Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening.

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

Anti-Cancer Activity of Buthus occitanus Venom on Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most dominant primary liver cancer, which can be caused by chronic hepatitis virus infections and other environmental factors. Resection, liver transplantation, and local ablation are only a few of the highly effective and curative procedures presently accessible. However, other complementary treatments can reduce cancer treatment side effects. In this present work, we evaluated the activity of Moroccan scorpion venom Buthus occitanus and its fractions obtained by chromatography gel filtration against HCC cells using a 3D cell culture model. The venom was fractionated by gel filtration chromatography, each fraction and the crude venom was tested on normal hepatocytes (Fa2N-4 cells). Additionally, the fractions and the crude venom were tested on MCTSs (multicellular tumor spheroids), and this latter was generated by cultivate Huh7.5 cancer cell line with WI38 cells, LX2 cells, and human endothelial cells (HUVEC). Our results indicate that Buthus occitanus venom toxin has no cytotoxic effects on normal hepatocytes. Moreover, it is reported that F3 fraction could significantly inhibit the MCTS cells. Other Protein Separation Techniques (High-performance liquid chromatography) are needed in order to identify the most active molecule.

3D Vehicle Trajectory Extraction Using DCNN in an Overlapping Multi-Camera Crossroad Scene.

The 3D vehicle trajectory in complex traffic conditions such as crossroads and heavy traffic is practically very useful in autonomous driving. In order to accurately extract the 3D vehicle trajectory from a perspective camera in a crossroad where the vehicle has an angular range of 360 degrees, problems such as the narrow visual angle in single-camera scene, vehicle occlusion under conditions of low camera perspective, and lack of vehicle physical information must be solved. In this paper, we propose a method for estimating the 3D bounding boxes of vehicles and extracting trajectories using a deep convolutional neural network (DCNN) in an overlapping multi-camera crossroad scene. First, traffic data were collected using overlapping multi-cameras to obtain a wide range of trajectories around the crossroad. Then, 3D bounding boxes of vehicles were estimated and tracked in each single-camera scene through DCNN models (YOLOv4, multi-branch CNN) combined with camera calibration. Using the abovementioned information, the 3D vehicle trajectory could be extracted on the ground plane of the crossroad by calculating results obtained from the overlapping multi-camera with a homography matrix. Finally, in experiments, the errors of extracted trajectories were corrected through a simple linear interpolation and regression, and the accuracy of the proposed method was verified by calculating the difference with ground-truth data. Compared with other previously reported methods, our approach is shown to be more accurate and more practical.

Discovery of thienothiazolocarboxamide analogues as novel anti-tubercular agent.

In order to identify anti-tubercular agents with a novel scaffold, commercial libraries of small organic compounds were screened against a fluorescent strain of Mycobacterium tuberculosis H37Rv, using a dual phenotypic assay. Compounds were assessed against bacteria replicating in broth medium, as well as inside macrophages, and thienothiazolocarboxamide (TTCA) scaffold was identified as hit in both assays, with submicromolar inhibitory concentrations. Derivatives of TTCA were further synthesized and evaluated for their inhibitory effects on M.tuberculosis H37Rv. In the present study we report the structure-activity relationship of these TTCA derivatives. Compounds 28, 32 and 42 displayed good anti-tubercular activities, as well as favorable ADME and PK properties. Compound 42 exhibited excellent oral bioavailability in mice with high distribution to lungs, within 1 h. It was found to be efficacious in a dose dependent manner in a murine model of M. tuberculosis infection. Hence, compound 42 is now under evaluation as a potential lead candidate for treatment of tuberculosis.

