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BackgroundTheADAMTS7locus was genome-wide significantly associated with coronary artery disease (CAD). Lack of the extracellular matrix (ECM) protease ADAMTS-7 was shown to reduce atherosclerotic plaque formation.ObjectiveTo identify molecular mechanisms and downstream targets of ADAMTS-7 mediating risk of atherosclerosis.MethodsTargets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence,in vitrodegradation assays, co-immunoprecipitation, and Förster resonance energy transfer (FRET)-based protein-protein interaction assays.ADAMTS7expression was measured in fibrous caps of human carotid artery plaques.ResultsIn humans,ADAMTS7expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of tissue inhibitor of metalloproteases 1 (Timp-1). In co-immunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7in vitro.ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target matrix metalloprotease 9 (MMP-9) As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. In order to facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim to decrease TIMP-1 degradation, we designed a FRET-based assay targeting the ADAMTS-7 catalytic site.ConclusionADAMTS-7,which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise genome-wide significantly associated with CAD. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for reduction of atherosclerosis-related events.

The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2/COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies., Measurement(s) cytotoxicity • killing by virus of host cell Technology Type(s) bright-field microscopy Factor Type(s) concentration • incubation time Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13562570

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2 / COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.

HIV-1 infection is a complex, multi-step process involving not only viral, but also multiple cellular factors. To date, drug discovery methods have primarily focused on the inhibition of single viral proteins. We present an efficient and unbiased approach, compatible with biosafety level 1 (BSL-1) conditions, to identify inhibitors of HIV-1 reverse transcription, intracellular trafficking, nuclear entry and genome integration. Starting with a fluorescent assay setup, we systematically improved the screening methodology in terms of stability, efficiency and pharmacological relevance. Stability and throughput were optimized by switching to a luciferase-based readout. BSL-1 compliance was achieved without sacrificing pharmacological relevance by using lentiviral particles pseudo-typed with the mouse ecotropic envelope protein to transduce human PM1 T cells gene-modified to express the corresponding murine receptor. The cellular assay was used to screen 26,048 compounds selected for maximum diversity from a 200,640-compound in-house library. This yielded z’ values greater than 0.8 with a hit rate of 3.3% and a confirmation rate of 50%. We selected 93 hits and enriched the collection with 279 similar compounds from the in-house library to identify promising structural features. The most active compounds were validated using orthogonal assay formats. The similarity of the compound profiles across the different platforms demonstrated that the reported lentiviral assay system is a robust and versatile tool for the identification of novel HIV-1 inhibitors.

To identify possible candidates for progression towards clinical studies against SARS-CoV-2, we screened a well-defined collection of 5632 compounds including 3488 compounds which have undergone clinical investigations (marketed drugs, phases 1 -3, and withdrawn) across 600 indications. Compounds were screened for their inhibition of viral induced cytotoxicity using the human epithelial colorectal adenocarcinoma cell line Caco-2 and a SARS-CoV-2 isolate. The primary screen of 5632 compounds gave 271 hits. A total of 64 compounds with IC50

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (1), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure–activity relationship studies around hit compound 1. The pivaloyl derivative (13) showed significant anti-T. brucei activity (EC50 = 1.1 μM) together with a selectivity index higher than 92. The early in vitro ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and hERG inhibition) were determined for compound 1 and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.

Aberrant activation of caspase-6 (C6) in the absence of other hallmarks of apoptosis has been demonstrated in cells and tissues from patients with Huntington disease (HD) and animal models. C6 activity correlates with disease progression in patients with HD and the cleavage of mutant huntingtin (mHTT) protein is thought to strongly contribute to disease pathogenesis. Here we show that the mHTT1-586 fragment generated by C6 cleavage interacts with the zymogen form of the enzyme, stabilizing a conformation that contains an active site and is prone to full activation. This shift toward enhanced activity can be prevented by a small-molecule inhibitor that blocks the interaction between C6 and mHTT1-586. Molecular docking studies suggest that the inhibitor binds an allosteric site in the C6 zymogen. The interaction of mHTT1-586 with C6 may therefore promote a self-reinforcing, feedforward cycle of C6 zymogen activation and mHTT cleavage driving HD pathogenesis.

