Candace Pert
Paradigms from Neuroscience: When Shift Happens
Candace Pert has spoken often in recent years about how lay audiences can incorporate the results of modern neuroscience into their own daily lives. Her lectures often touch on material from her 1997 book,Molecules of Emotion, now in its twelfth printing, and she in fact refers to her lecturing, somewhat exasperatedly, as “the book tour that never ends.” Given the frequency of her lectures, it is therefore somewhat disarming, as she sits down to talk, that she makes a voiced effort “to relax,” explaining that she is particularly excited about our interview, because unlike her “airy-fairy” lectures, she here feels that she is addressing “her people,” neuroscientists. She smiles proudly as she points out that she attended the very first meeting of the Society for Neuroscience some thirty years ago, and she is excited to be returning to the meeting in New Orleans this year to speak about the neurotherapeutic effects of a peptide that has recently shown dramatic efficacy in the treatment of AIDS. The peptide drug was in fact written up just last month in theWall Street Journal, and Pert is hoping that the clinical success will not only make a contribution to AIDS, but will further underscore the potential for peptide drugs in a wide variety of clinical and experimental settings. Pert’s interest in bioactive peptides began with her landmark studies, reported when she was still a graduate student in Sol Snyder’s laboratory, in which she first described the opiate receptor.
MI: You made such a tremendous splash in pharmacology even as you started out as a graduate student. How did you happen to get into the field?
CP: When I was an undergraduate, I was initially an English major. I was the editor of the yearbook and the literary magazine, and I was a poet. So, I have to give credit for entering into science to my first husband, Agu Pert, because he made me aware that I’d always been good at science. Agu was then—and still is—a psychologist, now at the NIMH. It was through my love of psychology that I had met him. And we developed a common dream, which basically was, together, to study the brain from two perspectives. We’d spend hours thinking about it, and I feel like I have an honorary degree in behavioral psychology because I used to go to all these great seminars and meet the great people that came to speak at Bryn Mawr, where I was an undergraduate at the time. And I want to say that Agu and I attended the very first meeting of the Society for Neuroscience, with only a few hundred people in attendance.
MI: And you said that you were going to be talking at the meeting this year?
CP: Yes! I’m very excited. I have a ten-minute oral presentation, and so I’m like a wreck. It’s frightening, though I’ve lectured all over the world.
MI: Can you say why you feel more stress when it comes to speaking to a scientific audience?
CP: It’s the difference between art and science. When I speak about hard experiments, there has to be the element of scientific rigor. I’m going to be talking about a new study, and it’s not just mine. I’ve had the privilege of working with Dr. Susan Wilt, a brilliant young woman scientist. I’m going to be talking about a very elegant rat model of neuroinflammatory disease, caused by murine leukemia virus, in which all of the components can be visualized and studied, at the cellular level. The virus causes profound neurodegeneration by infecting the endothelial cells of the brain and leading to activated microglial cells, which are very hot in neuroscience, and infiltrating neutrophils, so the animal has all kinds of rapidly progressive symptoms—tremors, ataxia, spasticity, and hindlimb weakness. It’s a beautiful model, because you’ve got a behavioral time course, you’ve got incredibly beautiful opportunities for visualization of cellular components. And we’re most excited that Peptide T, the drug that we’ve been developing in the distinct context of AIDS, delays the onset of the baby rat’s neurodegenerative symptoms by nineteen days!
So when I get up there, I have to honor my collaborator as well as the science itself, and I can’t make any mistakes. I have enormous respect for scientists and for the whole scientific culture and its truth seeking. It just has to be right. I’m looking forward to returning to the fold at the neuroscience meeting. It’s been a long time since I have presented at one. I’m particularly excited to be going back to Sol Snyder’s laboratory reunion, and seeing him and so many of my old friends and colleagues from over the years.
MI: Is this the first time that you’ve looked at the effects of Peptide T with regard to neurological disease per se?
