Dr Brigitte Kieffer & Dr Christopher Evans - 2014

“Cloning: twice in one day”

Dr Brigitte Kieffer (McGill University)

When a scientist receives an honour it is usually for a single major contribution to advancing the field, but for Brigitte Kieffer I would identify three major contributions to opioid science which have been world‐leading and have had a very significant impact on both INRC and on the wider scientific community. Firstly, her cloning of the delta opioid receptor (published in PNAS), secondly the development and characterization of the mu opioid receptor knockout mouse (published in Nature) and thirdly the development and characterization of the first conditional opioid receptor knockout mouse, with a fascinating behavioural phenotype that will be published shortly. In between there have been many other significant publications, including really elegant work on the role of opioids in emotional responses (in Nature Genetics) and in maternal attachment (in Science).

Brigitte first came to an INRC meeting in 1990 at Noordwijkerhout, and at that point several laboratories had spent nearly a decade trying to clone the first opioid receptor. In the 1980’s I listened to a lot of talks on the cloning of the opioid receptor that really had nothing to say! I felt sorry for a string of PhD students who had to write a thesis with precious little to show for their heroic efforts. So when Brigitte Kieffer and Chris Evans independently succeeded in cloning the first opioid receptor in 1992 these were landmark publications. Chris published in Science and Brigitte published in PNAS, though I know she originally submitted the manuscript to Nature. The editors foolishly said “it was just the cloning of another G‐protein coupled receptor” and rejected the manuscript. This paper is now cited over 850 times and I am sure the editors of Nature are kicking themselves. Nature did not make the same mistake twice and when Brigitte submitted her phenotypic characterization of the first opioid receptor knockout mouse in December 1996 the referee and editors reports were glowing. The citation count for this paper is now approaching four figures. She presented this seminal work at the INRC meeting in Long Beach earlier that year; a clearcut deletion of the mu opioid receptor and a remarkable loss of key behaviours that this receptor is involved in. I recall there was stunned silence in the room, as INRCers listened to this major field advance. Avram Goldstein who was standing next to me remarked “this is almost too good to be true”. But it was one of those set of experiments that we would all love to be involved with; I was privileged to be one of the collaborators on this work. Sometimes, experiments don’t work out as you had planned. Brigitte had set out to make a delta opioid receptor knockout mouse, and succeeded first in making a mu knockout – the kappa gene knockout came next (again a first) and the delta knockout last. Sometimes the direction of science is rather serendipitous.

There is a third landmark discovery to come and I suspect in her Founders lecture she might talk about her work in developing a conditional deletion of an opioid receptor to enable us to identify the role of these receptors in specific regions in the brain. This work and the previous development of knockout mice, have taken many years and stoic perseverance to come to fruition. It reflects Brigitte’s critical attention to detail, a characteristic I have seen when writing both grant applications and research publications with her. She has very many successful collaborations in both Europe and worldwide, and in looking at the author lists of her papers you might think she collaborates with everyone. This is not true – she chooses her collaborators very carefully. I remember her telling me when she cloned the delta opioid receptor that she gave the clones to everyone who asked; but far too many of those that asked did not do anything useful with them! So when the opioid knockouts were developed she took a much more cautious approach, and it has delivered over 200 high quality publications.

Brigitte is a person who is in love with her science and her success has been recognised by four national prizes from both French and US academies of science. She was appointed as Director of the world‐leading IGBMC in Strasbourg in 2012 where most of her opioid research has been carried out, but she always said that she must not allow leadership and management to compromise the important science she still had to do. So this year she moved to be scientific director of the Douglas Institute at McGill University in Canada – there is more to come, investigating opioid involvement in emotion.

For me, it has been an honour to collaborate with Brigitte for 20 years. There has been some great science and great friendship. In the early 1980’s you would have found Hans Kosterlitz (one of the founders of INRC) in the bar late at night at INRC meetings, working hard and playing hard. From 1992, you would find Chris Evans and Brigitte Kieffer (and me!) there too. As I said, great science and great friendship; it is what INRC is all about.

