Cell-Based Covalent-Capture Deubiquitinase Assay for Inhibitor Discovery.

Ubiquitination is a post-translational modification that elicits a variety of cellular responses. Deubiquitinases (DUBs) remove ubiquitin moieties from proteins and modulate cellular processes by counteracting the ubiquitin ligase activities. Ubiquitination and deubiquitination processes are tightly regulated by different mechanisms and their dysregulation is associated with many diseases. Discovery of DUB inhibitors could not only lead to therapeutics but also facilitate the understanding of ubiquitination/deubiquitination processes and their regulatory mechanisms. To enable the inhibitor discovery against DUBs, we developed a cell-based DUB assay that utilizes a cell-permeable ubiquitin probe, Biotin-cR10-Ub-PA, to covalently label DUBs in their native cellular environment. Amplified luminescent proximity homogeneous assay (Alpha, specifically AlphaLISA) is utilized to quantitatively assess the capture of the target DUB by the Biotin-cR10-Ub-PA probe. We demonstrated that this new cell-based DUB assay is robust and amenable to high-throughput screening. Human USP15 was selected as a DUB of interest and screened against a library of protease inhibitors as a proof of concept. In addition to the widely adopted pan-DUB inhibitor PR-619, several other DUB inhibitors from the library were also identified as hits. This new DUB assay can be readily adapted for inhibitor discovery against many other human DUBs to identify potent and cell-permeable inhibitors.

Small-Molecule Disruptors of the Interaction between Calcium- and Integrin-Binding Protein 1 and Integrin αIIbß3 as Novel Antiplatelet Agents.

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis. Platelets play crucial roles in both hemostasis and thrombosis. We have previously characterized calcium- and integrin-binding protein 1 (CIB1) as a key regulatory molecule that regulates platelet function. CIB1 interacts with several platelet proteins including integrin αIIbß3, the major glycoprotein receptor for fibrinogen on platelets. Given that CIB1 regulates platelet function through its interaction with αIIbß3, we developed a fluorescence polarization (FP) assay to screen for potential inhibitors. The assay was miniaturized to 1536-well and screened in quantitative high-throughput screening (qHTS) format against a diverse compound library of 14,782 compounds. After validation and selectivity testing using the FP assay, we identified 19 candidate inhibitors and validated them using an in-gel binding assay that monitors the interaction of CIB1 with αIIb cytoplasmic tail peptide, followed by testing of top hits by intrinsic tryptophan fluorescence (ITF) and microscale thermophoresis (MST) to ascertain their interaction with CIB1. Two of the validated hits shared similar chemical structures, suggesting a common mechanism of action. Docking studies further revealed promising interactions within the hydrophobic binding pocket of the target protein, particularly forming key hydrogen bonds with Ser180. The compounds exhibited a potent antiplatelet activity based on their inhibition of thrombin-induced human platelet aggregation, thus indicating that disruptors of the CIB1- αIIbß3 interaction could carry a translational potential as antithrombotic agents.

Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries.

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

Artificial Immune Cell, AI-cell, a New Tool to Predict Interferon Production by Peripheral Blood Monocytes in Response to Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles, or NANPs, rationally designed to communicate with the human immune system, can offer innovative therapeutic strategies to overcome the limitations of traditional nucleic acid therapies. Each set of NANPs is unique in their architectural parameters and physicochemical properties, which together with the type of delivery vehicles determine the kind and the magnitude of their immune response. Currently, there are no predictive tools that would reliably guide the design of NANPs to the desired immunological outcome, a step crucial for the success of personalized therapies. Through a systematic approach investigating physicochemical and immunological profiles of a comprehensive panel of various NANPs, the research team developes and experimentally validates a computational model based on the transformer architecture able to predict the immune activities of NANPs. It is anticipated that the freely accessible computational tool that is called an "artificial immune cell," or AI-cell, will aid in addressing the current critical public health challenges related to safety criteria of nucleic acid therapies in a timely manner and promote the development of novel biomedical tools.

