The Significance of HDAC Inhibitors
Histone deacetylases play critical roles in the regulation of gene transcription and cell signaling by catalyzing the deacetylation of histones and other important non-histone substrates
. Aberrant increases in HDAC enzyme activities are implicated in a number of human diseases, including cancer, metabolic disease and neurodegeneration. HDAC enzymes are attractive pharmacological targets because they are readily inhibited by small molecules
. In fact, several HDAC inhibitors (HDACi) have recently proceeded through or are close to FDA approval for the treatment of hematologic malignancies. However, the full promise of clinical HDACi therapy has been hampered by significant dose-limiting toxicities. These off-target effects have led to a renewed focus on basic HDAC biology and the development of isoenzyme-selective HDAC inhibitors, which could reduce or eliminate unintended toxicity. The selective HDAC-Glo™ Assays described in this article represent a new set of tools to characterize the selectivity profiles of novel HDAC inhibitors.
Chemistry and Selectivity of the HDAC-Glo™ Assay
In the HDAC-Glo™ reaction, HDAC enzymes remove the acetyl group on the luminogenic prosubstrate, which allows the peptide to be cleaved by a protease that liberates aminoluciferin. The free aminoluciferin product reacts with Ultra-Glo™ Recombinant Luciferase to produce a stable light signal in proportion to HDAC activity (Figure 1). Each of the three selective HDAC-Glo™ Assays uses a unique, optimized substrate. Selectivity and affinity for individual isoenzymes are summarized in Table 1.
Biochemical Isoenzyme Selectivity Panels
To characterize the selectivity of the three HDAC-Glo™ Assays, recombinant HDAC isoenzyme selectivity panels were performed for all three assays with 11 recombinant HDAC enzymes. As shown in Figure 2, the HDAC-Glo™ I/II Assay selectively measures activity from class I and IIb HDAC isoforms (HDACs 1, 2, 3, 6, 8 and 10). The HDAC-Glo™ Class IIa Assay selectively measures activity from class IIa HDAC isoforms (HDACs 4, 5, 7 and 9). Finally, the HDAC-Glo™ 2 Assay selectively measures activity from HDAC isoform 2 (HDAC 2).
Broad Linearity and Large Dynamic Range of Selective HDAC-Glo™ Assays
To examine the linear and dynamic ranges for the three selective HDAC-Glo™ Assays, recombinant HDAC isoenzyme titrations or cell titrations were performed. As shown in Figure 3, all three HDAC-Glo™ Assays produce broad linear responses (2–3 orders of magnitude) with large dynamic ranges in both a biochemical and cell-based (live cell and lytic) assay format. This means using less enzyme or fewer cells while still producing a large dynamic range, and translates into increased sensitivity to any effects on HDAC activity caused by HDAC inhibitors.
Simplified Cell-Based Profiling of HDAC Inhibitor Selectivity and Potency
Demonstrating HDAC inhibitor selectivity and potency in a biochemical format is a fairly straightforward and simple endeavor. However, conducting these experiments using a live-cell-based system has proven problematic with traditional methods. To demonstrate that the selective HDAC-Glo™ Assays can be used to investigate HDAC inhibitor selectivity and potency in a live-cell-based assay, we used two well-characterized selective HDAC inhibitors. Suberanilohydroxamic acid (SAHA) selectively inhibits HDAC class I and class IIb isoenzymes
while TMP269 provides selective inhibition of HDAC class IIa isoenzymes
. As shown in Figure 4, Panel A, when SAHA was titrated to determine potency using live K562 cells, the compound showed the expected strong inhibition and IC50 value against the HDAC-Glo™ I/II assay (class I/IIb selective) and was significantly less potent against the HDAC-Glo™ Class IIa assay (class IIa selective). In contrast, as shown in Figure 4, Panel B, when TMP269 was titrated to determine potency using live K562 cells, the compound strongly inhibited HDAC-Glo™ Class IIa assay (class IIa selective) and matched the expected IC50 value while observing no inhibition against the HDAC-Glo™ I/II assay (class I/IIb selective). These data reinforce the benefit of combining selective HDAC-Glo™ Assays to obtain more conclusive results when defining HDAC inhibitor selectivity in live cells.
