Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.     

Searching for the most effective screening system to identify cell-active inhibitors of beta-secretase

The beta-secretase BACE1 is an attractive drug target for reducing the level of the Alzheimer's disease-promoting Abeta peptide in the brain. Whereas potent peptidomimetic in vitro inhibitors of BACE1 have been designed, screening approaches to identify cell-permeable small molecule inhibitors have had limited success so far. In the present minireview we summarize existing screening methods, discuss their scope of application in the drug discovery process and compare them to a novel cell-based screening system to identify BACE1 inhibitors by a positive yeast growth selection.     

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K_i value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.     

Activities of multiple cancer-related pathways are associated with BRAF mutation and predict the resistance to BRAF/MEK inhibitors in melanoma cells

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.     

C57BL/6 congenic mouse NRASQ61K melanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo

RAS is frequently mutated in various tumors and known to be difficult to target. NRAS^Q61K/R are the second most frequent mutations found in human skin melanoma after BRAF^V600E. Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRAS^Q61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRAS^Q61K mouse melanoma that can be used in vitro and in vivo in an immune‐competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRAS^Q61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses.     

Virtual screening based identification of novel small-molecule inhibitors targeted to the HIV-1 capsid

The hydrophobic cavity of the C-terminal domain (CTD) of HIV-1 capsid has been recently validated as potential target for antiviral drugs by peptide-based inhibitors; however, there is no report yet of any small molecule compounds that target this hydrophobic cavity. In order to fill this gap and discover new classes of ant-HIV-1 inhibitors, we undertook a docking-based virtual screening and subsequent analog search, and medicinal chemistry approaches to identify small molecule inhibitors against this target. This article reports for the first time, to the best of our knowledge, identification of diverse classes of inhibitors that efficiently inhibited the formation of mature-like viral particles verified under electron microscope (EM) and showed potential as anti-HIV-1 agents in a viral infectivity assay against a wide range of laboratory-adapted as well as primary isolates in MT-2 cells and PBMC. In addition, the virions produced after the HIV-1 infected cells were treated with two of the most active compounds showed drastically reduced infectivity confirming the potential of these compounds as anti-HIV-1 agents. We have derived a comprehensive SAR from the antiviral data. The SAR analyses will be useful in further optimizing the leads to potential anti-HIV-1 agents.     

Searching new structural scaffolds for BRAF inhibitors. An integrative study using theoretical and experimental techniques

The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes.Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.     

Combinations of EGFR and MET inhibitors reduce proliferation and invasiveness of mucosal melanoma cells

Mucosal melanoma (MM) is a very rare and aggressive type of cancer for which immunotherapy or targeted therapy such as BRAF/MEK inhibitors, used in cutaneous melanoma, usually fail. Due to our earlier experience showing the high effectiveness of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) inhibitors in reducing the activation of the MAPK and PI3K/AKT signalling pathways, we aim to test whether these drugs would also be effective for mucosal melanoma. Cells representing two commercially available mucosal melanoma cell lines (GAK and HMVII) and one cell line obtained from a patient's vaginal melanoma were treated with MET or EGFR inhibitors, or combinations of these agents. The dual-inhibitor treatment strategy resulted in a decrease of cell proliferation, migration and invasion. Moreover, combinations of inhibitors led to reduction of pEGFR/EGFR and pMET/MET ratio and downregulation of PI3K/AKT and MEK/ERK1/2-based signalling pathways. Our findings indicate a potential therapeutic strategy based on EGFR and MET inhibitors in mucosal melanoma, which should be further evaluated in vivo and in clinical experiments. They also suggest that targeting multiple receptor tyrosine kinases may block signalling crosstalk and possibly delay the appearance of resistance to kinase inhibitors in mucosal melanoma cells.     