Venglustat Inhibits Protein N-Terminal Methyltransferase 1 in a Substrate-Competitive Manner.

Venglustat is a known allosteric inhibitor for ceramide glycosyltransferase, investigated in diseases caused by lysosomal dysfunction. Here, we identified venglustat as a potent inhibitor (IC50 = 0.42 µM) of protein N-terminal methyltransferase 1 (NTMT1) by screening 58,130 compounds. Furthermore, venglustat exhibited selectivity for NTMT1 over 36 other methyltransferases. The crystal structure of NTMT1-venglustat and inhibition mechanism revealed that venglustat competitively binds at the peptide substrate site. Meanwhile, venglustat potently inhibited protein N-terminal methylation levels in cells (IC50 = 0.5 µM). Preliminary structure-activity relationships indicated that the quinuclidine and fluorophenyl parts of venglustat are important for NTMT1 inhibition. In summary, we confirmed that venglustat is a bona fide NTMT1 inhibitor, which would advance the study on the biological roles of NTMT1. Additionally, this is the first disclosure of NTMT1 as a new molecular target of venglustat, which would cast light on its mechanism of action to guide the clinical investigations.

Allosteric Binders of ACE2 Are Promising Anti-SARS-CoV-2 Agents.

The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for acute treatment of the disease. We investigate whether compounds that bind the human angiotensin-converting enzyme 2 (ACE2) protein can decrease SARS-CoV-2 replication without impacting ACE2's natural enzymatic function. Initial screening of a diversity library resulted in hit compounds active in an ACE2-binding assay, which showed little inhibition of ACE2 enzymatic activity (116 actives, success rate ∼4%), suggesting they were allosteric binders. Subsequent application of in silico techniques boosted success rates to ∼14% and resulted in 73 novel confirmed ACE2 binders with K d values as low as 6 nM. A subsequent SARS-CoV-2 assay revealed that five of these compounds inhibit the viral life cycle in human cells. Further effort is required to completely elucidate the antiviral mechanism of these ACE2-binders, but they present a valuable starting point for both the development of acute treatments for COVID-19 and research into the host-directed therapy.

Allosteric binders of ACE2 are promising anti-SARS-CoV-2 agents.

The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for an acute treatment for the disease. We investigate whether compounds that bind the human ACE2 protein can interrupt SARS-CoV-2 replication without damaging ACE2’s natural enzymatic function. Initial compounds were screened for binding to ACE2 but little interruption of ACE2 enzymatic activity. This set of compounds was extended by application of quantitative structure-activity analysis, which resulted in 512 virtual hits for further confirmatory screening. A subsequent SARS-CoV-2 replication assay revealed that five of these compounds inhibit SARS-CoV-2 replication in human cells. Further effort is required to completely determine the antiviral mechanism of these compounds, but they serve as a strong starting point for both development of acute treatments for COVID-19 and research into the mechanism of infection.

Integrate mechanistic evidence from new approach methodologies (NAMs) into a read-across assessment to characterise trends in shared mode of action.

