Discovery and characterization of novel TRPML1 agonists.

Screening a library of >100,000 compounds identified the substituted tetrazole compound 1 as a selective TRPML1 agonist. Both enantiomers of compound 1 were separated and profiled in vitro and in vivo. Their selectivity, ready availability and CNS penetration should enable them to serve as the tool compounds of choice in future TRPML1 channel activation studies. SAR studies on conformationally locked macrocyclic analogs further improved the TRPML1 agonist potency while retaining the selectivity.

Implementation of an AI-assisted fragment-generator in an open-source platform.

We recently reported a deep learning model to facilitate fragment library design, which is critical for efficient hit identification. However, our model was implemented in Python. We have now created an implementation in the KNIME graphical pipelining environment which we hope will allow experimentation by users with limited programming knowledge.

Stabilization of the RAS:PDE6D Complex Is a Novel Strategy to Inhibit RAS Signaling.

RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.

Automated Generation of Novel Fragments Using Screening Data, a Dual SMILES Autoencoder, Transfer Learning and Syntax Correction.

Fragment-based hit identification (FBHI) allows proportionately greater coverage of chemical space using fewer molecules than traditional high-throughput screening approaches. However, effectively exploiting this advantage is highly dependent on the library design. Solubility, stability, chemical complexity, chemical/shape diversity, and synthetic tractability for fragment elaboration are all critical aspects, and molecule design remains a time-consuming task for computational and medicinal chemists. Artificial neural networks have attracted considerable attention in automated de novo design applications and could also prove useful for fragment library design. Chemical autoencoders are neural networks consisting of encoder and decoder parts, which respectively compress and decompress molecular representations. The decoder is applied to samples drawn from the space of compressed representations to generate novel molecules that can be scored for properties of interest. Here, we report an autoencoder model using a recurrent neural network architecture, which was trained using 486,565 fragments curated from commercial sources, to simultaneously reconstruct both SMILES and chemical fingerprints. To explore its utility in fragment design, we applied transfer learning to the fingerprint decoder layers to train a classifier using 66 frequent hitter fragments identified from our screening campaigns. Using a particle swarm optimization sampling approach, we compare the performance of this "dual" model to an architecture encoding SMILES only. The dual model produced valid SMILES with improved features, considering a range of properties including aromatic ring counts, heavy atom count, synthetic accessibility, and a new fragment complexity score we term Feature Complexity (FeCo). Additionally, we demonstrate that generative performance is further enhanced by use of a simple syntax-correction procedure during training, in which invalid and undesirable SMILES are spiked into the training set. Finally, we used the syntax-corrected model to generate a library of novel candidate privileged fragments.

Structure-based design, synthesis and biological evaluation of a novel series of isoquinolone and pyrazolo[4,3-c]pyridine inhibitors of fascin 1 as potential anti-metastatic agents.

Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies.

Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials.

Identification of a Selective G1-Phase Benzimidazolone Inhibitor by a Senescence-Targeted Virtual Screen Using Artificial Neural Networks.

Cellular senescence is a barrier to tumorigenesis in normal cells, and tumor cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning-based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~2M lead-like compounds. One hundred and forty-seven virtual hits were acquired for validation in growth inhibition and senescence-associated ß-galactosidase assays. Among the found hits, a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced senescence-associated ß-galactosidase activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1, and CDC25C. In addition, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long-term treatments. Preliminary structure-activity and structure clustering analyses are reported, and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor-like profile in normal cells, with different pathways affected in cancer cells.

Fragment-based hit identification: thinking in 3D.

The identification of high-quality hits during the early phases of drug discovery is essential if projects are to have a realistic chance of progressing into clinical development and delivering marketed drugs. As the pharmaceutical industry goes through unprecedented change, there are increasing opportunities to collaborate via pre-competitive networks to marshal multifunctional resources and knowledge to drive impactful, innovative science. The 3D Fragment Consortium is developing fragment-screening libraries with enhanced 3D characteristics and evaluating their effect on the quality of fragment-based hit identification (FBHI) projects.