CP: Well, no. It’s the first time in a long time because we’ve had our hands full, Dr. Michael Ruff and I, in developing Peptide T as an AIDS drug. But in 1996, we published, along with Gary Arendash in Peptides, a paper by Socci et al. showing very good reason to think that Peptide T might be useful in Alzheimer’s disease. We showed that when you lesion the basal forebrain nucleus of the rat—and to this day it is still true that this nucleus is damaged in every person with Alzheimer’s disease whether by virus, toxic insult, or head trauma—you see cortical shrinkage and neuronal loss that could be almost completely prevented by chronic post-lesion treatment with Peptide T. It is very dramatic, stopping the typical post-lesion shrinkage of the brain.
MI: Now, why would Peptide T be helping in a neuroinflammatory disorder? The genesis of Peptide T is that it was based on a protein from HIV, wasn’t it?
CP: Well, it’s all about chemokines, which are the hottest new class of peptidergic activity in brain and inflammation. They got hot after it was discovered in the late 90s that HIV uses chemokine receptors to enter and infect cells. We showed in Nature in 1988, with Doug Brenneman and Joanna Hill, the HIV envelope (gp120) alone can kill neurons —-you don’t need virus to kill neurons, and Peptide T prevents neurons from being killed by the envelope protein. We now know that Peptide T antagonizes chemokine receptors, which we were the first to show modulate neuronal survival in development and protect against gp120–induced neuronal cell death. Chemokines and their receptors were first found on immune cells—the name comes from chemo tactic cytokines. But they’re also on glial cells, and even neurons—— there are so many chemokines. And we know that neuronal degeneration itself is supported by chemokines. Peptide T is a mixed agonist/antagonist, probably on a number of receptors. But more important, work in neuroAIDS has shown that HIV—and probably many other viruses with envelope proteins shaped to fit receptors on the loose—- cause havoc not by actually infecting and destroying cells, but by taking up residence, in a tiny percentage of cells that can move from the periphery into the brain, and usurping cellular communication. And what we’re saying is that by targeting the receptors that viruses exploit, you can have very fine neutralizing drugs that act beyond merely stopping the spread of infection.
As to the genesis of Peptide T, it came out of one of the very first computer-assisted database searches. Now you could do such a search on a laptop, but at the time—around 1985—when we did it in the National Cancer Institute, the computer was bigger than a large room, and we had to go out for a long lunch while we waited for the printout. But what we were seeking was sequence homology between gp120 and any peptide in the database.
Based on our database search, I ordered four peptides to be made—I had learned a heck of a lot of peptide pharmacology from the endorphin-focused international meetings of the late 70s. I started my career as a “receptorologist,” and the way that you prove that something is really binding to a receptor and not to a piece of gunk is through structure-activity relationships. So, we did a binding assay with labeled gp120 versus each of the four peptides (including Peptide T). We found that three, but not the fourth, of the compounds were really potent inhibitors of gp120 binding! I was so excited, that I called up Frank Ruscetti, the expert virologist from the NCI who was collaborating with us then and is still crucially involved, and he had found that the same three peptides—and again, not the fourth—blocked the growth of HIV in the test tube. At that point, I was off and running, and it didn’t matter to me who would have said it was wrong, because I knew it was right. I knew it was right.
I say it didn’t matter to me who would have said we were wrong, because in fact there were several very well respected people who said they couldn’t repeat our results with Peptide T, and I think many people consequently thought that we were crazy and that we had gone off the deep end. But there was a great book review in Science magazine recently about “the premature discovery.” The premature discovery is something that comes along before the underlying supporting technology or scientific logic is available for us to make sense of it all, and so it seems crazy to people.
The idea at the time we discovered Peptide T was that the envelope protein used specific receptors to enter and infect cells, and in fact there was support for this idea in the literature at the time. So, we assumed that Peptide T was interfering with viral entry into the cell by binding to this receptor, namely, the T cell antigen CD4, thought then to be the only receptor for HIV. And we identified a band on a gel with a receptor binding assay, roughly with the same molecular weight as CD4, and concluded that Peptide T functioned by binding to the CD4 receptor. We now know of course that it was really the chemokine receptor—referred to now as the co-receptor for HIV—that had a molecular weight similar to CD4, and so we had the right idea, and the right peptide, but the wrong mechanism in our 1986 PNAS paper, eight years before the first chemokine was even discovered.
