Melding the best of two worlds: Cecil Pickett's work on cellular oxidative stress and in drug discovery and development

Many chemicals and cellular processes cause oxidative stress that can damage lipids, proteins, or DNA (1). To quickly sense and respond to this ubiquitous threat, organisms have evolved enzymes that neutralize harmful oxidants such as reactive oxygen species and electrophilic compounds (including xenobiotics and their breakdown products) in cells.

These antioxidant enzymes include GSH S-transferase (GST),² NADPH:quinone oxidoreductase 1, thioredoxin, hemeoxygenase-1, and others (2, 3). Many of these proteins are commonly expressed in cells exposed to oxidative stress.

The antioxidant response element (ARE) is a major regulatory component of this cellular stress response. The ARE is a conserved, 11-nucleotide-long DNA motif present in the 5′-flanking regions of many genes encoding antioxidant proteins. The laboratory of Cecil Pickett (Fig. 1) at the Merck Frosst Centre for Therapeutic Research in Quebec discovered ARE, a finding reported in the early 1990s in two JBC papers recognized as Classics here (4, 5).

Figure 1.

Cecil Pickett (pictured) and colleagues first described the ARE motif, present in the 5′ regions of many genes whose expression is up-regulated by oxidative stress and xenobiotics. Photo courtesy of Cecil Pickett.

ARE's discovery was spurred in large part by Pickett's career choice. After completing a PhD in biology and a 2-year postdoc at UCLA in the mid-1970s, he began to work in the pharmaceutical industry.

Recruited to Merck in 1978 by its then head of research and development (and later CEO), Roy Vagelos, “I became interested in how drug-metabolizing enzymes were induced by various xenobiotics,” Pickett says.

According to Pickett, Vagelos encouraged researchers at the company “to really start a program where we could build our research careers. But he also said, 'Keep in mind the long-term mission of Merck—and that is to discover novel medicines that could help people.'”

Pickett remembers joining Merck at a unique time in the pharmaceutical industry. “[Vagelos] really had an incredible vision for research and development.” Inspired by Vagelos' vision, Pickett launched a vigorous research program that attracted many talented scientists.

To find xenobiotic-metabolizing enzymes, Pickett and his team treated rats with various chemicals, including phenobarbital and 3-methylcholanthrene, to induce genes involved in xenobiotic metabolism and breakdown. To isolate these genes, the team used an in vitro system for translation of the mRNAs isolated from the livers of the animals.

This approach yielded promising results. “I noticed that the profile of the in vitro translated material from induced animals was very different from that of noninduced animals,” says Pickett. “And there tended to be a low-molecular-weight protein that was always induced.”

At first, it was unclear what this small protein might be. But a colleague at Merck, Anthony Lu, had a hunch that it might be GST, Pickett says.

Acting on Lu's idea, Pickett and his team applied a technique called polysomal immunoprecipitation. The researchers used GST-specific antibodies from Barbara Hales' laboratory at McGill University in Montreal to detect and isolate mRNA-ribosome complexes that contained GST-encoding mRNA sequences (6).

“That allowed us to synthesize cDNAs and isolate the structural genes for the GSTs,” says Pickett.

With the GST gene sequences in hand, the researchers could home in on cis-acting regulatory motifs in the 5′-flanking regions of these genes (reviewed by Pickett and Thomas Rushmore in Ref. 7).

ARE was one of the motifs the researchers discovered as being required for xenobiotics-induced activation of GST gene transcription. Its location in the promoter of the gene encoding the Ya subunit of GST was delineated by extensive deletion and point mutation analyses. These findings were reported in a series of articles (8, 9), including the two Classics papers (4, 5), the second of which reported the ARE consensus sequence.

The discovery of ARE by Pickett and colleagues caused a major shift in thinking about how xenobiotic-metabolizing genes are regulated. Up to that point, it was thought that the aryl hydrocarbon (Ah) receptor, a transcription factor that binds to the xenobiotic-responsive element (XRE) in gene promoters, activated all the genes encoding drug-metabolizing enzymes.