Inhibition of DNMT3B expression in activated hepatic stellate cells overcomes chemoresistance in the tumor microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a complex disease associated with a plethora of environmental and genetic/hereditary causative risk factors, more so than other oncological indications. Additionally, patients with HCC exhibit fibrosis, cirrhosis, and liver-related disease. This complicated etiology can affect the disease course and likely contributes to its poor prognosis. In this study, we aimed to improve HCC therapy by evaluating combination treatment using anti-cancer and anti-fibrosis drugs via identification of novel anti-fibrosis drugs. We performed high-throughput screening of 10,000 compounds to identify hepatic fibrosis inhibitors through morphometry analysis of multicellular hepatic spheroid (MCHS) models and identified CHIR-99021 as a candidate anti-fibrotic drug. Treatment with CHIR-99021 induced loss of cell-cell interactions and suppression of extracellular matrix-related protein expression via reprogramming of hepatic stellate cell (HSC) activation in MCHSs. In particular, CHIR-99021 regulated DNMT3B expression only in activated HSCs. Moreover, CHIR-99021 markedly improved the efficacy of sorafenib in HCC- multicellular tumor spheroids in vitro and through induction of apoptosis by decreasing DNMT3B expression in vivo. In summary, these findings suggest that targeting HSC reprogramming by attenuation of DNMT3B expression in the tumor environment might represent a promising therapeutic strategy for liver fibrosis and HCC.

Anthracene derivatives with strong spin-orbit coupling and efficient high-lying reverse intersystem crossing beyond the El-Sayed rule.

The attention to materials with hot exciton channel and triplet-triplet fusion (TTF) mediated high-lying reverse intersystem crossing (hRISC) has been raised for their ability to convert non-emissive 'dark' triplets into radiative singlet excitons. This spin conversion process results in high exciton utilization efficiency (EUE) that exceeds the theoretical limits. Notably, it is known that such spin conversion processes from the high-lying excited triplet to the singlet state are facilitated by the orthogonal orbital transition effect governed by the El-Sayed's rule. In this study, an anthracene derivative with indenoquinoline substituent 7,7-dimethyl-9-(10-(4-(naphthalen-1-yl)phenyl)anthracen-9-yl)-7H-indeno[1,2-f]quinoline (2MIQ-NPA) was synthesized and analyzed to investigate whether the hRISC process occurs in these molecules, even when the El-Sayed's rule is not followed. The hRISC channels of the emitter were fully unraveled through DFT calculations and experiments, which were quantitatively subdivided using transient electroluminescence measurements. The results showed that 2MIQ-NPA, which does not follow the El-Sayed's rule and has a relatively strong spin-orbit coupling matrix element of 0.116 cm-1 between the high-lying triplet state of T4 and the lowest singlet state of S1, effectively converted triplet excitons into singlet excitons with an EUE of 64.3%, contributed by a direct hot exciton channel of 19.2% and a TTF-mediated hot exciton channel of 15.1%. Despite the low outcoupling efficiency, the non-doped device with 2MIQ-NPA achieved an excellent device performance with an external quantum efficiency of 7.0%.

A combination screening to identify enhancers of para-aminosalicylic acid against Mycobacterium tuberculosis.

Para-aminosalicylic acid (PAS) is an antibiotic that was largely used for the multi-therapy of tuberculosis in the twentieth century. To try to overcome the inconvenience of its low efficacy and poor tolerance, we searched for novel chemical entities able to synergize with PAS using a combination screening against growing axenic Mycobacterium tuberculosis. The screening was performed at a sub-inhibitory concentration of PAS on a library of about 100,000 small molecules. Selected hit compounds were analyzed by dose-response and further probed with an intracellular macrophage assay. Scaffolds with potential additive effect with PAS are reported, opening interesting prospects for mechanism of action studies. We also report here evidence of a yet unknown bio-activation mechanism, involving activation of pyrido[1,2-a]pyrimidin-4-one (PP) derivatives through the Rv3087 protein.

Identification of hepatic fibrosis inhibitors through morphometry analysis of a hepatic multicellular spheroids model.

