In Memoriam: Norman S. Radin ( ) James A. Shayman. Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109

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1 In Memoriam: Norman S. Radin ( ) James A. Shayman Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan Address correspondence to: James Shayman, Department of Internal Medicine, 1150 West Medical Center Drive, Ann Arbor, Michigan Telephone: Fax: , E- mail: 1

2 Norman S. Radin, Ph.D. died peacefully on January 21 st in Cupertino, California. He was 92 years old. Norm Radin was Professor Emeritus of Neurochemistry and Research Scientist Emeritus at the University of Michigan. He joined the University of Michigan Mental Health Research Institute in 1960 and remained scientifically active for more than twenty years following his retirement. He was preceded in death by his wife Norma (Levinson), and is survived by his daughter Laurie, son Lon, daughter in law Hollis, grandsons Jesse and Max, and partner of later years Florence Abramson. Norm was a founding editor of the Journal of Lipid Research, authoring two papers in the Journal s inaugural issue (1, 2). It is therefore fitting that a memoriam be published in in this journal detailing his pioneering contributions to the field of sphingolipid biochemistry. Norm developed a fondness for chemistry at an early age. Norm credited this interest to having read the 1918 book Creative Chemistry by Edward Slosson. In this book, the chemistry of plastics was identified as particularly promising field. Norm liked to say that he was advised to get into the plastics fifty years before Dustin Hoffman in the film The Graduate. His father nurtured his passion for experimentation by installing a Bunsen burner in their basement and by allowing Norm to spend his hard earned allowance at Eimer and Amend, the chemical supply house that later became Fisher Scientific. This basement lab, Norm s first, was eventually shut down by his mother after he exploded too much hydrogen. Norm attended the renowned Stuyvesant High, a science and technology secondary school in Manhattan. He fondly recalled the many interesting courses and even more interesting teachers at Stuyvesant. He credits his biology teacher for convincing him of the tremendous future of biological chemistry, a discipline that was in its most nascent stage. An exceptional performance on the New York State Regents exam earned Norm a scholarship to Columbia University where he eventually completed both undergraduate and graduate studies. 2

3 Norm decided to pursue a degree in biochemistry and began graduate school in In order to support himself, Norm took a part-time job for the Association of American Soap and Glycerine Producers. He was assigned the task of performing library research scouring Chemical Abstracts and the patent literature for the industrial uses of soap. Much of this literature was focused on the technical uses of fatty acids, the primary components of soap. The dissolution properties of fatty acids in organic solvents was of particular interest to Norm, a property he put to good use in his later career as he developed numerous methods for lipid extraction. Norm s graduate work was interrupted by the onset of World War II. To aid in the war effort he found himself working in a rocket fuel research lab near Pittsburgh, run by the National Defense Research Council, the University of Pittsburgh, and the U.S. Bureau of Mines. His boss was Louis P. Hammett, famous for his studies of mechanisms in organic reactions. Among his tasks was the goal of finding ways to mold rocket fuel into pellets. Norm introduced the idea of lubricating the press molds for the solid fuel with a metallic soap, a solution unlikely to have been found by someone without a focus on lipid chemistry. Following the war Norm returned to Columbia to complete his graduate studies under David Rittenberg, who was known for his metabolic studies with heavy nitrogen and heavy hydrogen. Other faculty in the biochemistry department at that time included Erwin Chargaff and Konrad Bloch, both Nobel laureates. Norm pursued studies on the role of glycine and acetic acid in heme biosynthesis under the mentorship of Rittenberg and another biochemist David Shemin. This work was later recognized as a JBC classic (3, 4). Two of Norm s fellow graduate students were Isaac Asimov and Steve Zamenhof. Even while a graduate student Asimov had garnered some fame as a prolific writer of science fiction. Norm believed that a biochemistry textbook that Asimov had written was one of Azimov s most under-appreciated literary works. Zamenhof conducted much of the work 3