Real-Time Assessment of Cell Permeability and Inhibitor Association Rates for Compounds in a Live-Cell Format
While inhibitor selectivity and potency profiles continue to be key criteria for advancing compounds during HDACi development, recent reports suggest
that association and disassociation kinetics also affect the pharmacological utility of a drug. That is, the rate at which a drug diffuses or is transported into a cell and engages and disengages from its target (residence time) may influence functional potency and off-target effects. Because the HDAC-Glo™ Assays use cell-permeable substrates and produce a stable and proportional luminescent, real-time report of HDAC activity, we were able to examine these parameters.
We adapted the standard protocol by adding HDAC-Glo™ Reagents to live cells and incubating until a stable luminescent signal was achieved. Next, we applied serial dilutions of either SAHA or mocetinostat, which are known to have differential binding kinetics. Luminescence was measured in kinetic mode. As shown in Figure 5, Panel A, when measuring the inhibitory concentration (IC50) of mocetinostat in real time using live K562 cells and the selective HDAC-Glo™ 2 Assay, the test agent became more potent as a function of treatment time. These results suggest either a slow target engagement rate with its HDAC targets or poor cell permeability. In contrast, SAHA IC50 values did not change significantly over time (data not shown). This data indicates a fast association rate of SAHA with its target HDAC activities (occurring prior to the first luminescent measurement) and exceptional permeability. When the respective IC50 values are replotted versus time (Figure 5, Panel B), it is evident that mocetinostat has a much slower onset of action than SAHA, as expected. To address potential poor compound permeability, the same experiments were performed in a biochemical assay format using purified recombinant HDAC 2. The same association data profiles were observed (data not shown). This leads to the conclusion that the observed differences in association rates between the two compounds is most likely due to differences in target engagement rates and not cell permeability differences.
Mechanistic Cytotoxicity: Phenotypic Consequences of Selective HDAC Inhibition
Although measuring HDAC inhibitor potency and selectivity is important, it is also critical to measure the phenotypic consequences of selective HDAC inhibition. To do this, we multiplexed the luminescent HDAC-Glo™ 2 Assay with the fluorescent CellTox™ Green Cytotoxicity Assay. This probe is cell impermeant, nontoxic and selectively binds to DNA from dead cells
. As shown in Figure 6, Panel A, SAHA (class I/IIb HDAC inhibitor) produces a dose-dependent decrease in HDAC-Glo™ 2 luminescence as well as an increase in CellTox™ Green fluorescence. This indicates that HDAC class I/IIb inhibition is sufficient to produce cytotoxicity in K562 cells exposed to SAHA. In contrast, TMP269 (class IIa HDAC inhibitor) reduced the HDAC-Glo™ Class IIa Assay luminescence with no increase in CellTox™ Green fluorescence (Figure 6, Panel B). These results are consistent with use of TMP269 as an immune modulator.
The selective HDAC-Glo™ Assays offer significant advantages over existing plate-based methods. HDAC-Glo™ I/II and HDAC-Glo™ Class IIa Assays deliver isofamily selectivity, and HDAC-Glo™ 2 Assay delivers isoenzyme selectivity, for targeting and profiling isoenzyme-selective HDAC inhibitors. These versatile assays can be used in either biochemical or cell-based formats and are easy-to-use because of their add-mix-read, single reagent format. The assays are easily scalable into high-density plate formats and produce a steady luminescent signal. Because the assays produce broad linear responses (two to three orders of magnitude) with large dynamic ranges, the selective HDAC-Glo™ Assays offer enhanced sensitivity compared to current fluorescent-based formats. While these assays produce IC50 values that are comparable to published data
when using well characterized HDAC inhibitors, an additional benefit is the ability to assess differences in cell permeability and target association rates in live cells. Finally, the ability to multiplex the selective HDAC-Glo™ assays with fluorescent cytotoxicity assays (e.g., CellTox™ Green Cytotoxicity Assay) allows measurement of selective HDAC inhibition as well as the cytotoxic consequences of inhibitor activity on cell health.