Pickett's work made it clear that antioxidant genes such as GST have multiple regulatory elements in their promoters that are responsive to specific cellular stressors (Fig. 2). Subsequent work in Pickett's laboratory and by others helped identify the transcription factors that recognize the ARE and activate the transcription of GST and other oxidative stress response genes (2, 10, 11).

Figure 2.

An overview of the transcriptional elements in the promoter region of the GST Ya subunit gene. They include the ARE motif whose location and consensus sequence were first described by Pickett and colleagues (4, 5). Later work has shown that oxidative stress–induced NFE-related factors (NRF), after heterodimerizing with small MAF BZIP transcription factor (MAF) proteins (10), bind ARE to activate transcription (2). The XRE motif is required for activation of expression by the heterodimeric Ah receptor–Ah receptor nuclear translocator (ARNT) complex in response to many xenobiotics. GRE, glucocorticoid-responsive element; HNF1 and HNF4, hepatocyte nuclear factor-binding sites. Numbers indicate distance (in nucleotides) from the transcription start site of the GST gene. Adapted from Ref. 7. This research was originally published in the Journal of Biological Chemistry. Rushmore, T. H., and Pickett, C. B. Glutathione S-transferases, structure, regulation, and therapeutic implication. J. Biol. Chem. 1993; 268:11475–11478. © the American Society for Biochemistry and Molecular Biology.

One of these transcriptional regulators is NFE2-related factor 2 (NRF2), identified in 1994 by another research group (11). Because NRF2 binds ARE, suggesting that it helps activate oxidative stress responses, NRF2 soon became a major focus of Pickett's team, which helped uncover key aspects of how NRF2 itself is regulated by proteasomal degradation and protein kinases (2, 12, 13).

“The discovery of the ARE really laid the groundwork for much of the work that has been done in the field on NRF2,” Pickett notes.

Given the many fundamental discoveries coming out of Pickett's laboratory, one may be forgiven for thinking that his main focus was on fundamental research. But while running a very prolific research program, Pickett's talents for designing and overseeing drug discovery programs soon led him to also direct major drug development efforts at Merck.

This meant working with multidisciplinary research teams. “It was a large group of people—chemists, pharmacologists, molecular biologists, and biochemists,” says Pickett. “I oversaw a more integrated approach to how drugs need to be discovered and developed.”

Pickett also served on an internal committee that directed all of Merck's business in Canada; this unique vantage point further shaped his approach to drug discovery. Pickett's polymath talents and scientific background in the molecular bases of inflammatory diseases were key for the discovery and development of the anti-inflammatory medicine montelukast (Singulair), used chiefly to prevent asthma attacks and other inflammatory lung conditions.

In 1993, Pickett joined Schering–Plough in New Jersey as executive vice president of discovery research, eventually becoming president of the institute. “I had a very good balance between the more fundamental work and also the drug discovery work,” says Pickett of his time at Schering–Plough.

Pickett drove the development of several drugs for managing metabolic disorders and diseases, including cancer, fungal and viral infections, and hypercholesterolemia. He also continued basic research that further deciphered the role of the ARE-binding transcriptional regulator NRF2 in oxidative stress responses. This effort later helped advance drug development at Biogen Idec, a company Pickett joined as head of R&D in 2006.

“Work on NRF2 at Biogen began with some ideas that I had,” says Pickett. “Biogen's current small-molecule compound Tecfidera [dimethyl fumarate], which is used for the treatment of multiple sclerosis, activates the NRF2 pathway.”

Pickett is now formally retired, but he remains involved in R&D as a member on several advisory boards and committees of such organizations and companies as the American Association for Cancer Research and Zimmer Biomet.

Reflecting on the many challenges of directing both fundamental research and drug discovery efforts, Pickett emphasizes the synergy these diverse pursuits offered. “I think having my own lab, being engaged in that research, and reading the scientific literature helped me in making sure that the scientific thoughts in the [drug discovery] programs were solid.”