4 that led to Chargaff s seminal observations on the nucleotide content of DNA. Zamenhof had survived the Nazi occupation by swimming a long distance to safety. With this perspective he advised Norm that virtually all of the world s problems arose as a result of human stupidity and that the goal of a biochemist should be to find ways to make people smarter, presumably by better understanding the biochemistry of the brain. This was a challenge that Norm accepted and that eventually led him to pursue a career in neurochemistry and the study of brain sphingolipids. Following brief post-doctoral stints in Berkeley and Austin, Norm took his first independent position, running the radioisotope laboratory at the Hines VA Hospital in Chicago followed a few years later with a move to the biochemistry department at Northwestern University. Norm decided to focus on a poorly understood lipid termed cerebroside, a substance that comprised one percent of the total wet weight of the brain. Developing one of the first applications of chromatography to lipid biochemistry, Norm was able to demonstrate that Florisil could be used to isolate brain cerebroside with close to one hundred percent recovery and use this to report an analytical method for measuring its concentration (5). This early success encouraged Norm to pursue to a series of further studies defining the pathways for galactosylceramide metabolism and the identification of many of the associated enzymes. This work included the discoveries that uridine-diphosphate was a substrate for galactosylceramide, of the pathway for sulphatide synthesis, and of the degradation of sulfatide and galactosylceramide. Norm s training in chemistry, isotope tracer studies, and command of the literature in soap production positioned him to rapidly move the nascent field of sphingolipid biochemistry forward. This work was further aided by a series of talented and dedicated post-docs and graduate students all of whom went on to successful independent careers. Yasuo Kishimoto developed many innovative techniques for the analysis of the fatty acid composition of cerebrosides and other sphingolipids. He observed that cerebrosides were characterized by a large amount of odd numbered fatty acids (2). Amiya Hajra joined Norm as a 4

5 graduate student at Northwestern and later followed him to Ann Arbor. Together they discovered the one carbon degradative pathway for fatty acids, important in explaining the occurrence of 23-carbon and 25-carbon fatty acids in cerebrosides (6). Up to that point only the 2-carbon degradative pathway was known. These initial observations were followed by several radiolabeling studies that demonstrated a highly active metabolism of the fatty acids of cerebrosides including the action of an amidase, shortening and elongation pathways, and reutilization of the metabolized fatty acids back into cerebrosides. This highly elegant work established that cerebrosides underwent active metabolism in the brain and dispelled the commonly held belief that brain lipids were inert molecules. They also identified the existence of cerebroside esters. In 1960 Norm was recruited to join the Mental Health Research Institute at the University of Michigan led by Bernie Agranoff. Hajra followed him to Ann Arbor and with Krystyna Kopaczyk they discovered that radiolabeled galactosylceramide was metabolized in the brain to ceramide and the ceramide to sphingomyelin (7). This led to the characterization of the galactosidase that catabolizes both galactosylceramide and lactosylceramide, the same enzyme that Kuni Suzuki later demonstrated was deficient in children with Krabbe disease. The arrival of Pierre Morell to the lab changed the focus of the work to the pathways for cerebroside synthesis and the identification of the associated enzymes. Radin and Morell disproved that psychosine and stearoyl CoA were precursors for galactosylceramide formation, as had been previously reported. They established that galactosylceramide synthase utilized UDP-galactose and ceramide as substrates (8). Importantly, they also observed that ceramides containing hydroxylated fatty acyl groups were significantly better substrates for the synthase than ceramides without hydroxylated fatty acyl groups. Morell continued these studies with additional ones characterizing sphingosine and palmitoyl-coa as substrates for ceramide synthesis. Later on with David Ullman they demonstrated that the speed of the reaction was dependent on the fatty acid chain length, an important determinate of the ceramide composition found in brain and other organs (9). 5

