Blocking Genome Gatekeepers

A family of enzymes known as histone deacetylases (HDACs) helps to regulate how and when our genome blueprint is transcribed and translated into protein. Small-molecule inhibitors of these enzymes are under intense investigation as cancer therapeutics, as evidenced by research presented last month in Honolulu at the 5th International Chemical Congress of Pacific Basin Societies (Pacifichem). Scientists described their efforts to create HDAC inhibitors for use as both drugs and probes of HDAC biology.

Histone deacetylases remove acetyl groups from the histone-packing proteins around which genomic DNA is wrapped in the cell. The acetylation state of histones' lysine side chains helps orchestrate gene transcription: When acetylated, DNA-wrapped histones unravel to give the cell's transcription machinery access to the DNA. Acetylation also acts to recruit this machinery. When deacetylated, however, histones and DNA pack tightly together to prevent transcription.

HDAC inhibitors thus trigger an increase in gene expression that gives rise to their antitumor activity, noted Ronald Breslow of Columbia University, who invented the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). SAHA is now in late-stage Phase III clinical trials at Merck, reported symposium co-organizer Thomas Miller of Merck.

SAHA, which emerged from a search for a more potent version of an old cell-differentiation agent and was only later shown to be an HDAC inhibitor, was the first HDAC inhibitor to enter human trials. Merck previously has indicated that it plans to ask the Food & Drug Administration later this year to approve the drug to treat cutaneous T-cell lymphoma.

Nearly a dozen HDAC inhibitors are now undergoing human testing, and many of them contain the same hydroxamic acid scaffold. At Pacifichem, Lawrence Perez of Novartis reported that his company is testing the safety and efficacy of two hydroxamic acid-based HDAC inhibitors (LAQ824 and LBH589) in patients with leukemia or breast, prostate, and other cancers. South San Francisco-based Celera, San Diego-based Syrrx, Branford, Conn.-based Curagen, and Danish firm TopoTarget also have hydroxamic acid-based HDAC inhibitors in clinical trials for cancer.

Natural products also make use of the hydroxamic acid scaffold to inhibit HDACs. More than a decade ago, symposium co-organizer Minoru Yoshida of RIKEN, in Saitama, Japan, showed that the hydroxamic acid-containing microbial natural product trichostatin A inhibits histone deacetylase activity. This and other hydroxamic acid-based inhibitors do so by chelating the Zn²⁺ ion found in the active site of most HDAC enzymes, Yoshida noted. When an acetylated lysine binds to the enzyme, this Zn²⁺ ion facilitates hydrolytic attack of the enzyme's weakened acetamide bond by a water molecule. Hydroxamic acid chelation inactivates this metal ion.

Scientists figure that HDAC inhibitors kill cancer cells by enabling access to and transcription of genes that would otherwise remain unread, explained symposium co-organizer Robert Déziel of MethylGene, a small Montreal-based firm developing HDAC inhibitors. As a result, some scientists feared that HDAC inhibitors would have vicious side effects because they would trigger a willy-nilly increase in gene expression, Déziel told C&EN. "But, in fact, only a small percentage of genes seem to be activated by HDAC inhibitors," he added, including genes involved in blocking cell division and kick-starting apoptosis that are typically silenced in cancer cells.

Why HDAC inhibitors selectively regulate only a handful of key genes remains a mystery, Breslow noted. It could be that cancerous cells rely far more on acetylation-based gene regulation than healthy cells do, he suggested. The inhibitors' surprisingly restricted effects on gene expression may explain why they seem to have relatively few toxic side effects, he said.

In fact, much about the biology of this enzyme family remains mysterious, Perez noted. A dozen or so HDACs are known, but the specific cellular roles of each of these closely related enzymes or "isoforms" is not known, he observed. Certain isoforms don't seem to deacetylate histones at all. For example, HDAC6 is thought to deacetylate tubulin, a protein component of the cellular cytoskeleton.