We now know much more about HIV infectivity. Most HIV viruses use either CCR5 or CXCX4 receptors—two subtypes of chemokine receptors—and it turns out the CCR5, which is the one that Peptide T works on, is most important. In the early stages of the disease, for the first few years, the virus generally uses only CCR5; later on in the course of the disease, tropism depends on the CXCX4. The X4 lab-adapted strains of virus, which all labs other than Ruscetti’s used at the time, are virtually insensitive to Peptide T. So when I danced into the 1987 AIDS conference, used to being Miss Adorable from my graduate student days, and now a Section Chief of the NIMH, I was in for a very rude awakening: Everyone else was working with this X4 lab-adapted strain first isolated by Montagnier and Gallo, and of course they all said that they could not “repeat” our experiments. To get a really good R5-dependent virus, you need a primary isolate. Frank Ruscetti, who had worked with Gallo to discover IL-2, was using an R5-dependent virus that he had isolated himself. Back then, people didn’t know that there were two kinds of viruses, but the idea—that the drug binds to the receptor, and consequently, the virus can’t get in—was right. We have since verified the fact that Peptide T binds to CCR5 in all sorts of ways, using cell lines transfected with CCR5 receptors to show that Peptide T is a mixed agonist/antagonist at CCR5 receptors. And Michael Ruff went around to three different HIV laboratories and got them to replicate the potent Peptide T effect using the appropriate virus in cells that have the CCR5 receptor, publishing the collaborative paper in Anti-Viral Research in 2000. And I think that that’s the theme of life: You’ve got to get it right.
To this day, in 2003, we’re the only people who have envelope-derived peptides that block HIV infectivity at low concentrations.
MI: Having started out as a psychopharmacologist, what made you first enter research into AIDS and Peptide T?
CP: I had been very interested in the causes of schizophrenia, and I sensed that there was really an immunological link. I began spending time with immunologists, and one of them was so brilliant and so wonderful that I finally ended up marrying him: Dr. Michael Ruff. And we were fascinated by the continuity between the brain and the immune system. We published a paper in 1985 called “Neuropeptides and Their Receptors: A Psychosomatic Network.” We started exploring that neurons and immune cells have the same receptors and make the same peptides. People talk today about cellular traffic between the brain and the immune system, but there is more to it than just “traffic.” It’s not just brain stem cells that make new neurons in the adult, although this is an exciting finding that is finally accepted. But there may actually be bone marrow cells in your shinbone that can find their way into your brain and become neurons, and that is incredible: the psychoimmunoendocrine network. It’s the same cells, starting life in the body and winding up as neurons in the brain. It is truly a paradigm shift.
MI: Has Peptide T made a big splash in AIDS research?
CP: Well, Michael and I have been working on the development of Peptide T for over seventeen years, during which time we reported the things that I’ve been talking about. Now we’ve just published, in Peptides, several important findings from an eleven-person clinical trial, and the Wall Street Journal article is a great break in helping us to move it to the next level. I’m most excited about helping to create a business structure and raising the funds that will allow pivotal Peptide T phase II trials to start, so that the drug can get out. I’m also working on trial designs and on optimizing the formulation of the drug before we organize the next trial. I’ve spent seventeen years on this, and I’m prepared to spend seventeen years more, but I don’t think it’s going to take that long.
MI: Well, what did you report in the eleven-patient study?
