Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Deep Modeling of Regulating Effects of Small Molecules on Longevity-Associated Genes.

Aging is considered an inevitable process that causes deleterious effects in the functioning and appearance of cells, tissues, and organs. Recent emergence of large-scale gene expression datasets and significant advances in machine learning techniques have enabled drug repurposing efforts in promoting longevity. In this work, we further developed our previous approach-DeepCOP, a quantitative chemogenomic model that predicts gene regulating effects, and extended its application across multiple cell lines presented in LINCS to predict aging gene regulating effects induced by small molecules. As a result, a quantitative chemogenomic Deep Model was trained using gene ontology labels, molecular fingerprints, and cell line descriptors to predict gene expression responses to chemical perturbations. Other state-of-the-art machine learning approaches were also evaluated as benchmarks. Among those, the deep neural network (DNN) classifier has top-ranked known drugs with beneficial effects on aging genes, and some of these drugs were previously shown to promote longevity, illustrating the potential utility of this methodology. These results further demonstrate the capability of "hybrid" chemogenomic models, incorporating quantitative descriptors from biomarkers to capture cell specific drug-gene interactions. Such models can therefore be used for discovering drugs with desired gene regulatory effects associated with longevity.

Drugging the 'undruggable'. Therapeutic targeting of protein-DNA interactions with the use of computer-aided drug discovery methods.

Transcription factors (TFs) act as major oncodrivers in many cancers and are frequently regarded as high-value therapeutic targets. The functionality of TFs relies on direct protein-DNA interactions, which are notoriously difficult to target with small molecules. However, this prior view of the 'undruggability' of protein-DNA interfaces has shifted substantially in recent years, in part because of significant advances in computer-aided drug discovery (CADD). In this review, we highlight recent examples of successful CADD campaigns resulting in drug candidates that directly interfere with protein-DNA interactions of several key cancer TFs, including androgen receptor (AR), ETS-related gene (ERG), MYC, thymocyte selection-associated high mobility group box protein (TOX), topoisomerase II (TOP2), and signal transducer and activator of transcription 3 (STAT3). Importantly, these findings open novel and compelling avenues for therapeutic targeting of over 1600 human TFs implicated in many conditions including and beyond cancer.

Deep Docking: A Deep Learning Platform for Augmentation of Structure Based Drug Discovery.

Drug discovery is a rigorous process that requires billion dollars of investments and decades of research to bring a molecule "from bench to a bedside". While virtual docking can significantly accelerate the process of drug discovery, it ultimately lags the current rate of expansion of chemical databases that already exceed billions of molecular records. This recent surge of small molecules availability presents great drug discovery opportunities, but also demands much faster screening protocols. In order to address this challenge, we herein introduce Deep Docking (DD), a novel deep learning platform that is suitable for docking billions of molecular structures in a rapid, yet accurate fashion. The DD approach utilizes quantitative structure-activity relationship (QSAR) deep models trained on docking scores of subsets of a chemical library to approximate the docking outcome for yet unprocessed entries and, therefore, to remove unfavorable molecules in an iterative manner. The use of DD methodology in conjunction with the FRED docking program allowed rapid and accurate calculation of docking scores for 1.36 billion molecules from the ZINC15 library against 12 prominent target proteins and demonstrated up to 100-fold data reduction and 6000-fold enrichment of high scoring molecules (without notable loss of favorably docked entities). The DD protocol can readily be used in conjunction with any docking program and was made publicly available.

Rapid Identification of Potential Inhibitors of SARS-CoV-2 Main Protease by Deep Docking of 1.3†Billion Compounds.

The recently emerged 2019 Novel Coronavirus (SARS-CoV-2) and associated COVID-19 disease cause serious or even fatal respiratory tract infection and yet no approved therapeutics or effective treatment is currently available to effectively combat the outbreak. This urgent situation is pressing the world to respond with the development of novel vaccine or a small molecule therapeutics for SARS-CoV-2. Along these efforts, the structure of SARS-CoV-2 main protease (Mpro) has been rapidly resolved and made publicly available to facilitate global efforts to develop novel drug candidates. Recently, our group has developed a novel deep learning platform - Deep Docking (DD) which provides fast prediction of docking scores of Glide (or any other docking program) and, hence, enables structure-based virtual screening of billions of purchasable molecules in a short time. In the current study we applied DD to all 1.3 billion compounds from ZINC15 library to identify top 1,000 potential ligands for SARS-CoV-2 Mpro protein. The compounds are made publicly available for further characterization and development by scientific community.

ETS transcription factors as emerging drug targets in cancer.

