This story originally ran on Dec. 15.
A team led by scientists from the Scripps Research Institute has demonstrated the role of sulfenylation in regulating the kinase activity of epidermal growth factor receptor, a major anticancer drug target.
The research, which is detailed in a paper published this week in the online edition of Nature Chemical Biology, provides evidence for sulfenylation’s role as a global signaling mechanism similar to phosphorylation and has potentially significant implications for kinase inhibitor development, Kate Carroll, a Scripps researcher and leader of the study, told ProteoMonitor.
Protein sulfenylation is a post-translational modification generated via oxidation of cysteine residues to sulfenic acid by hydrogen peroxide. Traditionally, the modification has served as a biomarker of oxidative stress, but more recently, Carroll said, researchers have begun investigating its role in cellular signaling.
“Just on the basis of first principles, if you have enzymes that make hydrogen peroxide when you stimulate cells, then hydrogen peroxide must be reacting with something,” she said. “And the hypothesized target has been specific cysteine residues in signaling proteins. So this has been the model, and now [via the EGFR work] we’ve been able to show that this is actually the case.”
Key to this work was the development by Carroll’s lab of a chemoselective probe capable of selectively targeting protein sulfenylation in cells. Dubbed DYn-2, the reagent consists of a dimedone warhead – which specifically binds sulfenic acids – linked to a small azide chemical handle. Because the probe is able to permeate cells, it can measure protein sulfenylation states before cell homogenization, which can disrupt redox balances and skew results.
“With any probe you have to be really concerned about what happens when you add it to cells,” Carroll noted. “We’ve made sure that the reagents we’re using and getting across the cell membrane are reactive but not toxic to the cell; [and] that we’re not killing cells or disturbing redox homeostasis just by adding the probe to the cells.”
Using the probe, the researchers investigated the relationship between protein sulfenylation and EGFR signaling, a pathway selected, Carroll said, due to its high profile as a therapeutic target and past research linking it to redox signaling.
“It’s been known for some time that if you add EGF to cells containing EGFR you generate a bolus of hydrogen peroxide,” she said. “So we knew this, but the key question was, ‘What are these cellular targets?’”
In studies of A431 cells, the researchers found the addition of EGF increased cellular ROS levels and resulted in dynamic changes in protein sulfenylation. They also investigated the signaling phosphatases PTEN, PTP1B, and SHP2 – three components of the EGFR network – finding that each undergoes EGF-dependent oxidation. They further demonstrated that stimulation with EGF or exogenous hydrogen peroxide led to sulfenylation of EGRF, as well, and that this sulfenylation modulated the receptor’s kinase activity, with activity enhanced by moderate hydrogen peroxide concentrations and decreased by higher concentrations.
Via mass spec analysis on a Thermo Scientific LTQ-XL instrument, Carroll’s team identified Cys797 as the site of the EGF-induced sulfenylation modulating EGFR’s kinase activity. This, she noted, was a particularly interesting finding given that this cysteine is also the target of a number of irreversible kinase inhibitors currently in Phase III clinical trials. A better understanding of this sulfenylation site, she suggested, could improve the effectiveness of these drugs.
For these inhibitors to covalently modify EGFR, "the receptor has to be in the thiol [un-sulfenylated] form,” she said. “So we feel that a new and exciting area to explore would be targeting the most active form of the receptor, which is the oxidized form, the sulfenic acid form.”
Such an approach, Carroll said, could have several advantages. For instance, because sulfenylated proteins are significantly less abundant than thiol-containing proteins, a compound targeting EGFR’s sulfenylated form would suffer less from off-target interactions.
“In cells there’s a tripeptide called glutathione, for instance, that’s present at millimolar concentrations, and that thiol, although it’s not very reactive, can irreversibly react with these inhibitors,” she said, “whereas there’s no millimolar off-target source of sulfenic acid.”
“The other side of this,” Carroll added, “is that there are many disease states that are associated with really high levels of oxidative stress. We’ve profiled receptor oxidation among cancer cell lines, and it is quite high compared to normal growing cells.”
It’s possible "that we could design irreversible inhibitors that covalently modify the sulfenyl form of the receptor, which is going to be more prevalent in a tumor cell than a normal cell,” she said. “It basically would function as a biochemical basis for segregating cancer cells from normal cells.”
Carroll has contracts with several large pharma companies with which her lab is pursuing such work, she said, though she declined to name any specific firms. Currently a number of irreversible EGFR inhibitors – including Boehringer Ingelheim’s afatinib, Pfizer’s neratinib, and GlaxoSmithKline’s lapatinib – marketed as Tykerb – are in phase III clinical trials.
In addition to this drug development research, Carroll’s team is working to improve the quantitative capabilities of the DYn-2 probe used in the study by combining it with features of another probe for measuring oxidative stress that it introduced earlier this year (PM 1/21/11).
“The probe we just published on allows us to look at fold changes in sulfenic acid, but it doesn’t get at the thiol population [whereas the earlier probe] … allows us to look at both thiols and sulfenic acids,” she said. “So in the future we’re going to combine both technologies, and then we’re off to the races.”
The researchers are also investigating the role of redox signaling in other receptor tyrosine kinase pathways, specifically insulin signaling, Carroll said. The group also hopes to determine how general a mode of regulation cysteine oxidation of tyrosine kinases is.
“There are ten other kinases that have a cysteine at exactly the same position as EGRF does,” she said. “So are all those susceptible to oxidation? And, if so, what impact do they have on activity, and moreover, what other kinases have conserved cysteines within their active site and what role do those play in terms of redox regulation?”
“I think this study serves as a springboard to a lot of different areas,” she said.
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