CP: There are a number of positive changes in the patients. One of the important findings from the trial is that half of the patients treated had a statistically significant increase in their CD4 cells, which people are now thinking is the main solid indicator that clinically significant immunomodulation has occurred. There was also a sixfold increase in the number of γ-interferon–producing T cells, which are the cells that are charged to kill virus-infected cells. We showed that the actual cellular viral levels that you can isolate drops drastically, until after six months of treatment, we couldn’t isolate virus from any of the subjects’ cells. We used stringent molecular biology methods and found, for the majority of these eleven people, that Peptide T leads to undetectable virus levels in monocytes. This type of cellular “flushing” of virus is very big, because we already have so many great AIDS drugs that can make plasma levels of virus disappear, but none of them touch the virus that is sequestered within the cellular reservoirs. That’s the reason that the virus can mutate as soon as patients miss a few doses of their medication or comes roaring back within a few weeks of stopping medication. Peptide T is completely non-toxic as previously seen in phase I trials. So we’re hoping that cellular reservoirs of virus can be substantially flushed in placebo-controlled experiments.
MI: Is raising money for your next round of trials something that comes natural to you?
CP: It can be exhausting, and it is amazing what we all have to do to pursue our work, right? But the short answer is it comes neither naturally nor easily, but Michael and I are willing to do what needs to be done. This meant leaving a tenured position at the NIH, where I had an annual budget of two million dollars and a bunch of people working for me. We took the risk, and there have been some really lean times. I did an interview with someone who asked, “Why did you write Molecules of Emotion ?” And the answer was, “To make money!” The advance that I got put our daughter through college. So, at times, our work lives with Peptide T have been really challenging, and I have to say that Michael Ruff, my collaborator and husband through all of this, has always reminded me that science leads the way—I just have such respect for him as a scientist. There really is something about truth, and it’s just something when people discover reality together. It’s just extremely exciting, the power of science. And I love being a scientist more than anything.
MI: You speak about the breakthroughs that are finally happening with Peptide T. Can you talk about your first big breakthrough, that is, the discovery of opiate receptors? What was the key element from your perspective that made it work?
CP: The key in the breakthrough from a scientific perspective was when I read the work of Patton, the British pharmacologist, who had a theory that he called the ping-pong theory—I still don’t know if this theory is right or not, but it led me to do the right experiment. He claimed that antagonists tended to stay on the receptor, whereas agonists bumped on and off, which led me to say, “Aha! I need to get naloxone radiolabelled,” since I had tried many radiolabelled agonists—purifying them myself in the old days—- and none had worked. At the time, there were several papers in the literature that said, “There is no such thing as an opiate receptor because our lab tried to find it and we couldn’t do it.” But I was convinced by the pharmacological literature that there had to be an opiate receptor, and I kept at it, experiment after failed experiment, playing with the numerous variables as rationally as possible. When the big eureka moment finally came after many months it was incredibly exciting, and the privilege of collaborating with Sol was a true landmark in my life.
MI: What in your mind is the real value of the 1973 opiate receptor discovery that launched your remarkable career?
CP: You mean aside from landing us great staff positions at the NIMH?
MI: (laughter) Right!
CP: Well, it was the very first useful—simple and reproducible—- in vitro biochemical method for studying a drug receptor—and as it later turned out, after the discovery of endorphins—it actually was a neurotransmitter receptor. In our early papers, Sol and I collaborated with many renowned opiate pharmaceutical chemists and showed that the simple binding method could amazingly, perfectly, predict pharmacological activity, and so it could be used to design drugs, getting rapid feedback with tiny amounts of test compounds.
Actually, our discovery that intrinsic activity—- whether an opiate is an agonist, an antagonist, or something in the middle of the spectrum—-could be beautifully predicted from its “sodium response ratio” in the in vitro binding assay [Pert and Snyder, Molecular Pharmacology, 1974]. This assay was as important as the original discovery because it made it possible to peg a new chemical’s property in a few hours rather than in weeks or months of tedious and expensive in vivo testing. This was a big deal in the search for a non-addictive painkiller, which was believed to need mixed agonist-antagonist properties. It still astounds me how a binding assay in crude brain membranes can be used to make valid predictions. For example, an early paper published out of my NIMH lab correctly surmised that the valium receptor was coupled to the GABA receptor through the chloride ion channel well before good neurophysiological or any molecular biological data proved this in spades.
MI: So the opiate receptor binding assay was the jumping off point for other drug and neurotransmiter receptor assays?