Computational tools and resources for metabolism-related property predictions. 2. Application to prediction of half-life time in human liver microsomes.

BACKGROUND: The most important factor affecting metabolic excretion of compounds from the body is their half-life time. This provides an indication of compound stability of, for example, drug molecules. We report on our efforts to develop QSAR models for metabolic stability of compounds, based on in vitro half-life assay data measured in human liver microsomes. METHOD: A variety of QSAR models generated using different statistical methods and descriptor sets implemented in both open-source and commercial programs (KNIME, GUSAR and StarDrop) were analyzed. The models obtained were compared using four different external validation sets from public and commercial data sources, including two smaller sets of in vivo half-life data in humans. CONCLUSION: In many cases, the accuracy of prediction achieved on one external test set did not correspond to the results achieved with another test set. The most predictive models were used for predicting the metabolic stability of compounds from the open NCI database, the results of which are publicly available on the NCI/CADD Group web server ( http://cactus.nci.nih.gov ).

Computational tools and resources for metabolism-related property predictions. 1. Overview of publicly available (free and commercial) databases and software.

Metabolism has been identified as a defining factor in drug development success or failure because of its impact on many aspects of drug pharmacology, including bioavailability, half-life and toxicity. In this article, we provide an outline and descriptions of the resources for metabolism-related property predictions that are currently either freely or commercially available to the public. These resources include databases with data on, and software for prediction of, several end points: metabolite formation, sites of metabolic transformation, binding to metabolizing enzymes and metabolic stability. We attempt to place each tool in historical context and describe, wherever possible, the data it was based on. For predictions of interactions with metabolizing enzymes, we show a typical set of results for a small test set of compounds. Our aim is to give a clear overview of the areas and aspects of metabolism prediction in which the currently available resources are useful and accurate, and the areas in which they are inadequate or missing entirely.

Software and resources for computational medicinal chemistry.

Computer-aided drug design plays a vital role in drug discovery and development and has become an indispensable tool in the pharmaceutical industry. Computational medicinal chemists can take advantage of all kinds of software and resources in the computer-aided drug design field for the purposes of discovering and optimizing biologically active compounds. This article reviews software and other resources related to computer-aided drug design approaches, putting particular emphasis on structure-based drug design, ligand-based drug design, chemical databases and chemoinformatics tools.

Eriodictyol inhibits RSK2-ATF1 signaling and suppresses EGF-induced neoplastic cell transformation.

RSK2 is a widely expressed serine/threonine kinase, and its activation enhances cell proliferation. Here, we report that ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signaling plays an important role in EGF-induced neoplastic cell transformation. RSK2 phosphorylated ATF1 at Ser-63 and enhanced ATF1 transcriptional activity. Docking experiments using the crystal structure of the RSK2 N-terminal kinase domain combined with in vitro pulldown assays demonstrated that eriodictyol, a flavanone found in fruits, bound with the N-terminal kinase domain of RSK2 to inhibit RSK2 N-terminal kinase activity. In cells, eriodictyol inhibited phosphorylation of ATF1 but had no effect on the phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically suppresses RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation and tumor promoter-induced transformation of JB6 Cl41 cells. Eriodictyol or knockdown of RSK2 or ATF1 also suppressed Ras-mediated focus formation. Overall, these results indicate that RSK2-ATF1 signaling plays an important role in neoplastic cell transformation and that eriodictyol is a novel natural compound for suppressing RSK2 kinase activity.

Resveratrol, a red wine polyphenol, suppresses pancreatic cancer by inhibiting leukotriene A4hydrolase.

