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VCU's Donald J. Abraham was honored recently with a special invitation. Abraham, Ph.D., professor of medicinal chemistry and director of the Institute for Structural Biology and Drug Discovery, traveled to Cambridge, England, at the personal invitation of Vivien Perutz to give the closing remarks at a September memorial for Ms. Perutz's father and Abraham's friend and colleague - Nobel Laureate Max Ferdinand Perutz. Perutz, who shared the 1962 Nobel Prize for chemistry for his work on the structure of proteins, died in February of cancer. He was 87.

Shortly after Perutz's death, Abraham sat down at his computer at 4 a.m. one sleepless night to jot down a few thoughts about his mentor. The "few thoughts" became a career-full of reflections. These are Dr. Abraham's memories of his 30-year relationship with Max Perutz:

BACKGROUND: A TALE OF TWO VIENNESE
I did my post doctoral work with Professor Alfred Burger at the University of Virginia just before he retired. Professor Burger founded the field of medicinal chemistry, the Journal of Medicinal Chemistry and published the major reference work in our field, Burger's Medicinal Chemistry. I told Professor Burger that someday I wanted to design a drug from the three-dimensional structure of the receptor. He responded in 1964, "My boy, you probably will never see a receptor in your day." Working for Professor Burger not only introduced me to the field of medicinal chemistry but very much prepared me to work many years later with Max Perutz. Alfred received his degrees from the University of Vienna, and his cousin was Max's TA. He fled Hitler as Max did and came to the University of Virginia in the 1930s to work on analgesics by synthesizing different portions of the morphine molecule. Professor Burger's passion for his field, love of discovery, ability to write in English, seemingly poor health and effervescent interest in science (like Max's) continued until his death about a year and a half ago at the age of 96. Max very much wanted to meet Burger. On Max's last visit to us in Richmond VA, about a year before Burger's death, we had planned to drive the 90 miles to Charlottesville to see the Professor, but Max's sister was very ill and he had only a short two-day visit with us.

My wife and I hosted the professor and Mrs. Burger (she was 94, married to the professor for around 70 years and a very southern Virginia lady) at a pre-Christmas luncheon just before the millenium at their favorite restaurant overlooking the Blue Ridge Mountains near their retirement complex. The professor died a week after our luncheon. One of the professor's last words to me was that he wished that he had gotten to meet Max. Max's last note to me came on a Christmas card last December. It read, "Don: Hope your work is flourishing. By a quirk of fate I have solved the amyloid structure and have sent it off to PNAS. Yours, Max." Like Max's last note, Professor Burger was a scientist until the end. At the end of the luncheon, Professor Burger said, "Don, do we have enough chapters on computational chemistry? It is important now."

THE INVITATION TO COME TO CAMBRIDGE
I first heard Max give a seminar on hemoglobin at a Royal Society meeting in the early 1970s. I asked him why the vinyl group of the heme was out of the resonance plane. His eyes sparkled, and he said he didn't know. I was impressed that a famous scientist like Max would acknowledge that he didn't know something about the molecule he knew more about than anyone in the world.

I was fortunate to get an invitation to the 1971 Cold Spring Harbor Symposium, where the first protein structures were on display. Max held a wonderful afternoon free-time session at the beach with all of the hemoglobin enthusiasts attending. Although I had no idea that I too would join the collection of enthusiasts one day, I attended the session. It was the first time it really hit me that we could indeed see receptors now. After the Cold Spring Harbor meeting, I wrote the first paper (to present at Professor Burger's retirement symposium) on structure-based drug design. No one had yet taken a three-dimensional structure of a protein and designed, de nova, a drug molecule.

I next began to search the literature for protein structures that were involved in disease states. 1 It was clear that hemoglobin was the only candidate, and the disease was sickle cell anemia arising from a mutation of the 6th amino acid of the beta chains of hemoglobin. From 1975 to 1980 I failed miserably in making any progress in collecting hemoglobin data with small molecules bound. I believe it was the spring of 1980 that I was reading Nature, and there was a comment on a paper that said: "If you want to get hit by lightning, you have to go to where it storms." It was clear that I should go to Cambridge to work with Max Perutz, even though he was being honored with a retirement symposium at the Airlee House in Virginia. I took the chance that Max would not really retire and got one of the two last spots to attend the meeting.

