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Nov 1, 2010 (Vol. 30, No. 19)

Exploiting HDAC Inhibition

Cancer and Metabolic Diseases Represent Promising Applications

  • Interrogating Activity

    Click Image To Enlarge +
    Promega reports that its HDAC-Glo I/II can be used to interrogate deacetylase activity in either lytic or nonlytic cell-based formats and with extracts or recombinant enzymes. SAHA inhibition of K562 cells is shown.

    Discovery and characterization of new HDAC inhibitors requires sensitive and accurate high-throughput functional assays. According to Andrew Niles, senior research scientist at Promega, this has been a challenge.

    “We evaluated current commercial kits that utilize fluorescence readouts and found they required relatively large amounts of deacetylase enzyme and were difficult to miniaturize into high-density formats. Our approach was to develop a one-step, enzymatically coupled, bioluminescent assay reaction. This system employs an optimized acetylated peptide substrate as an indicator of deacetylase activity, with a proteolytic developer reagent and Ultra-Glo™ luciferase chemistry.”

    Niles says the assay is a simple add, mix, and measure format that typically only takes about 15 minutes to achieve a steady-state luminescent signal proportional to deacetylase activity. “The large signal windows and glow-type luminescence are what facilitates rapid high-throughput screening. In contrast, fluorescence methods require multiple steps, take up to two hours, and have less sensitivity.”

    Because HDACs consist of four families of isoforms, it is important that functional assays accurately interrogate the various HDAC classes. “Class III HDAC enzymes (sirtuins) utilize a NAD+ dependent deacetylase mechanism, whereas Class I/II enzymes operate in a zinc-dependent manner.” 

  • Click Image To Enlarge +
    Binding of pan-HDAC and HDAC8-isoform-specific inhibitors: The pan-HDAC inhibitor PCI-24781 is shown bound in the long narrow pocket of a HDAC1 model (A), in contrast to the HDAC8-specific inhibitor PCI-34051 bound in the shallower pocket of HDAC8 (B). [Pharmacyclics]

    Ultimately, advancing more HDAC inhibitors into viable therapeutics will require a more in-depth understanding, not only of the basic science of HDACs, but also their pharmacology, suggests Sriram Balasubramanian, Ph.D., senior director, translational research at Pharmacyclics.

    “We are finding that HDAC inhibitors work well for hematological malignancies such as lymphomas, leukemias, and multiple myeloma, but only partially for solid tumors, which represent the majority of cancers. So, a basic question is ‘how do we get them to work here?’ It is likely that HDAC inhibitors may function best in combination with other therapies. But, to answer these questions, we will need to elucidate basic mechanisms of how HDACs function. As that pool of knowledge grows, we will be better able to determine how to combine HDAC inhibitor therapy with other strategies.”

    Dr. Balasubramanian says another challenge is dealing with toxicity issues. “It is estimated that anywhere from 4–20 percent of the transcriptome can be altered as a result of using a single pan-HDAC inhibitor such as SAHA. Additionally, tens to hundreds of proteins may become acetylated after treatment with a pan-inhibitor. On the plus side, it appears that normal cells are more resistant to the apoptotic effects of HDAC inhibition. This may partly explain how clinical efficacy can be achieved at the tolerated doses.”

    Since all of the HDAC inhibitors currently marketed or in clinical development inhibit multiple isoforms, broader applications of HDAC inhibitors will require a more sophisticated understanding of how each isoform works.

    “There is clearly a role of specific isoforms in certain types of cancer, as well as in other diseases. But because of their similarity, it can be difficult to make selective compounds. This has been a real struggle in the field. Even with high-throughput screening, the progress has been limited by the structural information available,” Dr. Balasubramanian explains.

    “Since the binding pockets of different isoforms can be quite similar, high-resolution crystal structures are needed. HDAC isoforms have been hard to crystallize individually, partly because they are usually found in complexes and with few exceptions, not as single entities. What is needed is more structure-guided rational design and computational chemistry.”

    The exciting prospect of HDAC inhibitor therapeutics is clearly on the radar. Resolving specificity and toxicity issues should help launch even more candidates into clinical trials.


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