The anticancer effects of red wine have attracted considerable attention. Resveratrol (3,5,4'-trihydroxy-trans -stilbene) is a well-known polyphenolic compound of red wine with cancer chemopreventive activity. However, the basis for this activity is unclear. We studied leukotriene A(4) hydrolase (LTA(4)H) as a relevant target in pancreatic cancer. LTA(4)H knockdown limited the formation of leukotriene B(4) (LTB(4)), the enzymatic product of LTA(4)H, and suppressed anchorage-independent growth of pancreatic cancer cells. An in silico shape similarity algorithm predicted that LTA(4)H might be a potential target of resveratrol. In support of this idea, we found that resveratrol directly bound to LTA(4)H in vitro and in cells and suppressed proliferation and anchorage-independent growth of pancreatic cancer by inhibiting LTB(4) production and expression of the LTB(4) receptor 1 (BLT(1)). Notably, resveratrol exerted relatively stronger inhibitory effects than bestatin, an established inhibitor of LTA(4)H activity, and the inhibitory effects of resveratrol were reduced in cells where LTA(4)H was suppressed by shRNA-mediated knockdown. Importantly, resveratrol inhibited tumor formation in a xenograft mouse model of human pancreatic cancer by inhibiting LTA(4)H activity. Our findings identify LTA(4)H as a functionally important target for mediating the anticancer properties of resveratrol.

Role of NEK6 in tumor promoter-induced transformation in JB6 C141 mouse skin epidermal cells.

NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that overexpression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. Our data also show that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.

Anti-cancer gallotannin penta-O-galloyl-beta-D-glucose is a nanomolar inhibitor of select mammalian DNA polymerases.

Penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG) has been shown by us and others to inhibit the in vivo growth of human prostate cancer (PCa) xenografts in athymic nude mice and mouse lung cancer allograft in syngenic mice without evident adverse effect on their body weight. We observed a rapid inhibition of DNA synthesis in S-phase cells in PGG-exposed cancer cells and in PGG-treated isolated nuclei. The purpose of the present study was to test the hypothesis that PGG inhibits DNA replicative synthesis through a direct inhibition of one or more DNA polymerases (pols). Using purified pols, we show that PGG exhibited a selective inhibition against the activities of B-family replicative pols (alpha, delta and epsilon) and Y-family (eta, iota and kappa) of bypass synthesis pols, and the inhibitory effect of PGG on pol alpha was the strongest with IC(50) value of 13 nM. PGG also inhibited pol beta, but the potency was an order of magnitude less than against pol alpha. PGG inhibition of pol alpha and kappa activity was non-competitive with respect to the DNA template-primer and the dNTP substrate; whereas it inhibited pol beta competitively. Docking simulation on pol beta, which is the only mammalian pol with solved crystal structure, suggests several favorable interactions with the catalytic pocket/binding site for the incoming dNTP. These results support PGG as a novel inhibitor of select families of mammalian pols by distinct mechanisms, and suggest that the potent pol inhibition may contribute to its anti-cancer efficacy.

[6]-Gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase.

[6]-Gingerol, a natural component of ginger, exhibits anti-inflammatory and antitumorigenic activities. Despite its potential efficacy in cancer, the mechanism by which [6]-gingerol exerts its chemopreventive effects remains elusive. The leukotriene A(4) hydrolase (LTA(4)H) protein is regarded as a relevant target for cancer therapy. Our in silico prediction using a reverse-docking approach revealed that LTA(4)H might be a potential target of [6]-gingerol. We supported our prediction by showing that [6]-gingerol suppresses anchorage-independent cancer cell growth by inhibiting LTA(4)H activity in HCT116 colorectal cancer cells. We showed that [6]-gingerol effectively suppressed tumor growth in vivo in nude mice, an effect that was mediated by inhibition of LTA(4)H activity. Collectively, these findings indicate a crucial role of LTA(4)H in cancer and also support the anticancer efficacy of [6]-gingerol targeting of LTA(4)H for the prevention of colorectal cancer.

A regulatory mechanism for RSK2 NH(2)-terminal kinase activity.

