI acquisition. Each DTI scan was processed via atlas-based analysis using a WM parcellation map, and diffeomorphic mapping that shapes a template atlas to each individual subject space. Fractional anisotropy (FA), a measure of WM integrity was averaged over each of the 46 regions of the atlas. Eleven WM regions were examined based on previous reports of WM growth abnormalities in COS. RESULTS: Of those regions, patients with COS, and their healthy siblings had significantly lower mean FA in the left and right cuneus as compared to the healthy volunteers (P < .005). Together, these findings represent the largest DTI study in COS to date, and provide evidence that WM integrity is significantly impaired in COS. Shared deficits in their healthy siblings might result from increased genetic risk.

Identification of ligand efficient, fragment-like hits from an HTS library: structure-based virtual screening and docking investigations of 2H- and 3H-pyrazolo tautomers for Aurora kinase A selectivity.

Furanopyrimidine 1 (IC50 = 273 nM, LE = 0.36, LELP = 10.28) was recently identified by high-throughput screening (HTS) of an in-house library (125,000 compounds) as an Aurora kinase inhibitor. Structure-based hit optimization resulted in lead molecules with in vivo efficacy in a mouse tumour xenograft model, but no oral bioavailability. This is attributed to "molecular obesity", a common problem during hit to lead evolution during which degradation of important molecular properties such as molecular weight (MW) and lipophilicity occurs. This could be effectively tackled by the right choice of hit compounds for optimization. In this regard, ligand efficiency (LE) and ligand efficiency dependent lipophilicity (LELP) indices are more often used to choose fragment-like hits for optimization. To identify hits with appropriate LE, we used a MW cut-off <250, and pyrazole structure to filter HTS library. Next, structure-based virtual screening using software (Libdock and Glide) in the Aurora A crystal structure (PDB ID: 3E5A) was carried out, and the top scoring 18 compounds tested for Aurora A enzyme inhibition. This resulted in the identification of a novel tetrahydro-pyrazolo-isoquinoline hit 7 (IC50 = 852 nM, LE = 0.44, LELP = 8.36) with fragment-like properties suitable for further hit optimization. Moreover, hit 7 was found to be selective for Aurora A (Aurora B IC50 = 35,150 nM) and the possible reasons for selectivity investigated by docking two tautomeric forms (2H- and 3H-pyrazole) of 7 in Auroras A and B (PDB ID: 4AF3) crystal structures. This docking study shows that the major 3H-pyrazole tautomer of 7 binds in Aurora A stronger than in Aurora B.

The combination effects of licl and the active leflunomide metabolite, A771726, on viral-induced interleukin 6 production and EV-A71 replication.

Enterovirus 71 (EV-A71) is a neurotropic virus that can cause severe complications involving the central nervous system. No effective antiviral therapeutics are available for treating EV-A71 infection and drug discovery efforts are rarely focused to target this disease. Thus, the main goal of this study was to discover existing drugs with novel indications that may effectively inhibit EV-A71 replication and the inflammatory cytokines elevation. In this study, we showed that LiCl, a GSK3ß inhibitor, effectively suppressed EV-A71 replication, apoptosis and inflammatory cytokines production (Interleukin 6, Interleukin-1ß) in infected cells. Furthermore, LiCl and an immunomodular agent were shown to strongly synergize with each other in suppressing EV-A71 replication. The results highlighted potential new treatment regimens in suppressing sequelae caused by EV-A71 replication.

Ligand efficiency based approach for efficient virtual screening of compound libraries.

Here we report for the first time the use of fit quality (FQ), a ligand efficiency (LE) based measure for virtual screening (VS) of compound libraries. The LE based VS protocol was used to screen an in-house database of 125,000 compounds to identify aurora kinase A inhibitors. First, 20 known aurora kinase inhibitors were docked to aurora kinase A crystal structure (PDB ID: 2W1C); and the conformations of docked ligand were used to create a pharmacophore (PH) model. The PH model was used to screen the database compounds, and rank (PH rank) them based on the predicted IC50 values. Next, LE_Scale, a weight-dependant LE function, was derived from 294 known aurora kinase inhibitors. Using the fit quality (FQ = LE/LE_Scale) score derived from the LE_Scale function, the database compounds were reranked (PH_FQ rank) and the top 151 (0.12% of database) compounds were assessed for aurora kinase A inhibition biochemically. This VS protocol led to the identification of 7 novel hits, with compound 5 showing aurora kinase A IC50 = 1.29 µM. Furthermore, testing of 5 against a panel of 31 kinase reveals that it is selective toward aurora kinase A & B, with <50% inhibition for other kinases at 10 µM concentrations and is a suitable candidate for further development. Incorporation of FQ score in the VS protocol not only helped identify a novel aurora kinase inhibitor, 5, but also increased the hit rate of the VS protocol by improving the enrichment factor (EF) for FQ based screening (EF = 828), compared to PH based screening (EF = 237) alone. The LE based VS protocol disclosed here could be applied to other targets for hit identification in an efficient manner.