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Cellzome Study Suggests Epigenetic Inhibitors' Efficacy Depends on Protein-Complex Interactions

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By Adam Bonislawski

In a study published this week in Nature Biotechnology, researchers from the proteomics firm Cellzome demonstrated that the effect of small-molecule inhibitors on histone deacetylases, or HDACs, varies depending on the protein complex with which the target HDAC is associated.

The research suggests that HDAC inhibitors may be more selective than previously thought, a finding that could improve scientists' understanding of the biological pathways associated with the proteins and aid identification of potential epigenetic drug targets, Gerard Drewes, the company's vice president of discovery research and senior author on the paper, told ProteoMonitor.

HDACs help control protein lysine acetylation, a mechanism key to the epigenetic control of gene expression and the regulation of cell metabolism. As such, they are becoming a significant area of interest for pharmaceutical companies.

Two HDAC inhibitors, Merck's Zolinza and Celgene's Istodax, are currently sold as treatments for cutaneous T-cell lymphoma, and more than a dozen other HDAC inhibitors aimed at indications including various cancers and Alzheimer's disease are in some stage of clinical development.

In the body, HDACs serve as the catalytic core of megadalton complexes involved in chromatin modification and gene repression. Studies of these proteins, however, have traditionally been done in vitro using purified recombinant proteins. And, according to Drewes, under these conditions HDAC inhibitors have generally demonstrated low selectivity, with a given inhibitor often inhibiting "four or five or six or more" of the 11 known HDAC proteins.

This lack of selectivity raises the issue of potentially dangerous off-target effects, complicating drug-development efforts. It also limits the inhibitors' usefulness as tools for investigating the function of HDAC proteins.

Tested in vivo on HDACs situated within their characteristic protein complexes, however, these inhibitors appear to be significantly more selective. When the Cellzome researchers examined the activity of HDAC inhibitors in crude cell lysates, as opposed to purified recombinant proteins, they found that the inhibitors' affinities for the HDACs were influenced by the complex containing them.

"That was really the surprising outcome," Drewes said. "That some HDACs have differential sensitivity to inhibition [depending on] whether they're in one particular complex as opposed to another particular complex. It really demonstrates that the molecular environment that's shaped by the complex can really influence the inhibitor selectivity."

"It's a very interesting publication for the field," University of Freiburg professor Manfred Jung, who was not involved in the study, told ProteoMonitor. "The big question was always: 'How much can we learn from in vitro [HDAC] subtype selectivity profiling?'

"In vivo the [HDACs] occur in multi-protein complexes. That might affect the folding of the proteins; it might affect the binding itself," said Jung. "So you could maybe get two HDAC1-selective [inhibitors] that in vivo would bind with different affinity to different complexes that both contain HDAC1."

It suggests "that one has to think more about the [HDAC] complexes and not just the subtypes," he said.

In the study, the researchers, all of whom work for Cellzome, examined how 16 HDAC inhibitors affected 1,251 proteins. They added excess inhibitor to cell extract samples that they then passed over HDAC-specific probes that competed with the inhibitor for binding of the target HDAC.

Using TMT tagging and mass-spec analysis on Thermo Fisher LTQ Orbitrap and Orbitrap Velos machines, they quantified the reduction in protein capture due to inhibitor activity, allowing them to measure the binding of each inhibitor to the various HDAC complexes.

The research was done on Cellzome's chemoproteomic Episphere platform, which Drewes said can currently screen up to 100,000 compounds against native proteins. The platform consists of the probe technology and a number of non-proprietary mass-spec techniques. But the bulk of the intellectual property involved relates to the data analysis done on the back end.

"The way we filter, analyze, and quantify the spectra and actually turn this enormous amount of data into actual useful information — that is largely covered by proprietary software algorithms," he said.

The inhibitor-complex selectivity demonstrated by the work points to a possible way forward for HDAC research, Drewes suggested.

"At the moment there is no real data in the public domain that would, for instance, tell you that for [treating] a particular cancer you should target a particular HDAC," he said. "It's not like we could say, 'HDAC1 is the target for lung cancer.' That sort of data doesn't exist. The biology is still missing."

"The development of selective inhibitors will help move that research forward," he said. "The inhibitors can be used as probes in systems to actually tease out the specific biology of the different enzymes."

To Jung, the study "opens up a lot of questions that need to be addressed." Previously, "even if you could say that in vitro [an inhibitor] is selective for HDAC2 or HDAC4 or HDAC9, nobody knew how to see if that was relevant in animals, in plants, in patients," he said. "This opens up a way to monitor this in animals and cells and plants. Now the question is, 'Can we correlate this with phenotypic responses?'"

Based in Heidelberg, Germany, and Cambridge, UK, Cellzome has traditionally operated by forming collaborations with pharmaceutical firms, including Johnson & Johnson, Novartis, and GlaxoSmithKline.

In March 2010, in exchange for the potential to pocket more than $645 million in milestone payments, Cellzome signed an agreement with GSK to screen for epigenetic drug targets related to undisclosed immuno-inflammatory diseases (GWDN 3/10/2010).

The deal netted the company a $45 million upfront payment, and in December Cellzome reached its first research milestone, triggering a payment of an undisclosed amount.

The HDAC work is a proprietary effort by the company and not part of any pharma agreement, chief scientific officer David Simmons told ProteoMonitor. However, Cellzome "is open to collaborating with other companies in that space," he added.

The company is also developing probes to investigate other epigenetics-related proteins like methylases and demethylases, Drewes said.

"Basically, we're very interested in this area of epigenetics, which we believe offers new opportunities for drug discovery, and the pharmaceutical industry also has a growing interest in this area," he said. "[HDACs] are an entry into this area, but our interests go beyond that. It's not just the HDACs that are gaining momentum as novel [drug] targets. Enzymes like methylases may provide even more opportunities for drug development."


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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