Aim 1: Development of in vitro predictive assays to identify and characterize HRFD lead compounds.
Aim 2: Development of in vivo predictive translational HRFD models to enhance assessment of efficacy in preclinical testing.
Aim 3: Develop advanced HRFD-specific toxicology and safety screens.
Currently Available Resources
High throughput drug screening
- Hight throughput drug screening with primary and secondary assays
- The high throughput screening (HTS) will be performed in collaboration with The Southern Research Institute HTS Laboratory (http://www.southernresearch.org/life-sciences/lead-discovery-and-optimization/high-throughput-screening). SRI has a library of 450,000 compounds available for initial primary screening; they were selected from commercial libraries to fully represent chemical scaffolds for high throughput screening and include FDA approved drugs. Such screening typically identifies hit clusters and allows triage based on potency, efficacy, and chemistry.Secondary assays will confirm biology and/or evaluate toxicity/effects on proliferation. Systematic implementation of this strategy allows reduction of hits to numbers that can be screened in in vivo modes. Such follow up screening is available using HRFD models from Core D (Aim 2).
Preclinical testing of drug efficacy in HRFD models
- Longitudinal serial monitoring of HRFD progression
- While Core D can offer evaluation at a single (terminal) endpoint, it also offers longitudinal monitoring of HRFD progression. The longitudinal approach decreases the number of required animals by reducing unexplained variability at the terminal readout, i.e. the longitudinal prospective design allows adjustment of phenotypic severity at the final end-point to disease severity preceding experimental therapeutic intervention. In addition, obtaining data for three or more time-points allows comparison of individual disease progression trajectories (we described use of similar trajectories in human ADPKD studies. Core D's goal is to design a standardized in vivo evaluation of drug efficacy. Therefore, Core D will perform treatment and disease progression monitoring according to a schedule standardized for each model (e.g., Cys1cpk mouse and Pkhd1pck rat).
Figure 1. An example of longitudinal serial live imagin of Cys1cpk progression. Panel a: Longitudinal follow up of MRI-based total kidney volumes adjusted for body weight (bwTKV), (n=4 in each group); Panel b: Comparison of the bwTKV in Cys1cpk/cpk mice and wild type controls (wt); bars show SEM.
- Semiautomated histomorphometry analyses or renal and biliary phenotypes
- The histomorphometry analysis is often considered a golden standard for evaluation of HRFD renal and biliary (congenital hepatic fibrosis) phenotypes. To enhance precision and reduce bias and time required for evaluation of these() phenotypes from histopathology slides, Core D has developed and validated semi-automated tools for their assessment .
- Quality control studies aimed at establishing novel HRFD models for evaluation of drug efficacy
Preclinical testing of drug safety in HRFD models
- Toxicology and safety screens
- The recommendation for the type of biological sample collected at the endpoint of a treatment study will be individualized for each tested drug; however, it is expected to include blood, urine and relevant tissues. The safety and toxicology mouse/rat-specific study design will be informed and in part executed and interpreted by the Southern Research Institute Toxicology Laboratory (http://www.southernresearch.org/life-sciences/preclinical-development/toxicology) that routinely performs such evaluations. Comprehensive histopathology assessment and its interpretation will be done by the UAB Molecular and Morphological Analysis Facility (Core D co-director Dr. Gene P. Siegal has relevant decades long experience in animal studies).
Figure 2. An example of semiautomated histomorphometry analyses of renal and biliary HRFD phenotypes. Panel a: H&E staining of Cys1cpk kidney (P12); Panel b: Semi-automated selection of renal cystic areas; Panel c: Selection of medullary area; Panel d: Examples of corresponding renal cystic indices; Panel e: Pkhd1pck rat liver (P21); Panel f: Selection of congenital hepatic fibrosis regions; Panel g: Highlighted portal field area (blue line), dilated biliary ducts (red line) and portal vein (green line) (Detail from Panel b Spot 1); Panel h: Examples of biliary/congenital hepatic fibrosis phenotypes.
Requesting Resources and Services
Any member of the HRFD Center may request resources or services available from Core D. This is done by submitting a request to the Core Directors directly or through the HRFD Center website. Each member requesting Core D resources or services will be asked to submit: (i) a short description of the project and how samples are to be collected; (ii) anticipated impact on HRFD research; (iii) preferred shipping method (if applicable); and (iv) agreement to acknowledge Core D and the HRFD Core Center for generation of the resource when disseminating results that are based on the provided resources or services. After obtaining a request and completion of material transfer agreement (MTA; if required), relevant resources (e.g., an array of characterized cell lines with specific HRFD mutations) will be provided to the requestor. Specifically, arrays of cell lines assembled into optimized panels for therapeutic testing will be distributed by Core C with a detailed description (e.g., cell line origin, gene mutation, etc). The investigator receiving the resource will cover the costs of shipping.
Similarly, any member of the HRFD Core Center may suggest generation of new resources for therapeutics discovery and characterization (Aim 1; e.g., generation of specific HRFD mutations in a new cell line - this will require collaborative effort between Cores B and C to develop and characterize these cells and Core D to perform therapeutics development assays or services (e.g., in vivo drug efficacy/safety testing in an HRFD model that was not previously evaluated for such studies; Aim 2 and Aim 3). To request the generation of a new resource or service, the member will be also asked to submit: (i) a short description justifying the generation of the resources or services and anticipated impact on HRFD research; and (ii) agreement to share the resource with other investigators and define the embargo period prior to sharing the resource. Priority of requests for more intensive projects will be determined by Core Directors with input from the Scientific Advisory Committee (SAC) and advisors. Any potential conflicts that arise will be adjudicated by the SAC. Members will be made aware of any new resource through the HRFDCC webpage, at meetings, and through direct e-mail correspondence.