Each Early Stage Researcher will contribute to the DRUGtrain project through a tailored research project. Nine ESR-projects have been defined:
ESR PROJECT 1 'Integrated in silico selection of repurposing candidates'
ESR1: David Figueiredo Vidal
Supervisor: Gerard van Westen
Leiden University Medical Center / Leiden Amsterdam Centre for Drug Research, The Netherlands
Previous work in Leiden involved the screening of chemical libraries for activity towards inhibiting or reducing the development of cysts using 3D model systems. Preliminary cheminformatic analysis of obtained results has identified and validated several potential targetable proteins, including G Protein-Coupled Receptors (GPCRs). In addition, transcriptomics, metabolomics and proteomic datasets of PKD models have previously been generated and analysed separately.
In this project, results from this, a recent screening effort, and ‘omics’ analyses, including public data, will be thoroughly explored and combined using in silico techniques in order to obtain a shortlist of relevant protein targets for. Through a combination of mode-of-action analysis (cheminformatics, proteochemometrics, and ChEMBL), multi-omics correlation network, pathway analysis (bioinformatics and biostatistics) and machine learning (deep learning). Focus will be placed on known targets of approved drugs and a list of potentially relevant approved drugs that can be repurposed will be obtained. This list will be further explored to obtain an overview of on-targets, off-targets will be obtained through the use of literature and computational modelling. Subsequently, bioactivity spectra will be coupled to the SIDER database to link the bioactivity spectrum to known off-target effects. Relevant parts of the predicted bioactivity spectra can be validated in vitro and/or in vivo. Collaboration with Certara and Univeristy of Manchester should allow the modelling of pharmacokinetics for selected drugs to optimize the link between efficacy on protein targets and concentration in relevant tissues.
- Increased understanding of PKD relevant interactions between small molecules and proteins.
- List of approved drugs potentially interesting for repurposing based on identified new drug targets for PKD.
- Prioritised set of approved drugs interesting for in vitro/in vivo testing.
ESR PROJECT 2, 'Target identification and drug validation in (preclinical) in model system'
ESR2: Nicolas Olalde Hightower
Supervisor: Dorien Peters
Leiden University Medical Center, The Netherlands
A complex network of signalling pathways is driving cyst formation, but the exact contribution of different pathways, and how they are interconnected is poorly understood. Sophisticated Pkd1del mouse-models will be used to identify/test therapeutic interventions.
Available transcriptomics, metabolomics and proteomic datasets of PKD models, with and without drug treatments, will be related to compound databases (CHEMBL) to identify druggable pathways. Selected targets and related pathways will be validated and further characterised in early and advanced disease progression. Analyses include Quantitative PCR, western blotting and immunohistochemistry as well as assays for intracellular signalling on renal tissues as well as 3D cultured renal organoids with and without CRISPR/CAS based gene disruption. Efficacy of selected drugs will be tested in organoids and in the PKD-deletion mice, and will be validated in different models. In mice with short exposure the (direct) effects of drugs on the signalling network will be studied using different methods described above.
- Identification and validation of druggable targets in PKD.
- Insight into the early and late effects of drug treatments on the signalling network and PKD.
ESR PROJECT 3, 'High-throughput phenotypic drug testing for polycystic kidney disease, using 3D cultured renal organoids'
ESR3: Margarita Iljin
Supervisors: Leo Price and Hester Bange
Crown Bioscience / OcellO B.V., The Netherlands
OcellO recently developed a 3D cell based assay to simulate cyst formation and swelling, using a PKD1 knockout mouse cell line. Applied in a high throughput 384-well plate format and using automated 3D microscopy and image analysis to detect changes in cyst characteristics, this assay has been used to screen compound libraries, resulting in the identification of lead compounds that have been effective in subsequent animal models of PKD. These data will be used with the partners to identify new targets/compounds for treatment. For further translation additional cell models/assays are required.
In this project, the 3D cyst screening assays using human kidney organoid cultures derived from patient biopsies will be expanded. In addition, cultures cysts will be characterised for signalling pathways (B1-LUMC, B4-USC, B7-UM). Library screenings and candidate drug testing of compounds and compound/drug formulations will be performed.
- A validated in vitro phenotypic drug screening based on human cells that can be exploited for commercial drug discovery and for mechanistic research into the effects of the different patient mutations on drug sensitivity.
- Characterisation of 3D renal organoid cultures
- New compounds identified and/or validated at different doses and in different organoids.