CP: Yes! In Sol’s lab, after the opiate receptor showed what was possible, it was one receptor after another with different ligands and slightly modified conditions. The brilliant students that he had attracted and trained, who read like a list of Who’s Who in Psychoneuropharmacology, were starting the “receptor-a-month club.” Anne Young, the president-elect of the Society for Neuroscience, kicked things off back then by finding the glycine receptor with its radiolabelled antagonist, strychnine. Later, my NIMH lab focused on finding neuropeptide receptors like bombesin, VIP, and the f-met-leu-phe receptor on immune cells.
MI: So what were your NIH days like?
CP: Pure heaven. To quote Joni Mitchell, “You don’t know what you’ve got til it’s gone.” I got to learn about biological psychiatry from Biff Bunney. Agu and I got to work on Biff’s theory that lithium works in bipolar disorder by damping oscillations in neurotransmitter receptor sensitivity—- this needs to be revisited with modern techniques. Tom Insel, NIMH director, among other scientifically based psychiatrists, actually passed through my lab to learn the latest peptide and receptor techniques! And oh the pleasure of working with the great scientists that I had as postdocs, who have gone on to pharmaceutical and neuroscientfic glory—Remi Quirion, Sandy Moon-Edley, Stafford McLean—to name three. Of course, the greatest thing about NIH was the chance to collaborate without strings—for the sheer science of it all—with the greats, like Jesse Roth, Steve Paul, and Elliot Schiffman.
My most fun long-term collaboration (excluding Agu and Michael, of course) was with Miles Herkenham to develop in vitro receptor autoradiography —taking it to practically an art form [see the cover of this issue] and studying the detailed distribution pattern of neuropeptides and their receptors. This later provided a scientific rationale for calling them the “molecules of emotion,” and led to my theory of emotions. You know, mapping receptors is where biochemistry meets behavior.
MI: What was it that brought you to your present position at Georgetown University?
CP: The reason we came to Georgetown is that Dr. Mike Lumpkin, who’s an expert on GHRH [growth hormone releasing hormone], was at a talk Michael and I gave, and he noticed a close sequence homology between Peptide T and GHRH. And that led to an invitation to join his department and a series of collaborations, related to the wasting and endocrine effects seen in AIDS, that started with rat, published in PNAS, where we wound up showing that gp120, at least pharmacologically, seems to compete with GHRH receptors and block GH release both at the hypothalamic and pituitary level. And Peptide T restores growth hormone secretion, not just in rats, but also in a pilot study of children with AIDS. And Georgetown has been so wonderful to us and I love the medical school there—it has great science. Georgetown is leading the way for integrative medicine. Our physiology department has a major grant from the Complementary and Alternative Medicine people of the NIH, and there are medical students who are actually being studied who are doing meditation and doing new-paradigm practices. CAM is now part of the curriculum, and all departments are starting to contribute. This all appeals to my own interdisciplinary interests—which is why pharmacology is so interesting in the first place. And then also, Georgetown carries the Jesuit mission to do good in the world. It’s a wonderful place, and they’ve been really good to us.
MI: So what do you hope to be doing in the future?
CP: My dream is to be hanging out at an esthetically gorgeous Institute for New Medicine at the Georgetown School of Medicine where, funded by the successful commercialization of peptide T for AIDS, a team of blissful scientists will be working on cures for diseases by designing other short, stable receptor-active peptides from the sequences of polypeptides and viral envelope proteins that form the intercellular psychosomatic communication network that modulates health and disease. Whew! That’s a mouthful of a vision! I’d love to be able to personally focus on schizophrenia and autism, two neurodevelopmental diseases with a lot of interesting implicatioins. I also have a plan to make a relaxation/wellness CD that harnesses the twin transformative powers of great music and sound scientific conditioning principles. And last but not least, I hope to one day be bouncing a grandchild on my knee when Evan, Vanessa, or Brandon—my children—decide to cooperate!
- © American Society for Pharmacology and Experimental Theraputics 2010