"Histone deacetylases should be renamed protein deacetylases," Yoshida said in an interview. He noted that HDAC inhibitors prevent the deacetylation of proteins other than histones, including certain "chaperone" proteins that stabilize cancer-promoting oncogenic proteins in tumor cells. With their acetyl groups intact, these chaperone proteins are rendered unable to protect their oncogenic wards from degradation.

"Isoform-specific HDAC inhibitors would be strongly desirable as tool compounds for understanding the biology of regulating histone acetylation and as pharmaceutical treatments for diseases that are the result of abnormal gene expression," Perez told C&EN. In addition to cancer, tempting targets include diabetes and inflammation, Déziel suggested.

A number of academic and industrial scientists are exploring isoform-specific HDAC inhibitors. Stuart Schreiber's lab at Harvard University previously designed a hydroxamic acid bearing a long tail that selectively inhibits HDAC6. This molecule has been used to help clarify HDAC6's biological role. In Hawaii, Ellen Leahy of Celera described her company's efforts to provide a structural basis for the design of isoform-specific inhibitors. Her team has solved the crystal structures of human HDAC8 with SAHA, trichostatin A, and other nonspecific hydroxamic acid-based inhibitors as well as its own proprietary inhibitors. This and other structural information should eventually allow scientists to rationally design versions specific for individual isoforms or pools of isoforms, she said.

Others hunting for isoform-specific inhibitors are betting on non-hydroxamic acid-based scaffolds. For instance, Schering AG has an o-aminoanilide-based inhibitor that's specific for a handful of HDAC isoforms in clinical trials for cancer. At Pacifichem, Jeffrey Besterman of MethylGene presented his firm's efforts to design a panel of o-aminoanilide-based isoform-specific HDAC inhibitors that might be useful both as therapeutics and for teasing apart HDAC biology. One such selective inhibitor (MGCD0103) is now in Phase II trials for certain types of cancer. And Yoshida and Norikazu Nishino of Japan's Kyushu Institute of Technology described their collaborative efforts to develop hydroxamic acid analogs of the cyclic tetrapeptide natural product trapoxin B that are isoform specific.

"A chemical toolbox of isoform-specific inhibitors holds great promise for sorting out histone deacetylase biology," Yoshida said in an interview. A better understanding of the biology of deacetylase enzymes should help drug designers pinpoint the optimal isoform profiles for fighting cancer and other diseases, he added.

PROTEIN DEACETYLATION

Sirtuins: Deacetylases That Target Multiple Cellular Proteins

Another class of deacetylase enzymes, the sirtuins, is piquing the interest of biologists and drug designers alike, according to work presented in mid-December at Pacifichem. The sirtuins appear to deacetylate not only histone substrates but also other cellular proteins, including ones involved in cell-cycle arrest and in the cell's response to DNA damage.

In contrast to zinc-dependent histone deacetylases, sirtuins use a nicotinamide adenine dinucleotide cofactor to strip acetyl groups from protein lysines. This reaction produces the corresponding deacetylated lysine residue as well as nicotinamide, 2'-O-acetyl-ADP-ribose.

"The consequences of sirtuin inhibition are not yet clear," noted Manfred Jung of Albert-Ludwigs University, Freiburg, Germany, "in part because potent and stable inhibitors of these enzymes have not been available."

Hoping to shed some light on the biological role of these protein deacetylases, scientists are hunting for potent and specific inhibitors of individual members of the sirtuin family. Julian A. Simon of Fred Hutchinson Cancer Research Center in Seattle described splitomicin, an inhibitor of a yeast sirtuin. He and Jung each reported splitomicin analogs that inhibit specific human sirtuins.

For instance, Jung's team has developed a selective inhibitor of a sirtuin that reportedly inhibits the deacetylation of a viral protein required for HIV transcription. In addition to clarifying its target's biological role, "our inhibitor could lead to the development of novel therapeutics for HIV," Jung told C&EN. Simon hopes that his selective inhibitors of sirtuins implicated in aging and cancer may help clarify the biological mechanisms of these diseases and perhaps someday guide the way to therapeutics.