ESR PROJECT 4, 'Integrated in silico selection of repurposing candidates'
ESR4: Bola Khalil
Supervisors: Herman van Vlijmen and Natalia Dyubankova
Janssen Pharmaceutica N.V., Belgium
Improve methods for the in silico identification of secondary targets for compounds, with a focus on using the deep learning software system DeepChem. This work will build on the expertise in compound activity prediction that has been developed at Janssen in collaboration with academic partners in several projects (Leiden, Leuven, Linz). To develop the best possible models we will combine ligand-based experimental input data (e.g. Janssen and public pharmacological activities) with 3D structural information of protein targets.
These predicted target activities will be combined with internal Janssen gene expression and high content cellular imaging data to identify the most interesting diseases to which the compounds can be repurposed. The gene expression and cellular imaging data have been collected on individual compounds for several cell lines, and this information can be matched to gene expression and/or cellular imaging data of disease phenotypes. For instance, compounds that have an inverse phenotype to the disease phenotype (e.g. PKD) are interesting candidates for experimentally testing in an in vitro disease model. A survey of drugs/compounds that already have applications in multiple diseases will be done to compile a list that can be used to validate our approach. For prospective applications, Janssen has a collection of internal compounds that have not made it to the market (for various reasons, including internal strategic direction changes) but have passed preclinical or even clinical tox experiments. These are therefore very interesting candidates for identification of alternative uses. We will apply our developed predictive models and insights to this set of compounds and find ways to experimentally validate our predictions in disease-relevant models in collaboration with OcellO.
- Improved methods for in silico identification of secondary targets for chemical compounds, using ligand-based and target-based information.
- Identification and validation of druggable targets in PKD and other diseases of interest that can be addressed by known drugs or internal Janssen compounds.
- Prioritised list of compounds that can be tested in relevant disease models.
ESR PROJECT 5, 'In vitro target-based screening for identifying drug-target combinations'
ESR5: Shannon Jenkins
Supervisors: Mabel Loza Garcia and Pepo Brea
Universidad de Santiago de Compostela, Spain
The major goal of this project is identifying novel drug-target combinations useful for the treatment of PKD. The program will begin from the previous data already identified in a phenotypic screening by LUMC and OcellO and preliminary cheminformatics analysis that has identified several targetable proteins. Furthermore, the selected targets will be also studied by screening the repurposing libraries already available already available at USC in order to increase the data and enrich the predictions coming from LUMC and OcellO.
The aims are:
- To develop miniaturised assays for the targets and evaluate the currently identified drug-target combinations.
- To evaluate repurposing libraries on these targets in order to identify novel drug-target combinations for current drugs that may be employed in PKD.
- To explore the novel drug-target combinations detected in WP1. Thus, when needed, novel assays will be developed and then the in silico predicted drugs will be screened.
- The novel drugs-target interactions detected will then be confirmed in renal organoids together with OcellO and will be tested for cellular toxicity in Konstanz.
- Assays developed for druggable targets.
- Repurposing drugs identified at each studied target.
- Characterisation of the repurposing drugs in in vitro/vivo.
ESR PROJECT 6, 'Off-target toxicity in human and mouse renal epithelial cell systems/organoids using in vitro transwell systems: in vitro to in vivo extrapolations'
Supervisor: Daniel Dietrich
Universität Konstanz, Germany
Unrecognized off-target toxicity is a serious issue in the safety assessment of human drugs, primarily as a result of poor translatability of findings in surrogate species or in vitro test systems to the true patient situation. The latter issues are addressed by an in vitro transwell testing system using Tert (telomerase) transformed human renal proximal tubule epithelial cells (RPTEC) either as a single cell assays or in transwell co-culture with human Tert-fibroblasts or -endothelial cells, allowing for directional transport of drugs, drug metabolism and detection of off-target toxicity. Using physiological oxygen (10%) conditions eliminates artefacts observed under routine normox culturing (19-21% O2). Culture systems will be stablished with 1) primary human RPTEC from PKD patients and 2) mouse RPTEC, fibroblasts and endothelial cells using normal and PKD mice (LUMC). Treatment of PKD and normal mice in vivo with identified drugs or formulated drugs for detection of efficacy and toxicity will allow translation of in vivo findings to in vitro observations and vice versa. The use of the murine system (in vitro / in vivo) in combination with the in vitro renal PKD and healthy human systems ensures direct translatability of drug toxicity, and specific drug efficacy, to the PKD patient. Read-outs in the human renal transwell systems: membrane integrity (TEER); cell death (LDH leakage, MTT, Caspase assay), cell proliferation (cell type/number via cytotracker), cell architecture (confocal microscopy), water and drug transport; metabolism (Phase I &II enzyme expression at mRNA/protein levels, metabolic rates), mitochondrial integrity & ROS production (JC10; fluorescent ROS probes). Quantification of enzymes and transporter proteins in renal tissues/cells will be done in collaboration with University Manchester. Chem- and bioinformatic in silico tools will be heavily employed to define the most-likely off-target effect(s) that could be expected and thus to identify the read-outs required.