Professor Ian Kitchen University of Surrey, UK April 2014

Dr Christopher Evans (University of California Los Angeles)

When I was asked by the organizers of this year INRC to write a short text to acknowledge Chris Evans’ contribution to our society, I was wondering where has the time gone. It seems like yesterday that Chris and I first met, at the 1981 Kyoto meeting, when INRC was at its first golden age. It was at the time when endogenous opioid peptides were being identified and the society experienced a large influx of scientists whose research focuses deviated from the traditional approaches used by the anatomists, biochemists, medical chemists, pharmacologists and physiologists that populated the society at that time. Chris is one of those scientists who joined INRC during that time and his scientific footprints since then have been tremendous. His thesis training was as a peptide chemist with Derek Smyth at MRC England. Chris then went to Stanford for a postdoctoral position with Jack Barchas where, in close collaboration with Eckard Weber, he isolated several new opioid peptides, including metorphamide, and developed antibody probes for

studying the different processing products of proenkephalin, prodynorphin and proopiomelanocortin.

Everyone in the society knows that Chris was instrumental in the cloning of the opioid receptors. His initial report on the successful cloning of delta-opioid receptor by expression cloning in 1992 has been cited >1000 times. Together with Brigitte Kieffer, who independently reported the cloning of delta-opioid receptor at the same time, they launched INRC into its second golden age. Subsequent successful attempts to clone other opioid receptors and the Orphanin F/Q receptor are based on the two cDNA sequences reported by Chris and Brigitte. But few know that before his successful attempt, Chris was involved in a failed attempt to clone the receptor by expression cloning. I was one member on the team that failed. To his credit and persistency, an ingenious method of synthesizing 125I‐labeled peptide for high affinity binding and autoradiography to identify clones, and a library generated in collaboration with Rob Edwards that eliminated secondary mRNA structure thus enabling complete reverse transcription of the receptor mRNA, Chris and his talented technician Duane Keith, were successful while many of us failed in cloning the receptor. For this singular achievement, Chris’ contribution to the society is unparalleled.

In addition to the cloning of the receptor, another significant contribution that alters the course of INRC is Chris’ work on biased agonism. Together with Mark von Zastrow, based on my initial observations that morphine is a partial agonist in the delta-opioid receptor and will not internalize the receptor, Chris demonstrated unequivocally that biased agonism exists in the opioid receptor systems. Chris’ initial reports on the differential regulation of receptor trafficking laid the foundation for biased agonism, probable mechanisms for differential opiate tolerance and addiction, and probable drug development that one day might eliminate the many side‐effects of opiate analgesics, the holy grail of opioid receptor research.

Chris’ contribution to the society is not limited to his scientific accomplishments. Chris has been an active participant in yearly INRC meetings, has served on the Executive Committee, and local organizing committees. But I think the other major impact that Chris has on INRC is his mantra for collaborative research and out of the box ideas. I am sure many of us have benefited from Chris’ generosity in providing reagents and clones. Our studies on the opioid genes transcription and generation of the μ‐opioid receptor knockout mice would not be possible if not for his initial willingness to share the yet to be reported delta-opioid receptor cDNA clone. Sometimes, I still have problems comprehending his ideas, seemingly come out from the right field, to paraphrase an American baseball idiom. A case and point is his continued enthusiasm and efforts on the phylogenetic studies of the opioid receptors and peptides. I guess one of these days, another high impact study will come out of Chris’ group that will change the course of how we think about the biological functions of opioids.

What I have attempted does not detail all Chris’ contributions to INRC. What I have summarized is Chris’ contributions that change the course of INRC, contributions that rank with those of INRC founders, such as Avram Goldstein, Eric Simon and Hans Kosterlitz. For these contributions, Chris definitely deserves the honor of being one of this year Founder’s Lecture speakers. I am honored to have the opportunity to write this tribute and called Chris as my friend and collaborator.

Professor Ping‐Yee Law University of Minnesota, USA May 2014