The ETS family of proteins consists of 28 transcription factors, many of which have been implicated in development and progression of a variety of cancers. While one family member, ERG, has been rigorously studied in the context of prostate cancer where it plays a critical role, other ETS factors keep emerging as potential hallmark oncodrivers. In recent years, numerous studies have reported initial discoveries of small molecule inhibitors of ETS proteins and opened novel avenues for ETS-directed cancer therapies. This review summarizes the state of the art data on therapeutic targeting of ETS family members and highlights the corresponding drug discovery strategies.

DeepCOP: deep learning-based approach to predict gene regulating effects of small molecules.

MOTIVATION: Recent advances in the areas of bioinformatics and chemogenomics are poised to accelerate the discovery of small molecule regulators of cell development. Combining large genomics and molecular data sources with powerful deep learning techniques has the potential to revolutionize predictive biology. In this study, we present Deep gene COmpound Profiler (DeepCOP), a deep learning based model that can predict gene regulating effects of low-molecular weight compounds. This model can be used for direct identification of a drug candidate causing a desired gene expression response, without utilizing any information on its interactions with protein target(s). RESULTS: In this study, we successfully combined molecular fingerprint descriptors and gene descriptors (derived from gene ontology terms) to train deep neural networks that predict differential gene regulation endpoints collected in LINCS database. We achieved 10-fold cross-validation RAUC scores of and above 0.80, as well as enrichment factors of >5. We validated our models using an external RNA-Seq dataset generated in-house that described the effect of three potent antiandrogens (with different modes of action) on gene expression in LNCaP prostate cancer cell line. The results of this pilot study demonstrate that deep learning models can effectively synergize molecular and genomic descriptors and can be used to screen for novel drug candidates with the desired effect on gene expression. We anticipate that such models can find a broad use in developing novel cancer therapeutics and can facilitate precision oncology efforts. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computer-Aided Discovery of Small Molecule Inhibitors of Thymocyte Selection-Associated High Mobility Group Box Protein (TOX) as Potential Therapeutics for Cutaneous T-Cell Lymphomas.

Cutaneous T-cell lymphomas (CTCL) are the most common primary lymphomas of the skin. We have previously identified thymocyte selection-associated high mobility group (HMG) box protein (TOX) as a promising drug target in CTCL; however, there are currently no small molecules able to directly inhibit TOX. We aimed to address this unmet opportunity by developing anti-TOX therapeutics with the use of computer-aided drug discovery methods. The available NMR-resolved structure of the TOX protein was used to model its DNA-binding HMG-box domain. To investigate the druggability of the corresponding protein-DNA interface on TOX, we performed a pilot virtual screening of 200,000 small molecules using in silico docking and identified 'hot spots' for drug-binding on the HMG-box domain. We then performed a large-scale virtual screening of 7.6 million drug-like compounds that were available from the ZINC15 database. As a result, a total of 140 top candidate compounds were selected for subsequent in vitro validation. Of those, 18 small molecules have been characterized as selective TOX inhibitors.

Toxic Colors: The Use of Deep Learning for Predicting Toxicity of Compounds Merely from Their Graphic Images.

The majority of computational methods for predicting toxicity of chemicals are typically based on "nonmechanistic" cheminformatics solutions, relying on an arsenal of QSAR descriptors, often vaguely associated with chemical structures, and typically employing "black-box" mathematical algorithms. Nonetheless, such machine learning models, while having lower generalization capacity and interpretability, typically achieve a very high accuracy in predicting various toxicity endpoints, as unambiguously reflected by the results of the recent Tox21 competition. In the current study, we capitalize on the power of modern AI to predict Tox21 benchmark data using merely simple 2D drawings of chemicals, without employing any chemical descriptors. In particular, we have processed rather trivial 2D sketches of molecules with a supervised 2D convolutional neural network (2DConvNet) and demonstrated that the modern image recognition technology results in prediction accuracies comparable to the state-of-the-art cheminformatics tools. Furthermore, the performance of the image-based 2DConvNet model was comparatively evaluated on an external set of compounds from the Prestwick chemical library and resulted in experimental identification of significant and previously unreported antiandrogen potentials for several well-established generic drugs.

Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer.

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.

Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1.

The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis.

Identification of a potent antiandrogen that targets the BF3 site of the androgen receptor and inhibits enzalutamide-resistant prostate cancer.

There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1H-indol-2-yl)-1H-indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

Orphan nuclear receptors as drug targets for the treatment of prostate and breast cancers.

Nuclear receptors (NRs), a family of 48 transcriptional factors, have been studied intensively for their roles in cancer development and progression. The presence of distinctive ligand binding sites capable of interacting with small molecules has made NRs attractive targets for developing cancer therapeutics. In particular, a number of drugs have been developed over the years to target human androgen- and estrogen receptors for the treatment of prostate cancer and breast cancer. In contrast, orphan nuclear receptors (ONRs), which in many cases lack known biological functions or ligands, are still largely under investigated. This review is a summary on ONRs that have been implicated in prostate and breast cancers, specifically retinoic acid-receptor-related orphan receptors (RORs), liver X receptors (LXRs), chicken ovalbumin upstream promoter transcription factors (COUP-TFs), estrogen related receptors (ERRs), nerve growth factor 1B-like receptors, and ''dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1'' (DAX1). Discovery and development of small molecules that can bind at various functional sites on these ONRs will help determine their biological functions. In addition, these molecules have the potential to act as prototypes for future drug development. Ultimately, the therapeutic value of targeting the ONRs may go well beyond prostate and breast cancers.

Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer.

The androgen receptor (AR) is a transcription factor that has a pivotal role in the occurrence and progression of prostate cancer. The AR is activated by androgens that bind to its ligand-binding domain (LBD), causing the transcription factor to enter the nucleus and interact with genes via its conserved DNA-binding domain (DBD). Treatment for prostate cancer involves reducing androgen production or using anti-androgen drugs to block the interaction of hormones with the AR-LBD. Eventually the disease changes into a castration-resistant form of PCa where LBD mutations render anti-androgens ineffective or where constitutively active AR splice variants, lacking the LBD, become overexpressed. Recently, we identified a surfaced exposed pocket on the AR-DBD as an alternative drug-target site for AR inhibition. Here, we demonstrate that small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length and splice variant AR forms at low to sub-micromolar concentrations. The inhibition is lost when residues involved in drug interactions are mutated. Furthermore, the compounds did not impede nuclear localization of the AR and blocked interactions with chromatin, indicating the interference of DNA binding with the nuclear form of the transcription factor. Finally, we demonstrate the inhibition of gene expression and tumor volume in mouse xenografts. Our results indicate that the AR-DBD has a surface site that can be targeted to inhibit all forms of the AR, including enzalutamide-resistant and constitutively active splice variants and thus may serve as a potential avenue for the treatment of recurrent and metastatic prostate cancer.

Prioritizing disease-linked variants, genes, and pathways with an interactive whole-genome analysis pipeline.

Whole-genome sequencing (WGS) studies are uncovering disease-associated variants in both rare and nonrare diseases. Utilizing the next-generation sequencing for WGS requires a series of computational methods for alignment, variant detection, and annotation, and the accuracy and reproducibility of annotation results are essential for clinical implementation. However, annotating WGS with up to date genomic information is still challenging for biomedical researchers. Here, we present one of the fastest and highly scalable annotation, filtering, and analysis pipeline-gNOME-to prioritize phenotype-associated variants while minimizing false-positive findings. Intuitive graphical user interface of gNOME facilitates the selection of phenotype-associated variants, and the result summaries are provided at variant, gene, and genome levels. Moreover, the enrichment results of specific variants, genes, and gene sets between two groups or compared with population scale WGS datasets that is already integrated in the pipeline can help the interpretation. We found a small number of discordant results between annotation software tools in part due to different reporting strategies for the variants with complex impacts. Using two published whole-exome datasets of uveal melanoma and bladder cancer, we demonstrated gNOME's accuracy of variant annotation and the enrichment of loss-of-function variants in known cancer pathways. gNOME Web server and source codes are freely available to the academic community (http://gnome.tchlab.org).

The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.

gSearch: a fast and flexible general search tool for whole-genome sequencing.

BACKGROUND: Various processes such as annotation and filtering of variants or comparison of variants in different genomes are required in whole-genome or exome analysis pipelines. However, processing different databases and searching among millions of genomic loci is not trivial. RESULTS: gSearch compares sequence variants in the Genome Variation Format (GVF) or Variant Call Format (VCF) with a pre-compiled annotation or with variants in other genomes. Its search algorithms are subsequently optimized and implemented in a multi-threaded manner. The proposed method is not a stand-alone annotation tool with its own reference databases. Rather, it is a search utility that readily accepts public or user-prepared reference files in various formats including GVF, Generic Feature Format version 3 (GFF3), Gene Transfer Format (GTF), VCF and Browser Extensible Data (BED) format. Compared to existing tools such as ANNOVAR, gSearch runs more than 10 times faster. For example, it is capable of annotating 52.8 million variants with allele frequencies in 6 min. AVAILABILITY: gSearch is available at http://ml.ssu.ac.kr/gSearch. It can be used as an independent search tool or can easily be integrated to existing pipelines through various programming environments such as Perl, Ruby and Python.