One afternoon during a poster session, I saw Max, approached and said, "I am Don Abraham. We met briefly a couple of times previously." I told him I was reading one of his papers and had an idea of how we might design anti-sickling molecules de nova. Max was all ears. In his paper on growing hemoglobin crystals (J. Crystal Growth), someone had placed a question mark on the photocopy I had next to toluene. Toluene is needed to grow oxy crystals, but why? Knowing that phenyl alanine was an anti-sickling agent, I offered that if we could find the toluene-binding site, we could start de nova design from the phenyl alanine site. Max's instant reply was that Joyce Baldwin did not observe toluene molecules in her density maps. I was prepared for this question and handed him a paper on the HPLC analysis of oxy crystals showing 10 moles of toluene for every mole of hemoglobin. I speculated that the toluene was probably disordered. I added that I could synthesize a toluene derivative with a mercury atom sandwiched between the methyl group and aromatic ring. This derivative would bypass the surface sulfhydryls (beta Cys 93) that Max used to be the first to solve the phase problem for a protein and might bind to the aromatic site occupied by phenylalanine. Immediately, and without reservation, Max said, "Come to Cambridge.". My spirits rose to the heavens. I was invited to the best laboratory in the world, not with recommendation letters or CV, but on an idea. This I learned was a hallmark of Max's genius. Good ideas, whether his or others, were treasured. Formalities were not to stand in the way of good ideas.

CAMBRIDGE FROM 1980 TO 1988
On my first trip in 1980 to Max's laboratory, we found several aromatic heavy atom molecules that bound to hemoglobin in the oxy state. One event during that first trip stood out. It was near the end of my stay and 3 a.m. I was in the basement taking photographs of hemoglobin crystals grown in the presence of phenylalanine, a well-established anti-sickling agent. I had just come out of the dark room and was holding up the photograph looking for spot difference. When I saw several intensity shifts from the native crystal, I was dancing around the basement and couldn't wait to show Max the next morning. Suddenly, as if seeing a ghost, I was surprised to see the smiling face of Bernal, Max's mentor, shining down on me from his picture on the wall. The next day Max looked at the film and said, "Don, you must come back to Cambridge and finish this work!" Again my spirits rose to the heavens. From 1980 through 1988 I made 15 research trips to work with Max.

By 1982, Max, Judd Fermi, Simon Phillips and I had worked out the basic features of a molecule that would bind to hemoglobin with antisickling (antigelling) properties. It had to have a substituted aromatic ring with a short chain ending with an acid moiety. We had discovered and tested in vitro many such molecules that were active in my laboratory in Pittsburgh, but all were too toxic for human use. Anemic sickle cell patients have around 320 grams of sickle cell hemoglobin to treat every day of their lives. This requires high aspirin-like doses. I asked a colleague of mine what drug on the market is given in high doses that contains a halogen, aromatic ring and an acid group. He responded immediately that the anitlipidemic agent clofibric acid was such a molecule. The in vitro tests were outstanding, and I was sure that we had a drug to treat sickle cell anemia.

WITH HIS OWN BLOOD
Just before the Christmas 1982, I started growing hemoglobin crystals with clofibric acid added, called Max and told him I would come with crystals and exciting news. I arrived after New Year's on a Friday afternoon, as most always. I would give the crystals to Max who would immediately take a five angstrom set of data and have it ready for analysis on Saturday, always a big day in the lab for Max. We were all excited. After a few weeks I asked Max if we shouldn't look at the effect of clofibric acid on the oxygen-binding curve of hemoglobin. I thought this might be interesting to observe, and the FDA might require this as it would be given to sickle cell anemia patients as well those already taking the drug for high lipid levels. This was on a Friday, and Max immediately went to the storeroom to find the glass tonometer and accessories to do the experiment. Another hallmark of Max's passion for science was to never let any good idea languish. Max said, "Don, come in on Monday and have some blood drawn as we need blood from a non smoker to avoid CO contamination on the hemoglobin." I was relieved to have the weekend before having to have my arm punctured with a needle. I showed up as usual on Saturday morning in the lab when Max sees me and comes running out of his office with a tube of blood and said "Don, let's get cracking." I asked whose blood he had and he said, "Mine." The initial oxygen binding experiments that laid the ground work for discovering a future drug were conducted with Max Perutz's blood.