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 mM for Leishmania infantum, 3.4 mM for L. donovani, 6.7 mM for L. major), Trypanosoma cruzi (EC50 7.5 mM) and T. brucei (EC50 0.8 mM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

[Image: see text] Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (1), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure–activity relationship studies around hit compound 1. The pivaloyl derivative (13) showed significant anti-T. brucei activity (EC(50) = 1.1 μM) together with a selectivity index higher than 92. The early in vitro ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and hERG inhibition) were determined for compound 1 and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6] uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>10(7)M(-1)) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters (k(cat) = 0.12 s(-1) and K-M = 24 mu M) and inhibition constants of the two ODC inhibitors alpha-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

An important part of the beneficial effects of calorie restriction (CR) on healthspan and lifespan is mediated through regulation of protein synthesis that is under control of the mechanistic target of rapamycin complex 1 (mTORC1). As one of its activities, mTORC1 stimulates translation into the metabolic transcription factor CCAAT/Enhancer Binding Protein β (C/EBPβ) isoform Liver-specific Inhibitory Protein (LIP). Regulation of LIP expression strictly depends on a translation re-initiation event that requires a conserved cis-regulatory upstream open reading frame (uORF) in the C/EBPβ-mRNA. We showed before that suppression of LIP in mice, reflecting reduced mTORC1-signaling at the C/EBPβ level, results in CR-type of metabolic improvements. Hence, we aim to find possibilities to pharmacologically down-regulate LIP in order to induce CR-mimetic effects. We engineered a luciferase-based cellular reporter system that acts as a surrogate for C/EBPβ-mRNA translation, emulating uORF-dependent C/EBPβ-LIP expression under different translational conditions. By using the reporter system in a high-throughput screening (HTS) strategy we identified drugs that reduce LIP. The drug Adefovir Dipivoxil passed all counter assays and increases fatty acid β-oxidation in a hepatoma cell line in a LIP-dependent manner. Therefore, these drugs that suppress translation into LIP potentially exhibit CR-mimetic properties.

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)-compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni(2+)-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

As part of an international research project, the marine fungal strain collection of the Helmholtz Centre for Ocean Research (GEOMAR) research centre was analysed for secondary metabolite profiles associated with anticancer activity. Strain MF458 was identified as Tolypocladium geodes, by internal transcribed spacer region (ITS) sequence similarity and its natural product production profile. By using five different media in two conditions and two time points, we were able to identify eight natural products produced by MF458. As well as cyclosporin A (1), efrapeptin D (2), pyridoxatin (3), terricolin A (4), malettinins B and E (5 and 6), and tolypocladenols A1/A2 (8), we identified a new secondary metabolite which we termed tolypocladenol C (7). All compounds were analysed for their anticancer potential using a selection of the NCI60 cancer cell line panel, with malettinins B and E (5 and 6) being the most promising candidates. In order to obtain sufficient quantities of these compounds to start preclinical development, their production was transferred from a static flask culture to a stirred tank reactor, and fermentation medium development resulted in a nearly eight-fold increase in compound production. The strain MF458 is therefore a producer of a number of interesting and new secondary metabolites and their production levels can be readily improved to achieve higher yields.

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2’-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3–4.2 μM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-β-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z'), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

The nuclear receptors are transcription factors involved in the regulation of a variety of physiological processes whose activity can be modulated by binding to relevant small molecule ligands. Their dysfunction has been shown to play a role in disease states such as diabetes, cancer, inflammatory diseases, and hormonal resistance ailments, which makes them interesting targets for drug discovery. The nuclear receptor REV-ERBα is involved in regulating the circadian rhythm and metabolism. Its natural ligand is heme and there is significant interest in identifying novel synthetic modulators to serve as tools to characterize its function and to serve as drugs in treating metabolic disorders. To do so, we established a mammalian cell-based two-hybrid assay system capable of measuring the interaction between REV-ERBα and its co-repressor, nuclear co-repressor 1. This assay was validated to industry standard criteria and was used to screen a subset of the LOPAC®1280 library and 29568 compounds from a diverse compound library. Profiling of the primary hits in a panel of counter and selectivity assays confirmed that REV-ERBα activity can be modulated pharmacologically and chemical scaffolds have been identified for optimization.

Protein phosphatases (PP) are interesting drug targets. However, their ubiquitous presence and involvement in different, partially opposing signal pathways suggest that specificity may be achieved rather by targeting their interaction with subunits determining substrate specificity than the enzyme itself. An interesting subunit is phosphatase inhibitor-1 (I-1), which, in its protein kinase A-phosphorylated form (I-1(P)), inhibits the catalytic subunit of type 1 phosphatase (PP1c). In the current study, we established a colorimetric and a fluorescence-based assay system for the identification of compounds interfering with the inhibitory effect of I-1(P) on PP1c. The fluorescence assay exhibited 500-fold higher sensitivity toward PP1c. A nine-residue peptide containing the PP1c-binding motif (RVxF) of I-1 stimulated PP1c activity in the presence of I-1(P) (EC50 27 µM and 2.3 µM in the colorimetric and fluorescence assay, respectively). This suggests that the peptide interfered with the inhibitory effect of I-1(P) on PP1c and represents a proof-of-principle. The calculated Z' factor for PP1c (0.84) and the PP1c-I-1(P) complex (0.73) confirmed the suitability of the fluorescence assay for high-throughput screenings (HTS). By testing several thousand small molecules, we suggest the advantages of kinetic measurements over single-point measurements using the fluorescence-based assay in an HTS format. peerReviewed