Read-across approaches often remain inconclusive as they do not provide sufficient evidence on a common mode of action across the category members. This read-across case study on thirteen, structurally similar, branched aliphatic carboxylic acids investigates the concept of using human-based new approach methods, such as in vitro and in silico models, to demonstrate biological similarity. Five out of the thirteen analogues have preclinical in vivo studies. Three out of them induced lipid accumulation or hypertrophy in preclinical studies with repeated exposure, which leads to the read-across hypothesis that the analogues can potentially induce hepatic steatosis. To confirm the selection of analogues, the expression patterns of the induced differentially expressed genes (DEGs) were analysed in a human liver model. With increasing dose, the expression pattern within the tested analogues got more similar, which serves as a first indication of a common mode of action and suggests differences in the potency of the analogues. Hepatic steatosis is a well-known adverse outcome, for which over 55 adverse outcome pathways have been identified. The resulting adverse outcome pathway (AOP) network, comprised a total 43 MIEs/KEs and enabled the design of an in vitro testing battery. From the AOP network, ten MIEs, early and late KEs were tested to systematically investigate a common mode of action among the grouped compounds. The targeted testing of AOP specific MIE/KEs shows that biological activity in the category decreases with side chain length. A similar trend was evident in measuring liver alterations in zebra fish embryos. However, activation of single MIEs or early KEs at in vivo relevant doses did not necessarily progress to the late KE "lipid accumulation". KEs not related to the read-across hypothesis, testing for example general mitochondrial stress responses in liver cells, showed no trend or biological similarity. Testing scope is a key issue in the design of in vitro test batteries. The Dempster-Shafer decision theory predicted those analogues with in vivo reference data correctly using one human liver model or the CALUX reporter assays. The case study shows that the read-across hypothesis is the key element to designing the testing strategy. In the case of a good mechanistic understanding, an AOP facilitates the selection of reliable human in vitro models to demonstrate a common mode of action. Testing DEGs, MIEs and early KEs served to show biological similarity, whereas the late KEs become important for confirmation, as progression from MIEs to AO is not always guaranteed.

Hybrid In Silico Approach Reveals Novel Inhibitors of Multiple SARS-CoV-2 Variants.

The National Center for Advancing Translational Sciences (NCATS) has been actively generating SARS-CoV-2 high-throughput screening data and disseminates it through the OpenData Portal (https://opendata.ncats.nih.gov/covid19/). Here, we provide a hybrid approach that utilizes NCATS screening data from the SARS-CoV-2 cytopathic effect reduction assay to build predictive models, using both machine learning and pharmacophore-based modeling. Optimized models were used to perform two iterative rounds of virtual screening to predict small molecules active against SARS-CoV-2. Experimental testing with live virus provided 100 (∼16% of predicted hits) active compounds (efficacy > 30%, IC50 ≤ 15 µM). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further investigation resulted in the identification of allosteric binders to host receptor angiotensin-converting enzyme 2; these compounds were then shown to inhibit the entry of pseudoparticles bearing spike protein of wild-type SARS-CoV-2, as well as South African B.1.351 and UK B.1.1.7 variants.

GenoVault: a cloud based genomics repository.

GenoVault is a cloud-based repository for handling Next Generation Sequencing (NGS) data. It is developed using OpenStack-based private cloud with various services like keystone for authentication, cinder for block storage, neutron for networking and nova for managing compute instances for the Cloud. GenoVault uses object-based storage, which enables data to be stored as objects instead of files or blocks for faster retrieval from different distributed object nodes. Along with a web-based interface, a JavaFX-based desktop client has also been developed to meet the requirements of large file uploads that are usually seen in NGS datasets. Users can store files in their respective object-based storage areas and the metadata provided by the user during file uploads is used for querying the database. GenoVault repository is designed taking into account future needs and hence can scale both vertically and horizontally using OpenStack-based cloud features. Users have an option to make the data shareable to the public or restrict the access as private. Data security is ensured as every container is a separate entity in object-based storage architecture which is also supported by Secure File Transfer Protocol (SFTP) for data upload and download. The data is uploaded by the user in individual containers that include raw read files (fastq), processed alignment files (bam, sam, bed) and the output of variation detection (vcf). GenoVault architecture allows verification of the data in terms of integrity and authentication before making it available to collaborators as per the user's permissions. GenoVault is useful for maintaining the organization-wide NGS data generated in various labs which is not yet published and submitted to public repositories like NCBI. GenoVault also provides support to share NGS data among the collaborating institutions. GenoVault can thus manage vast volumes of NGS data on any OpenStack-based private cloud.

Discovery of TMPRSS2 Inhibitors from Virtual Screening as a Potential Treatment of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted researchers to pivot their efforts to finding antiviral compounds and vaccines. In this study, we focused on the human host cell transmembrane protease serine 2 (TMPRSS2), which plays an important role in the viral life cycle by cleaving the spike protein to initiate membrane fusion. TMPRSS2 is an attractive target and has received attention for the development of drugs against SARS and Middle East respiratory syndrome. Starting with comparative structural modeling and a binding model analysis, we developed an efficient pharmacophore-based approach and applied a large-scale in silico database screening for small-molecule inhibitors against TMPRSS2. The hits were evaluated in the TMPRSS2 biochemical assay and the SARS-CoV-2 pseudotyped particle entry assay. A number of novel inhibitors were identified, providing starting points for the further development of drug candidates for the treatment of coronavirus disease 2019.

Development of Robust Quantitative Structure-Activity Relationship Models for CYP2C9, CYP2D6, and CYP3A4 Catalysis and Inhibition.

Cytochrome P450 enzymes are responsible for the metabolism of >75% of marketed drugs, making it essential to identify the contributions of individual cytochromes P450 to the total clearance of a new candidate drug. Overreliance on one cytochrome P450 for clearance levies a high risk of drug-drug interactions; and considering that several human cytochrome P450 enzymes are polymorphic, it can also lead to highly variable pharmacokinetics in the clinic. Thus, it would be advantageous to understand the likelihood of new chemical entities to interact with the major cytochrome P450 enzymes at an early stage in the drug discovery process. Typical screening assays using human liver microsomes do not provide sufficient information to distinguish the specific cytochromes P450 responsible for clearance. In this regard, we experimentally assessed the metabolic stability of ∼5000 compounds for the three most prominent xenobiotic metabolizing human cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4, and used the data sets to develop quantitative structure-activity relationship models for the prediction of high-clearance substrates for these enzymes. Screening library included the NCATS Pharmaceutical Collection, comprising clinically approved low-molecular-weight compounds, and an annotated library consisting of drug-like compounds. To identify inhibitors, the library was screened against a luminescence-based cytochrome P450 inhibition assay; and through crossreferencing hits from the two assays, we were able to distinguish substrates and inhibitors of these enzymes. The best substrate and inhibitor models (balanced accuracies ∼0.7), as well as the data used to develop these models, have been made publicly available (https://opendata.ncats.nih.gov/adme) to advance drug discovery across all research groups. SIGNIFICANCE STATEMENT: In drug discovery and development, drug candidates with indiscriminate cytochrome P450 metabolic profiles are considered advantageous, since they provide less risk of potential issues with cytochrome P450 polymorphisms and drug-drug interactions. This study developed robust substrate and inhibitor quantitative structure-activity relationship models for the three major xenobiotic metabolizing cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4. The use of these models early in drug discovery will enable project teams to strategize or pivot when necessary, thereby accelerating drug discovery research.

Large-Scale Modeling of Multispecies Acute Toxicity End Points Using Consensus of Multitask Deep Learning Methods.

Computational methods to predict molecular properties regarding safety and toxicology represent alternative approaches to expedite drug development, screen environmental chemicals, and thus significantly reduce associated time and costs. There is a strong need and interest in the development of computational methods that yield reliable predictions of toxicity, and many approaches, including the recently introduced deep neural networks, have been leveraged towards this goal. Herein, we report on the collection, curation, and integration of data from the public data sets that were the source of the ChemIDplus database for systemic acute toxicity. These efforts generated the largest publicly available such data set comprising > 80,000 compounds measured against a total of 59 acute systemic toxicity end points. This data was used for developing multiple single- and multitask models utilizing random forest, deep neural networks, convolutional, and graph convolutional neural network approaches. For the first time, we also reported the consensus models based on different multitask approaches. To the best of our knowledge, prediction models for 36 of the 59 end points have never been published before. Furthermore, our results demonstrated a significantly better performance of the consensus model obtained from three multitask learning approaches that particularly predicted the 29 smaller tasks (less than 300 compounds) better than other models developed in the study. The curated data set and the developed models have been made publicly available at https://github.com/ncats/ld50-multitask, https://predictor.ncats.io/, and https://cactus.nci.nih.gov/download/acute-toxicity-db (data set only) to support regulatory and research applications.

Discovery of TMPRSS2 inhibitors from virtual screening.

The SARS-CoV-2 pandemic has prompted researchers to pivot their efforts to finding antiviral compounds and vaccines. In this study, we focused on the human host cell transmembrane protease serine 2 (TMPRSS2), which plays an important role in the viral life cycle by cleaving the spike protein to initiate membrane fusion. TMPRSS2 is an attractive target and has received attention for the development of drugs against SARS and MERS. Starting with comparative structural modeling and binding model analysis, we developed an efficient pharmacophore-based approach and applied a large-scale in silico database screening for small molecule inhibitors against TMPRSS2. The hits were evaluated in the TMPRSS2 biochemical assay and the SARS-CoV-2 pseudotyped particle (PP) entry assay. A number of novel inhibitors were identified, providing starting points for further development of drug candidates for the treatment of COVID-19.

Combining In Vivo Data with In Silico Predictions for Modeling Hepatic Steatosis by Using Stratified Bagging and Conformal Prediction.

Hepatic steatosis (fatty liver) is a severe liver disease induced by the excessive accumulation of fatty acids in hepatocytes. In this study, we developed reliable in silico models for predicting hepatic steatosis on the basis of an in vivo data set of 1041 compounds measured in rodent studies with repeated oral exposure. The imbalanced nature of the data set (1:8, with the "steatotic" compounds belonging to the minority class) required the use of meta-classifiers-bagging with stratified under-sampling and Mondrian conformal prediction-on top of the base classifier random forest. One major goal was the investigation of the influence of different descriptor combinations on model performance (tested by predicting an external validation set): physicochemical descriptors (RDKit), ToxPrint features, as well as predictions from in silico nuclear receptor and transporter models. All models based upon descriptor combinations including physicochemical features led to reasonable balanced accuracies (BAs between 0.65 and 0.69 for the respective models). Combining physicochemical features with transporter predictions and further with ToxPrint features gave the best performing model (BAs up to 0.7 and efficiencies of 0.82). Whereas both meta-classifiers proved useful for this highly imbalanced toxicity data set, the conformal prediction framework also guarantees the error level and thus might be favored for future studies in the field of predictive toxicology.

Outbreak Prediction of COVID-19 for Dense and Populated Countries Using Machine Learning.

The Coronavirus Disease-2019 (COVID-19) pandemic persists to have a mortifying impact on the health and well-being of the global population. A continued rise in the number of patients testing positive for COVID-19 has created a lot of stress on governing bodies across the globe and they are finding it difficult to tackle the situation. We have developed an outbreak prediction system for COVID-19 for the top 10 highly and densely populated countries. The proposed prediction models forecast the count of new cases likely to arise for successive 5 days using 9 different machine learning algorithms. A set of models for predicting the rise in new cases, having an average accuracy of 87.9%  ± 3.9% was developed for 10 high population and high density countries. The highest accuracy of 99.93% was achieved for Ethiopia using Auto-Regressive Moving Average (ARMA) averaged over the next 5 days. The proposed prediction models used by us can help stakeholders to be prepared in advance for any sudden rise in outbreak to ensure optimal management of available resources.

Propafenone analogue with additional H-bond acceptor group shows increased inhibitory activity on P-glycoprotein.

P-glycoprotein (P-gp) is an ATP-dependent efflux pump that has a marked impact on the absorption, distribution, and excretion of therapeutic drugs. As P-gp inhibition can result in drug-drug interactions and altered drug bioavailability, identifying molecular properties that are linked to inhibition is of great interest in drug development. In this study, we combined chemical synthesis, in vitro testing, quantitative structure-activity relationship analysis, and docking studies to investigate the role of hydrogen bond (H-bond) donor/acceptor properties in transporter-ligand interaction. In a previous work, it has been shown that propafenone analogs with a 4-hydroxy-4-piperidine moiety exhibit a generally 10-fold higher P-gp inhibitory activity than expected based on their lipophilicity. Here, we specifically expanded the data set by introducing substituents at position 4 of the 4-phenylpiperidine moiety to assess the importance of H-bond donor/acceptor features in this region. The results suggest that indeed an H-bond acceptor, such as hydroxy and methoxy, increases the affinity by forming a H-bond with Tyr310.

In Silico Approaches to Predict Drug-Transporter Interaction Profiles: Data Mining, Model Generation, and Link to Cholestasis.

Transport proteins play a crucial role in drug distribution, disposition, and clearance by mediating cellular drug influx and efflux. Inhibition of these transporters may lead to drug-drug interactions or even drug-induced liver injury, such as cholestasis, which comprises a major challenge in drug development process. Thus, computer-based (in silico) models that can predict the pharmacological and toxicological profiles of these small molecules with respect to liver transporters may help in the early prioritization of compounds and hence may lower the high attrition rates. In this chapter, we provide a protocol for in silico prediction of cholestasis by generating validated predictive models. In addition to the two-dimensional molecular descriptors, we include transporter inhibition predictions as descriptors and evaluate the influence of the same on the performance of the cholestasis models.

Integrative Data Mining, Scaffold Analysis, and Sequential Binary Classification Models for Exploring Ligand Profiles of Hepatic Organic Anion Transporting Polypeptides.

Hepatocellular organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are important for proper liver function and the regulation of the drug elimination process. Understanding their roles in different conditions of liver toxicity and cancer requires an in-depth investigation of hepatic OATP-ligand interactions and selectivity. However, such studies are impeded by the lack of crystal structures, the promiscuous nature of these transporters, and the limited availability of reliable bioactivity data, which are spread over different data sources in the open domain. To this end, we integrated ligand bioactivity data for hepatic OATPs from five open data sources (ChEMBL, the UCSF-FDA TransPortal database, DrugBank, Metrabase, and IUPHAR) in a semiautomatic KNIME workflow. Highly curated data sets were analyzed with respect to enriched scaffolds, and their activity profiles and interesting scaffold series providing indication for selective, dual-, or pan-inhibitory activity toward hepatic OATPs could be extracted. In addition, a sequential binary modeling approach revealed common and distinctive ligand features for inhibitory activity toward the individual transporters. The workflows designed for integrating data from open sources, data curation, and subsequent substructure analyses are freely available and fully adaptable. The new data sets for inhibitors and substrates of hepatic OATPs as well as the insights provided by the feature and substructure analyses will guide future structure-based studies on hepatic OATP-ligand interactions and selectivity.

Interspecies comparison of putative ligand binding sites of human, rat and mouse P-glycoprotein.

Prior to the clinical phases of testing, safety, efficacy and pharmacokinetic profiles of lead compounds are evaluated in animal studies. These tests are primarily performed in rodents, such as mouse and rats. In order to reduce the number of animal experiments, computational models that predict the outcome of these studies and thus aid in prioritization of preclinical candidates are heavily needed. However, although computational models for human off-target interactions with decent quality are available, they cannot easily be transferred to rodents due to lack of respective data. In this study, we assess the transferability of human P-glycoprotein activity data for development of in silico models to predict in vivo effects in rats and mouse using a structure-based approach. P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that transports xenobiotic compounds such as toxins and drugs out of cells and has a broad substrate and inhibitor specificity. Being mostly expressed at barriers, it influences the bioavailability of drugs and thus contributes also to toxicity. Comparison of the binding site interaction profiles of human, rat and mouse P-gp derived from docking studies with a set of common inhibitors suggests that the inhibitors share potentially similar binding modes. These findings encourage the use of in vitro human P-gp data for predicting in vivo effects in rodents and thus contributes to the 3Rs (Replace, Reduce and Refine) of animal experiments.