- Established murine and human renal transwell systems (PKD and healthy mono- and/or co-culture).
- Testing compounds identified from WP1 & 2 for predicted off-target effects and general toxicity with the human renal transwell system at variable physiological O2.
- Testing of compounds for toxicity and off-target effects in murine and human healthy and PKD renal transwell systems including read-outs and comparison to renal organoids.
- Refinement of limited number of drugs (including delivery systems (B8-UKA) via direct comparison of renal organoids with healthy and PKD mouse in vivo and murine and human healthy and PKD transwell systems with regard to toxicity and efficacy.
ESR PROJECT 7, 'Pharmaceutical profiling and intracellular bioavailability of repurposing lead candidates'
ESR7: Alina Meyer
Supervisor: Per Artursson
Uppsala Universitet, Sweden
You will first investigate our new repurposing candidate molecules at our profiling platform under the supervision of an experienced drug discovery team. Here, you will apply a series human cellular, subcellular and physicochemical (ADME) assays and analyze the results with state-of-the-art mass spectrometry. You will then participate in the selection of the best performing candidate molecules for further studies. Since the target proteins identified by the partners are localized inside the target cells, you will investigate the intracellular compound concentration available for target interaction.
The results will be correlated to the pharmacological response in 3D cultured kidney organoids during a secondment at one of our partners. Then, the global proteomes of the target cells will be quantified and subcellular target and off-target engagement will be investigated using total proteome profiling. Here, you will learn state-of-the-art proteomics, bioinformatics and high-resolution mass spectrometry. Finally, results will be merged with those of other DRUGtrain partners, used in PBPK modelling and the best drug candidate(s) for repurposing will be selected.
- Pharmaceutical profiling of lead candidates that identify the best repurposing candidates for further optimisation.
- Relationship between intracellular drug concentration and pharmacological effect for lead PKD-candidates.
- Understanding subcellular target and off target engagement of lead PKD-candidates.
ESR PROJECT 8, 'Quantification of drug-metabolising enzymes and transporters in healthy and diseased kidney, and the development of physiologically-based pharmacokinetic models of the kidney'
ESR8: Annika Tillmann
Supervisors: Amin Rostami and Jill Barber
University of Manchester, United Kingdom
To quantify the ADME (enzymes and transporters) proteins of mouse kidney by LC-MS/MS (tandem mass spectrometry) using untargeted (label-free) methods. Similar studies will be carried out using healthy and diseased human tissue, which will be supplemented with targeted approaches based on labelled standard peptides. Correlations between the different enzymes and transporters will be determined. Quantification of these proteins will enable to develop PBPK (physiologically-based pharmacokinetic) models of drug metabolism and transport in both rodent and human kidney.
In addition, the effect of polycystic disease on the ADME proteins in mouse models and in human kidney will be determined. In silico modelling will allow the effects of drugs, most especially their doses, to be estimated.
- A profile of the proteins of the mouse kidney, especially the ADME proteins, which we will analyse in detail.
- A corresponding profile for human kidney proteins.
- A model of mouse and human kidney drug metabolism and transport, which may be used to estimate the effects of drugs in the kidney.
- A model of disease in the mouse kidney.
ESR PROJECT 9, 'Delivery systems to improve the therapeutic index of repurposed drugs'
ESR9: Vedangi Kulkarni
Supervisor: Twan Lammers
Universitätsklinikum Aachen, Germany
The overall objective is to identify optimal delivery systems for kidney-specific delivery of selected repurposed drugs. The delivery systems will be based on linear PHPMA polymers, polymeric micelles based on PEG-PHPMA (for hydrophobic drugs) and PEGylated liposomes (for hydrophilic drugs). These representative and clinically relevant drug delivery systems can – upon functionalisation with contrast agents – be assessed for kidney targeting ability using various imaging techniques, including multimodal and multiscale optical imaging (going from the whole-body to organ (kidney) to cellular to subcellular level). Multiple previous studies exemplify the use of optical imaging, as well as magnetic resonance imaging, for kidney specific targeting and imaging (see relevant publications). In addition, anatomical, functional and molecular imaging, as well as extensive nephro-pathology, has been and will be employed for kidney disease treatment monitoring. Concrete objectives will include:
- To establish drug delivery systems with high encapsulation efficiency, high loading capacity and sustained release profiles.
- To evaluate the pharmacokinetics, biodistribution, efficacy and toxicity of drugs and drug delivery systems in appropriate models.
- To employ advanced imaging techniques to non-invasively and quantitatively monitor target site accumulation and therapeutic efficacy.
- Reformulated drugs with higher efficacy and lower toxicity.
- Tools and technologies to non-invasively assess treatment response.