Emerging frontiers in virtual drug discovery: From quantum mechanical methods to deep learning approaches

Virtual screening-based approaches to discover initial hit and lead compounds have the potential to reduce both the cost and time of early drug discovery stages, as well as to find inhibitors for even challenging target sites such as protein–protein interfaces. Here in this review, we provide an overview of the progress that has been made in virtual screening methodology and technology on multiple fronts in recent years. The advent of ultra-large virtual screens, in which hundreds of millions to billions of compounds are screened, has proven to be a powerful approach to discover highly potent hit compounds. However, these developments are just the tip of the iceberg, with new technologies and methods emerging to propel the field forward. Examples include novel machine-learning approaches, which can reduce the computational costs of virtual screening dramatically, while progress in quantum-mechanical approaches can increase the accuracy of predictions of various small molecule properties.     

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors

Background

Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies.

Predicting inactive conformations of protein kinases using active structures: conformational selection of type-II inhibitors

Protein kinases have been found to possess two characteristic conformations in their activation-loops: the active DFG-in conformation and the inactive DFG-out conformation. Recently, it has been very interesting to develop type-II inhibitors which target the DFG-out conformation and are more specific than the type-I inhibitors binding to the active DFG-in conformation. However, solving crystal structures of kinases with the DFG-out conformation remains a challenge, and this seriously hampers the application of the structure-based approaches in development of novel type-II inhibitors. To overcome this limitation, here we present a computational approach for predicting the DFG-out inactive conformation using the DFG-in active structures, and develop related conformational selection protocols for the uses of the predicted DFG-out models in the binding pose prediction and virtual screening of type-II ligands. With the DFG-out models, we predicted the binding poses for known type-II inhibitors, and the results were found in good agreement with the X-ray crystal structures. We also tested the abilities of the DFG-out models to recognize their specific type-II inhibitors by screening a database of small molecules. The AUC (area under curve) results indicated that the predicted DFG-out models were selective toward their specific type-II inhibitors. Therefore, the computational approach and protocols presented in this study are very promising for the structure-based design and screening of novel type-II kinase inhibitors.     

Sirtuin deacetylases: A new target for melanoma management

Melanoma continues to cause more deaths than any other skin cancer, necessitating the development of new avenues of treatment. One promising new opportunity comes in the form of mechanism-based therapeutic targets. We recently reported the overexpression and delocalization of the class III histone deacetylase SIRT1 in melanoma, and demonstrated that its small molecule inhibition via Tenovin-1 decreased cell growth and viability of melanoma cells, possibly by a p53 mediated induction of p21. Here, we support our data using additional SIRT inhibitors, viz. Sirtinol and Ex-527, which suggests possible benefits of concomitantly inhibiting more than one Sirtuin for an effective cancer management strategy. This “Extra View” paper also includes a discussion of our results in the context of similar recent and concurrent studies. Furthermore, we expand upon our findings in an analysis of new research that may link the cellular localization and growth effects of SIRT1 with the PI3K signaling pathway.     

Des-A-ring benzothiadiazines: inhibitors of HCV genotype 1 NS5B RNA-dependent RNA polymerase

In our program to discover non-nucleoside, small molecule inhibitors of genotype 1 HCV polymerase, we investigated a series of promising analogs based on a benzothiadiazine screening hit that contains an ABCD ring system. After demonstrating that a methylsulfonylamino D-ring substituent increased the enzyme potency into the low nanomolar range, we explored a minimum core required for activity by truncating to a three-ring system. Described herein are the syntheses and structure-activity relationship of a set of inhibitors lacking the A-ring of an ABCD ring system. We observed that small aromatic rings and alkenyl groups appended to the 5-position of the B-ring were optimal, resulting in inhibitors with low nanomolar potencies.     

Laboratory scale structural genomics

At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.     

Purification of Ribonuclease L from Aspergillus sp. by Affinity Chromatography

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3′-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

Novel screening method for potential skin-whitening compounds by a luciferase reporter assay

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high‐throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard‐of‐care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma.     

Enhancing the evaluation of PI3K inhibitors through 3D melanoma models

Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two‐ and three‐dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti‐invasive potential of PI3K inhibitors and that drugs such as PX‐866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E‐BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.