Our previous findings indicated that RSK2 plays a critical role in proliferation and cell transformation induced by tumor promoters, such as epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate, and that kaempferol, a natural compound found in edible plants, selectively inhibits RSK2 activity. However, the molecular mechanism for RSK2 activation is unclear. Herein, we provide evidence showing that NH(2)-terminal kinase domain (NTD) activation of RSK2 is required for the activation of the extracellular signal-regulated kinase-mediated COOH-terminal kinase domain (CTD). We also found that the NTD plays a key role in substrate phosphorylation and that kaempferol binds with the NTD but not the CTD in both the active and inactive forms. Homology modeling of the RSK2 NH(2)-terminal domain and small-molecule docking, validated by mutagenesis experiments, clearly showed that Val(82) and Lys(100) are critical amino acids for kaempferol binding and RSK2 activity. Furthermore, immunohistofluorescence and Western blot results indicated that the RSK2 protein level is markedly higher in cancer cell lines as well as cancer tissues compared with nonmalignant cell lines or normal tissues. In addition, kaempferol inhibited proliferation of malignant human cancer cell lines, including A431, SK-MEL-5 and SK-MEL-28, and HCT-116. These results indicate that targeting RSK2 with natural compounds, such as kaempferol, might be a good strategy for chemopreventive or chemotherapeutic application.

Cyclin-dependent kinase-3-mediated c-Jun phosphorylation at Ser63 and Ser73 enhances cell transformation.

c-Jun is a component of the activator protein-1 (AP-1) complex, which plays a crucial role in the regulation of gene expression, cell proliferation, and cell transformation, as well as cancer development. Herein, we found that cyclin-dependent kinase (Cdk)-3, but not Cdk2 or c-Jun NH(2)-terminal kinase, is a novel kinase of c-Jun induced by stimulation with growth factors such as epidermal growth factor (EGF). Cdk3 was shown to phosphorylate c-Jun at Ser63 and Ser73 in vitro and ex vivo. EGF-induced Cdk3 activation caused c-Jun phosphorylation at Ser63 and Ser73, resulting in increased AP-1 transactivation. Ectopic expression of Cdk3 resulted in anchorage-independent cell transformation of JB6 Cl41 cells induced by EGF and foci formation stimulated by constitutively active Ras (Ras(G12V)), which was mediated by AP-1 in NIH3T3 cells. These results showed that the Cdk3/c-Jun signaling axis plays an important role in EGF-stimulated cell proliferation and cell transformation.

The resveratrol analogue 3,5,3',4',5'-pentahydroxy-trans-stilbene inhibits cell transformation via MEK.

Resveratrol, present in grapes and red wine, is reported to be a natural chemopreventive agent against cancer. However, the concentrations required to exert these effects may be difficult to achieve by drinking only 1 or 2 glasses of red wine a day. Therefore, developing more potent, nontoxic analogues of resveratrol may provide a feasible means of achieving an effective physiologic concentration. Here we report that the resveratrol analogue, 3,5,3',4',5'-pentahydroxy-trans-stilbene (RSVL2), inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation in JB6 P+ mouse epidermal cells. Further, we identified MEK/ERK signaling as the direct molecular target for the anticancer effects of RSVL2 and demonstrated that RSVL2 inhibited MEK1, but not Raf1 or ERK2 kinase activity. RSVL2 also dose-dependently suppressed MEK1 kinase activity induced by TPA and the inhibition of H-Ras-induced cell transformation was much stronger for RSVL2 than for PD098059 or resveratrol. Both in vitro and ex vivo pull-down assays indicated that RSVL2, but not resveratrol, directly bound with GST-MEK1, but did not compete with ATP for binding. Docking data indicated that the low inhibitory activity of resveratrol might be due to the lack of the hydroxyl group at the meta position of the B ring, thereby preventing resveratrol from forming a hydrogen bond with the backbone amide group of Ser212, which is the key interaction for stabilizing the inactive conformation of the activation loop.

(-)-Epigallocatechin gallate regulates CD3-mediated T cell receptor signaling in leukemia through the inhibition of ZAP-70 kinase.

The zeta chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 micromol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cgamma1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells.