Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

A Patient-Derived Cell Atlas Informs Precision Targeting of Glioblastoma.

Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells. We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.

High content drug screening for Fanconi anemia therapeutics.

BACKGROUND: Fanconi anemia is a rare disease clinically characterized by malformations, bone marrow failure and an increased risk of solid tumors and hematologic malignancies. The only therapies available are hematopoietic stem cell transplantation for bone marrow failure or leukemia, and surgical resection for solid tumors. Therefore, there is still an urgent need for new therapeutic options. With this aim, we developed a novel high-content cell-based screening assay to identify drugs with therapeutic potential in FA. RESULTS: A TALEN-mediated FANCA-deficient U2OS cell line was stably transfected with YFP-FANCD2 fusion protein. These cells were unable to form fluorescent foci or to monoubiquitinate endogenous or exogenous FANCD2 upon DNA damage and were more sensitive to mitomycin C when compared to the parental wild type counterpart. FANCA correction by retroviral infection restored the cell line's ability to form FANCD2 foci and ubiquitinate FANCD2. The feasibility of this cell-based system was interrogated in a high content screening of 3802 compounds, including a Prestwick library of 1200 FDA-approved drugs. The potential hits identified were then individually tested for their ability to rescue FANCD2 foci and monoubiquitination, and chromosomal stability in the absence of FANCA. CONCLUSIONS: While, unfortunately, none of the compounds tested were able to restore cellular FANCA-deficiency, our study shows the potential capacity to screen large compound libraries in the context of Fanconi anemia therapeutics in an optimized and cost-effective platform.

Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin.

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.

A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family.

The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.

Diverse heterocyclic scaffolds as dCTP pyrophosphatase 1 inhibitors. Part 1: Triazoles, triazolopyrimidines, triazinoindoles, quinoline hydrazones and arylpiperazines.

A high-throughput screening campaign using a commercial compound library (ChemBridge DiverSET) revealed diverse chemotypes as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). Triazole, triazolopyrimidine, triazinoindole, quinoline hydrazone and arylpiperazine hits were clustered, confirmed by IC50 determinations, and their preliminary structure-activity-relationships (SAR) and ligand efficiency scores are discussed in this letter.

Diverse heterocyclic scaffolds as dCTP pyrophosphatase 1 inhibitors. Part 2: Pyridone- and pyrimidinone-derived systems.

Two screening campaigns using commercial (Chembridge DiverSET) and proprietary (Chemical Biology Consortium Sweden, CBCS) compound libraries, revealed a number of pyridone- and pyrimidinone-derived systems as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). In this letter, we present their preliminary structure-activity-relationships (SAR) and ligand efficiency scores (LE and LLE).

Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1.

The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.

Case-specific potentiation of glioblastoma drugs by pterostilbene.

Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Comparative drug pair screening across multiple glioblastoma cell lines reveals novel drug-drug interactions.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults, and despite state-of-the-art treatment, survival remains poor and novel therapeutics are sorely needed. The aim of the present study was to identify new synergistic drug pairs for GBM. In addition, we aimed to explore differences in drug-drug interactions across multiple GBM-derived cell cultures and predict such differences by use of transcriptional biomarkers. METHODS: We performed a screen in which we quantified drug-drug interactions for 465 drug pairs in each of the 5 GBM cell lines U87MG, U343MG, U373MG, A172, and T98G. Selected interactions were further tested using isobole-based analysis and validated in 5 glioma-initiating cell cultures. Furthermore, drug interactions were predicted using microarray-based transcriptional profiling in combination with statistical modeling. RESULTS: Of the 5 × 465 drug pairs, we could define a subset of drug pairs with strong interaction in both standard cell lines and glioma-initiating cell cultures. In particular, a subset of pairs involving the pharmaceutical compounds rimcazole, sertraline, pterostilbene, and gefitinib showed a strong interaction in a majority of the cell cultures tested. Statistical modeling of microarray and interaction data using sparse canonical correlation analysis revealed several predictive biomarkers, which we propose could be of importance in regulating drug pair responses. CONCLUSION: We identify novel candidate drug pairs for GBM and suggest possibilities to prospectively use transcriptional biomarkers to predict drug interactions in individual cases.

Localisation of nursery areas based on comparative analyses of the horizontal and vertical distribution patterns of juvenile Baltic cod (Gadus morhua).

Knowledge of the spatial distribution of juvenile cod is essential for obtaining precise recruitment data to conduct sustainable management of the eastern and western Baltic cod stocks. In this study, the horizontal and vertical distribution and density patterns of settled juvenile 0- and 1-group Baltic cod are determined, and their nursery areas are localised according to the environmental factors affecting them. Comparative statistical analyses of biological, hydrographic and hydroacoustic data are carried out based on standard ICES demersal trawl surveys and special integrated trawl and acoustic research surveys. Horizontal distribution maps for the 2001-2010 cohorts of juvenile cod are further generated by applying a statistical log-Gaussian Cox process model to the standard trawl survey data. The analyses indicate size-dependent horizontal and distinct vertical and diurnal distribution patterns related to the seabed topography, water layer depth, and the presence of hydrographic frontal zones (pycnoclines) as well as intraspecific patterns in relation to the presence of adult cod. The extent of the nursery areas also depends on the cod year class strength. Juvenile cod (≥3 cm) are present in all areas of the central Baltic Sea (CBS), showing broad dispersal. However, their highest density in the Baltic Basins is found at localities with a 40-70 m bottom depth in waters with oxygen concentrations above 2 ml O2.l⁻¹ and temperatures above 5°C. The smallest juveniles are also found in deep sea localities down to a 100 m depth and at oxygen concentrations between 2-4 ml O2.l⁻¹. The vertical, diurnally stratified and repeated trawling and hydroacoustic target strength-depth distributions obtained from the special surveys show juvenile cod concentrations in frontal zone water layers (pycnocline). However, the analyses indicate that in the CBS, juvenile cod of all sizes do not appear to aggregate in dense schooling patterns, which differs from what has been reported from the North Sea.

Retinoic acid receptor alpha is associated with tamoxifen resistance in breast cancer.

About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen.

Shadow effects in simulated ultrasound images derived from computed tomography images using a focused beam tracing model.

Simulation of ultrasound images based on computed tomography (CT) data has previously been performed with different approaches. Shadow effects are normally pronounced in ultrasound images, so they should be included in the simulation. In this study, a method to capture the shadow effects has been developed, which makes the simulated ultrasound images appear more realistic. The method using a focused beam tracing model gives diffuse shadows that are similar to the ones observed in measurements on real objects. Ultrasound images of a cod (Gadus morhua) were obtained with a BK Medical 2202 ProFocus ultrasound scanner (BK Medical, Herlev, Denmark) equipped with a dedicated research interface giving access to beamformed radio frequency data. CT images were obtained with an Aquilion ONE Toshiba CT scanner (Toshiba Medical Systems Corp., Tochigi, Japan). CT data were mapped from Hounsfield units to backscatter strength, attenuation coefficients, and characteristic acoustic impedance. The focused beam tracing model was used to create maps of the transmission coefficient and scattering strength maps. Field II was then used to simulate an ultrasound image of 38.9 × 55.3 × 4.5 mm, using 10(6) point scatterers. As there is no quantitative method to assess quality of a simulated ultrasound image compared to a measured one, visual inspection was used for evaluation.

A proteomics approach to the study of absorption, distribution, metabolism, excretion, and toxicity.

A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.

The expression of cytosolic and mitochondrial type II acyl-CoA thioesterases is upregulated in the porcine corpus luteum during pregnancy.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to free fatty acids and CoASH, thereby regulating fatty acid metabolism. This activity is catalyzed by numerous structurally related and unrelated enzymes, of which several acyl-CoA thioesterases have been shown to be regulated via the peroxisome proliferator-activated receptor alpha, strongly linking them to fatty acid metabolism. Two protein families have recently been characterized, the type I acyl-CoA thioesterase gene family and the type II protein family, which are expressed in cytosol, mitochondria and peroxisomes. Still, only little is known about regulation of their expression and precise functions in vivo. In the present study, we have investigated the activity and expression of acyl-CoA thioesterase in the porcine ovary during different phases of the estrus cycle. The activity was low in homogenates obtained during the immature and follicular phases, increasing nearly 4-fold during the luteal phase, with the highest activity being found in the pregnant corpus luteum (about 7-fold higher than in immature follicles). The increase in homogenate activity in corpus luteum from pregnant pigs was due to a moderate increase in the cytosolic activity, and an approximately 20-25-fold increase in the mitochondrial fraction. Western blot analysis showed no detectable expression of the type I acyl-CoA thioesterases (CTE-I and MTE-I) and revealed that the increased activity in cytosol and mitochondria is due to increased expression of the type II acyl-CoA thioesterases (CTE-II and MTE-II). This apparent hormonal regulation of expression of the type II acyl-CoA thioesterase may provide new insights into the functions of these enzymes in the mammalian ovary.

Oxidation of protein tyrosine phosphatases as a pharmaceutical mechanism of action: a study using 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione.

Growth factor and insulin signal transduction comprise series of protein kinases and protein phosphatases whose combined activities serve to propagate the growth factor signal in a regulated fashion. It was shown previously that such signaling cascades generate hydrogen peroxide inside cells. Recent work has implied that one function of this might be to enhance the feed-forward signal through the reversible oxidation and inhibition of protein tyrosine phosphatases (PTPs). We identified compound 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione (BVT.948) as an agent that is able to inhibit PTP activity in vitro noncompetitively, a mechanism involving oxidation of the catalytic cysteine residue. We investigated the pharmaceutical utility of this compound by examining its effects in a series of in vitro cellular and in vivo assays. Results showed that BVT.948 was able to enhance insulin signaling in cells, although it did not increase tyrosine phosphorylation globally. Furthermore, the compound was active in vivo, enhancing insulin tolerance tests in ob/ob mice, therefore apparently enhancing insulin sensitivity. BVT.948 was able to inhibit several other PTPs tested and also was efficient at inhibiting several cytochrome P450 (P450) isoforms in vitro. The data suggest that inhibitors of PTPs that display noncompetitive kinetics must be viewed with caution because they may oxidize the enzyme irreversibly. Furthermore, although such compounds display interesting biological effects in vitro and in vivo, their general pharmaceutical utility may be limited due to undesired effects on P450 enzymes.

Comparison of radiance and polarization values observed in the Mediterranean Sea and simulated in a Monte Carlo model.

Measurements of the radiance and degree of polarization made in 1971 in the Mediterranean Sea are presented along with the simulation of all observed quantities by a Monte Carlo technique. It is shown that our independent scattering treatment utilizing a Stokes vector formalism to describe the polarization state of the light field produces remarkably good agreement with those values measured in situ.