A chronic, local inflammatory milieu can cause tissue fibrosis that results in epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition (EndMT), increased abundance of fibroblasts, and further acceleration of fibrosis. In this study, we aimed to identify potential mechanisms and inhibitors of fibrosis using 3D model-based phenotypic screening. We established liver fibrosis models using multicellular tumor spheroids (MCTSs) composed of hepatocellular carcinoma (HCC) and stromal cells such as fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVEC) seeded at constant ratios. Through high-throughput screening of FDA-approved drugs, we identified retinoic acid and forskolin as candidates to attenuate the compactness of MCTSs as well as inhibit the expression of ECM-related proteins. Additionally, retinoic acid and forskolin induced reprogramming of fibroblast and cancer stem cells in the HCC microenvironment. Of interest, retinoic acid and forskolin had anti-fibrosis effects by decreasing expression of α-SMA and F-actin in LX2 cells and HUVEC cells. Moreover, when sorafenib was added along with retinoic acid and forskolin, apoptosis was increased, suggesting that anti-fibrosis drugs may improve tissue penetration to support the efficacy of anti-cancer drugs. Collectively, these findings support the potential utility of morphometric analyses of hepatic multicellular spheroid models in the development of new drugs with novel mechanisms for the treatment of hepatic fibrosis and HCCs.

Anti-Cancer Effect of Moroccan Cobra Naja haje Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

Comparison of Antibody and T Cell Responses Induced by Single Doses of ChAdOx1 nCoV-19 and BNT162b2 Vaccines.

There are limited data directly comparing humoral and T cell responses to the ChAdOx1 nCoV-19 and BNT162b2 vaccines. We compared Ab and T cell responses after first doses of ChAdOx1 nCoV-19 vs. BNT162b2 vaccines. We enrolled healthcare workers who received ChAdOx1 nCoV-19 or BNT162b2 vaccine in Seoul, Korea. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S1 protein-specific IgG Abs (S1-IgG), neutralizing Abs (NT Abs), and SARS-CoV-2-specific T cell response were evaluated before vaccination and at 1-wk intervals for 3 wks after vaccination. A total of 76 persons, comprising 40 injected with the ChAdOx1 vaccine and 36 injected with the BNT162b2 vaccine, participated in this study. At 3 wks after vaccination, the mean levels (±SD) of S1-IgG and NT Abs in the BNT162b2 participants were significantly higher than in the ChAdOx1 participants (S1-IgG, 14.03±7.20 vs. 6.28±8.87, p<0.0001; NT Ab, 183.1±155.6 vs. 116.6±116.2, p=0.035), respectively. However, the mean values of the T cell responses in the 2 groups were comparable after 2 wks. The humoral immune response after the 1st dose of BNT162b2 developed faster and was stronger than after the 1st dose of ChAdOx1. However, the T cell responses to BNT162b2 and ChAdOx1 were similar.

Inhibitors of Na+/K+ ATPase exhibit antitumor effects on multicellular tumor spheroids of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), one of the most common malignant cancers worldwide, is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in patients with HCC. The multicellular tumor spheroid (MCTS) model is a powerful method for anticancer research because of its ability to mimic the complexity and heterogeneity of tumor tissue, the three-dimensional cellular context of tumor tissue, and the pathophysiological gradients of in vivo tumors. However, it is difficult to obtain meaningful results from the MCTS model without considering the conditions of clinical tumors. We, therefore, provided a proof of concept to determine whether spheroid models simulate in vivo tumor microenvironments. Through a high-throughput screening for HCC therapy using the MCTS model, we selected inhibitors of Na+/K+-ATPase (ouabain and digoxin) that could suppress cell growth and migration via inhibition of the epithelial-mesenchymal transition of HCC in vivo and in vitro. The results showed that this model provides a new paradigm for high-throughput drug screening and will significantly improve the efficiency of identifying new drugs for HCC treatment. Through utilization of MCTS models, here we found that inhibitors of Na+/K+-ATPase may be feasible as a novel target to sensitize HCC cells.

Identification of radiation-induced EndMT inhibitors through cell-based phenomic screening.

Radiation-induced pulmonary fibrosis (RIPF) triggers physiological abnormalities. Endothelial-to-mesenchymal transition (EndMT) is the phenotypic conversion of endothelial cells to fibroblast-like cells and is involved in RIPF. In this study, we established a phenomic screening platform to measure radiation-induced stress fibers and optimized the conditions for high-throughput screening using human umbilical vein endothelial cells (HUVECs) to develop compounds targeting RIPF. The results of screening indicated that CHIR-99021 reduced radiation-induced fibrosis, as evidenced by an enlargement of cell size and increases in actin stress fibers and α-smooth muscle actin expression. These effects were elicited without inducing serious toxicity in HUVECs, and the cytotoxic effect of ionizing radiation (IR) in nonsmall cell lung cancer was also enhanced. These results demonstrate that CHIR-99021 enhanced the effects of IR therapy by suppressing radiation-induced EndMT in lung cancer.

Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages.

A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains.

High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system.

The massive epidemic of Ebola virus disease (EVD) in West Africa, followed in recent months by two outbreaks in the Democratic Republic of the Congo, underline the importance of this severe disease. Because Ebola virus (EBOV) must be manipulated under biosafety level 4 (BSL4) containment, the discovery and development of virus-specific therapies have been hampered. Recently, a transient transfection-based transcription- and replication competent virus-like particle (trVLP) system was described, enabling modeling of the entire EBOV life cycle under BSL2 conditions. Using this system, we optimized the condition for bulk co-transfection of multiple plasmids, developed a luciferase reporter-based assay in 384-well microtiter plates, and performed a high-throughput screening (HTS) campaign of an 8,354-compound collection consisting of U.S. Food & Drug Administration (FDA) -approved drugs, bioactives, kinase inhibitors, and natural products in duplicates. The HTS achieved a good signal-to-background ratio with a low percent coefficient of variation resulting in Z' = 0.7, and data points were reproducible with R2 = 0.89, indicative of a robust assay. After applying stringent hit selection criteria of ≥70% EBOV trVLP inhibition and ≥70% cell viability, 381 hits were selected targeting early, entry, and replication steps and 49 hits targeting late, maturation, and secretion steps in the viral life cycle. Of the total 430 hits, 220 were confirmed by dose-response analysis in the primary HTS assay. They were subsequently triaged by time-of-addition assays, then clustered and ranked according to their chemical structures, biological functions, therapeutic index, and maximum inhibition. Several novel drugs have been identified to very efficiently inhibit EBOV. Interestingly, most showed pharmacological activity in treatments for central nervous system-related diseases. We developed and screened an HTS assay using the novel EBOV trVLP system. Newly identified inhibitors are useful tools to study the poorly understood EBOV life cycle. In addition, they also provide opportunities to either repurpose FDA-approved drugs or develop novel viral interventions to combat EVD.

High-Content Screening of Raw Actinomycete Extracts for the Identification of Antituberculosis Activities.

The feasibility and relevance of screening a library of raw actinomycete extracts (ECUM library) for the identification of antituberculosis activities was assessed on 11,088 extracts using a multiple-screening approach. Each extract was first tested at two concentrations against noninfected macrophages as a control, then against Mycobacterium tuberculosis growing in broth medium as well as infecting murine macrophages. The screening results indicated a library of good quality with an apparent low proportion of cytotoxic extracts. A correlation was found between both bacterial assays, but the intracellular assay showed limitations due to low rates of cell survival. Several extracts of interest were highlighted by this multiple screening. A focus on the strain producing the two most effective revealed similarities with known producers of active molecules, suggesting the possibility of selecting relevant extracts using this strategy.

Kinome siRNA screen identifies novel cell-type specific dengue host target genes.

Dengue is a global emerging infectious disease, with no specific treatment available. To identify novel human host cell targets important for dengue virus infection and replication, an image-based high-throughput siRNA assay screening of a human kinome siRNA library was conducted using human hepatocyte cell line Huh7 infected with a recent dengue serotype 2 virus isolate BR DEN2 01-01. In the primary siRNA screening of 779 kinase-related genes, knockdown of 22 genes showed a reduction in DENV-2 infection. Conversely, knockdown of 8 genes enhanced viral infection. To assess host cell specificity, the confirmed hits were tested in the DENV-infected monocytic cell line U937. While the expression of EIF2AK3, ETNK2 and SMAD7 was regulated in both cell lines after infection, most kinases were hepatocyte-specific. Monocytic cells represent initial targets of infection and an antiviral treatment targeting these cells is probably most effective to reduce initial viral load. In turn, infection of the liver could contribute to pathogenesis, and the novel hepatocyte-specific human targets identified here could be important for dengue infection and pathogenesis.