I called my wife that January and said I am delayed here in Cambridge to do some oxygen binding experiments. I spent that whole winter measuring one point at a time, something that is now done in minutes using a machine. To our great surprise, we found that clofibric acid right shifted the oxygen binding curve like the natural allosteric effector DPG (2,3-diphosphoglyceric acid). Many anti-sickling agents do the opposite, left shifting the curve to produce more oxy-Hb which is less sensitive to polymerizing. Unfortunately clofibric acid bound to serum albumin over 98%, no matter how much was in plasma negating it as potential anti-sickling drug.

However, Max immediately recognized that an allosteric effector that right shifts the curve could be of great value to treat hypoxic diseases or problems such as angina, stroke, blood loss, transplant surgery, and blood storage. He immediately started searching for other anitlipidemic agents that might act better than clofibric acid and came up with bezafibrate. I found gemfibrozil, but it was weaker than bezafibrate as an allosteric effector.

A NEW HYDROGEN BOND
We published our initial finding with clofibric acid and other antigelling agents in Proc. Natl. Acad. Sci. USA in 1983, Vol. 80, pages 324-328. Max Perutz and Claude Poyart from INSERM in Paris found that bezafibrate was a much better allosteric effector than clofibric acid.4 I grew the Hb-bezafibrate crystals while in Cambridge, Max took the data and I calculated and contoured the electron density maps. It was around 8 p.m. when I called Max at home when I saw the first bezafibrate binding site interactions. I said, "Max, you have to see this very unusual hydrogen bond. The NH of Asp 108 beta is sticking right into the center of the pi electron density of the drug's terminal aromatic ring." He said "I will be right over!" That is another great characteristic of Max, you knew you could call or see him at any time and he would be as excited as you were, if not more. We published a new paper that I believe Max considered as one of his many memorable ones "Hemoglobin as a Receptor of Drugs and Peptides: Stereochemistry of Binding" J.Am.Chem.Soc., 108:1064-1078 (1986).

Max later got Michael Levitt (J. Mol. Biol., 1988, Vol. 201, 751) to compute the energy of this new hydrogen bond, and they published a paper that showed the hydrogen bond to be stronger than many of us expected. Greg Petsko (Burley & Petsko, FEBS Lett. 1986 Jul 28;203(2):139-43 and Science. 1985 Jul 5;229 (4708):23-8. Review) picked up on this new hydrogen bond from a lecture Max gave showing its importance in protein structure and others in receptor interaction. The amazing thing was that it was there all along, but none of us realized it in native structures. It took a bound drug molecule to illustrate it.

DEVELOPMENT OF A HEMOGLOBIN ALLOSTERIC EFFECTOR FOR THE RADIATON TREATMENT OF CANCER
To my great surprise, the medical specialists that first followed up on our 1983 paper in PNAS were not in cardiology but in radiation oncology. They had a theory that delivery of oxygen to hypoxic tumors would enhance their kill since it was the oxygen singlet radical and not the x-ray radiation that was effective.

Almost immediately after I moved to Virginia Commonwealth University, we discovered two agents that not only were very good allosteric effectors but also entered red cells in a sufficient manner to have a significant shift in the oxygen binding curve. All of these agents retained their low toxicity profiles, which is what we had hoped for. The new and entrepreneurial president of Virginia Commonwealth University, Dr. Eugene P. Trani, encouraged me to start a company to develop these agents as the large pharmaceutical house were not interested in allosteric effectors of hemoglobin. The new company, Allos Therapeutics, was able to take our results, develop the drug and run the Phase I trials at VCU for about $2 million -- a record I believe. Allos has raised $40 million in private funds and $90 million on NASDAQ. That is sufficient funds to bring us to the end of our Phase III clinical trials to treat metastatic brain cancer. Over 500 people have been treated with the drug so far. We are one step from FDA approval, and Max's and my dream to discover a drug based on the three-dimensional structure of hemoglobin. Many experts believe that the drug, if approved, will be used for numerous other radiation treatments of tumors and may be used for certain surgeries that cool down patients and organs left shifting the oxygen-binding curve.

