A Comparative Study of Target Engagement Assays for HDAC1 Inhibitor Profiling.

Histone deacetylases (HDACs) are epigenetic modulators linked to diseases including cancer and neurodegeneration. Given their therapeutic potential, highly sensitive biochemical and cell-based profiling technologies have been developed to discover small-molecule HDAC inhibitors. Ultimately, the therapeutic action of these inhibitors is dependent on a physical engagement with their intended targets in cellular and tissue environments. Confirming target engagement in the cellular environment is particularly relevant for HDACs since they function as part of cell type-specific multiprotein complexes. Here we implemented two recently developed high-throughput target engagement technologies, NanoBRET and SplitLuc CETSA, to profile 349 compounds in the Epigenetic-Focused collection for HDAC1 binding. We found that the two HDAC1 target engagement assays correlated well with each other and with orthogonal activity-based assays, in particular those carried out in cellular environments rather than with isolated HDAC proteins. The assays detected a majority of the previously described HDAC1 inhibitors in the collection and, importantly, triaged HDAC inhibitors known to target other HDACs.

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.

Keeping It Clean: The Cell Culture Quality Control Experience at the National Center for Advancing Translational Sciences.

Quality control monitoring of cell lines utilized in biomedical research is of utmost importance and is critical for the reproducibility of data. Two key pitfalls in tissue culture are 1) cell line authenticity and 2) Mycoplasma contamination. As a collaborative research institute, the National Center for Advancing Translational Sciences (NCATS) receives cell lines from a range of commercial and academic sources, which are adapted for high-throughput screening. Here, we describe the implementation of routine NCATS-wide Mycoplasma testing and short tandem repeat (STR) testing for cell lines. Initial testing identified a >10% Mycoplasma contamination rate. While the implementation of systematic testing has not fully suppressed Mycoplasma contamination rates, clearly defined protocols that include the immediate destruction of contaminated cell lines wherever possible has enabled rapid intervention and removal of compromised cell lines. Data for >2000 cell line samples tested over 3 years, and case studies are provided. STR testing of 186 cell lines with established STR profiles revealed only five misidentified cell lines, all of which were received from external labs. The data collected over the 3 years since implementation of this systematic testing demonstrate the importance of continual vigilance for rapid identification of "problem" cell lines, for ensuring reproducible data in translational science research.

Transient cellular adhesion on poly(ethylene-glycol)-dimethacrylate hydrogels facilitates a novel stem cell bandage approach.

We discovered a transient adhesion property in poly(ethylene glycol) dimethacrylate (PEG-DMA) hydrogels and employed it to develop a novel "stem cell bandage" model of cellular delivery. First, we cultured human mesenchymal stromal cells (MSCs) on the surface of PEG-DMA hydrogels with high amounts of arginine-glycine-aspartic acid (RGD) adhesive peptides (RGD++) or without RGD (RGD-). On day 1, MSCs underwent an initial adhesion to RGD- hydrogels that was not significantly different over 13 days (n = 6). In addition, cells appeared to be well spread by day 3. Significantly fewer cells were present on RGD- hydrogels on day 15 compared to day 9, suggesting that RGD- hydrogels allow for an initial cellular adhesion that is stable for multiple days, but transient over longer periods with a decrease by day 15. This initial adhesion is especially surprising considering that PEG-DMA does not contain any biological adhesion motifs and is almost chemically identical to poly(ethylene glycol) diacrylate (PEG-DA), which has been shown to be non-adhesive without RGD. We hypothesized that MSCs could be cultured on RGD- PEG-DMA hydrogels and then applied to a wound site to deliver cells in a novel approach that we refer to as a "stem cell bandage". RGD- donor hydrogels were successfully able to deliver MSCs to PEG-DMA acceptor hydrogels with high RGD content (RGD++) or low amounts of RGD (RGD+). Our novel "bandage" approach promoted cell delivery to these model surfaces while preventing cells from diffusing away. This stem cell delivery strategy may provide advantages over more common stem cell delivery approaches such as direct injections or encapsulation and thus may be valuable as an alternative tissue engineering approach.

A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase.

Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.