"THE SPIRIT IN MAX'S LABORATORY"

• I spent a number of trips still trying to find an anti-sickling agent. Kiyoshi Nagai, Ben Luisi and I shared the little two-bench laboratory with Max, so it was crowded. I think we did our best work at night and often had night supper at 3 a.m. as it was the freshest food of the day. One day after a late-night session, Max came in around 10 a.m. and wanted to know where everyone was. I told him Kiyoshi, Ben and I were working until 4 a.m. This story ended up in Nature (without names) in Max's short discourse on "A cycle ride to Stockholm" reviewing Krebs' work. Max was extolling the Cambridge MRCLMB freedom to the more-structured German laboratory environment.
• Max came often to Pittsburgh to lecture and a few times to my new home at Virginia Commonwealth University. He usually preferred to stay at our home. Finding ripe bananas for Max - who had diet restrictions -- in the winter in Pittsburgh was always an iffy situation. On one trip, we could only find green ones, so I ripened them in the oven, thinking that they looked just fine. As soon as Max took a bite he said, "These were ripened in the oven, I can taste the alcohol"! A good mind in science analyzes everything.
• I was a terrible writer when I first visited Max in 1980. He would take my papers (without his name on them) and without any qualm cut words and rearranged sentences into short statements. Later when I let a paper go out from my laboratory by one of my co-workers that I did not rewrite, one of the referees responded, "Don Abraham writes much better than this." I learned how to write from Max.
• I also learned form Max not to drop any ideas because I couldn't do the experiments myself. Once, when we thought clofibric acid would be an anti-sickling drug, Max said. "Let's look at it in dogs." I said, "We can't do that at the MRCLMB, can we?" "Of course not," he responded. "There is a professor in Cambridge who I believe can do that for us." It was with this spirit that I asked two toxicologists at Virginia Commonwealth University to see if the drug we wanted to forward to clinical trials was suitable, and it was!
• On one of my first trips to Max's laboratory, the cleaning lady came in with Max's special crystal growing tubes and said, "These are for the little guy with the glasses (meaning Max of course)." The collegial spirit he established at the MRCLMB is probably unequaled anywhere.
• Finally, on one trip Max made a statement to me that I treasure above all else. I was going back to Pittsburgh that day and was short of money, without time to go to Barclays Bank for a withdrawal. Max handed me 20 pounds, and I told him I couldn't pay him back for some time. He said, "Don't worry, you are a member of the lab." I will carry that statement to my grave.

1 In the late 1960's I became a small molecule crystallographer using crystallography to better understand drug design and action in the cancer field. When I was 10, my grandfather died of cancer, and I dreamed one day of discovering a drug to treat the disease.

2 There was only one problem; I had no funds to take a sabbatical trip to Cambridge. Through a most unlikely source did such funds appear. After failing for five years with four NIH proposals turned down to study structure-based drug design for sickle cell anemia. I had lost my group and was depressed. During a short three- story elevator ride to my laboratories in Salk Hall at the University of Pittsburgh, a chemical-delivery man, Bob Heflin, noticed my depression and asked me what as wrong. I told him I had lost all my grants trying to pursue designing a drug to treat sickle-cell anemia. Bob said he could help me. Not wanting to be unkind, I took him down to my office and showed him a 5,000-atom structure of hemoglobin and picked up a peptide. I told Bob to pretend the hemoglobin was the moon and the peptide a spaceship. If we could design the spaceship to land at this point on the moon, we could beat this disease. He said again, "I can help." I said, "How?" Bob introduced me to the Pittsburgh Sickle Cell Anemia organization, and I had in one day a Heinz fellowship to go work with Max. And -- I ended up in the Pittsburgh Pirate (baseball) dugout to receive the first of $20,000 for research from Willie Stargell, the famous first baseman who had organized the black athletes in the National Baseball League.

3 My poor wife stayed at home through a very cold and snowy winter taking care of everything, including transporting one of my three high school aged sons 18 miles each way three days a week to train with the university swim team.

4 Another amazing example of Max's mentorship and love forwarding science happened in 1982. I was on my way home to Pittsburgh when Max said, "Here is a ticket to go to Paris and meet Claude Poyart. You should start collaborating with him." This collaboration turned out to be helpful in the development of a clinically viable allosteric effector. Unfortunately, Claude passed away about a year before Max.

5 Steve Hoffman, the founding CEO of Allos Therapeutics (the company started to advance our hemoglobin allosteric effector), has told me on several occasions that Max changed his life profoundly but is disappointed he never got to tell Max how much he influenced his life. Steve and another professor from the University of Colorado started the blood substitute company, Somatogen, in the early to mid 1980s. It is now owned by Baxter. Somatogen was founded using the technology that came out across the bench from me via Kiyoshi Nagai. So two new biotechnology companies came from Max's two small benches!