6 In 1971 Norm proposed a novel strategy for the treatment of Krabbe disease, a condition of lysosomal galactosylceramide excess. He postulated the use of small molecule inhibitors of galactosylceramide synthase as opposed to the replacement of the defective galactosidase. This approach was based on the assumption that other metabolic pathways for galactosylceramide or sulfatide would be partially intact. Although a few compounds were identified with limited inhibitory activity, this project had limited success. Upon the realization that Gaucher disease was more prevalent than Krabbe disease, Norm redirected his attention to the identification of inhibitors of glucosylceramide synthase, an enzyme discovered by Subash Basu and Saul Roseman working across the street from Norm in Ann Arbor. Working with a succession of three organic chemists in his lab, Ramesh Arora, Radhey Misra, and Ranga Vunnam, over 100 compounds were designed and synthesized. The most promising compounds were modeled after chloramphenicol (10). 1-Phenyl-2- decanoylamino-3-morpholino-propanol, PDMP, was the most promising compound, acting as a reversible inhibitor of glucosylceramide synthase with an IC 50 of 20 M. Jinichi Inokuchi joined the lab and was able to demonstrate that the D-threo enantiomer was the active compound (11). While PDMP lacked the sensitivity and specificity to be considered as a drug candidate, it was immediately recognized as being a valuable reagent for probing the biological role of glucosylceramide-based glycosphingolipids. Norm generously made this inhibitor available to glycobiologists around the world, eventually leading to the publication of hundreds of papers using this reagent and the discovery and elucidation of a wide variety of functions for glycosphingolipids. I was fortunate to begin a long-term scientific collaboration and enduring friendship with Norm in We decided to better define the pharmacophore required for inhibition of glucosylceramide synthase inhibition. This was followed by the design and synthesis of a series of second and third generation PDMP analogues eventually leading to the identification of compounds with low nanomolar activity and significantly higher specificity. Eventually, an ethylenedioxyphenyl homologue was characterized and chosen 6

7 as a clinical lead compound. This drug, now known as eliglustat tartrate, has been the basis for seven clinical trials for the treatment of type I Gaucher disease (12). Recently, two phase 3 trials have reported that eliglustat is comparable to or superior to enzyme replacement therapy with recombinant -glucocerebrosidase for the treatment of newly diagnosed Gaucher patients. Thus 42 years after Norm first proposed the use of inhibitors of glucosylceramide synthesis for lysosomal storage disease, positive clinical proof is now available. Fortunately, he lived long enough to realize that his synthesis inhibition theory had been proven. Norm was a serious scientist who had little patience for those scientists who did not design experiments properly or express their results cogently. He would often surprise his colleagues by sending unsolicited comments on papers they had published. These comments were greatly valued as they were motivated by only the best of intentions and promoted discourse in the field. Norm also had a dry wit and droll sense of humor. As a graduate student he penned a spoof inspired by his department chairman s books that summarized the effort during the war to bring penicillin to the battlefield. His satirical version of this was The isolation and characterization of plentisillin. This article described the chemical synthesis and testing of a completely nonantibiotic substance that was noted mainly for the ethanol produced as a side-product in its isolation. On a lark he submitted it for publication to Science. Remarkably, the editors of which decided to publish the article as a special edition of Science, with its own cover page and imaginary volume number ( ). Norm formally retired in 1995, moving from Ann Arbor to Palo Alto to care for his wife Norma in closer proximity to his children. Norma had contracted multiple myeloma, the same disease that ended his mother s life. Norm s scientific curiosity and writing remained unabated. He published over 40 papers after age seventy, many directed toward the hypothesis that inhibition of glycosphingolipid synthesis is a viable strategy for the treatment of myeloma and other malignancies. In his later years Norm rued his own inability to test this hypothesis and the lack of commitment of many in the oncology 7

8 community to pursue this concept. Hopefully, these papers will be rediscovered and we will know whether yet another of Norm s theories will be confirmed. With Norm s passing the field of lipid biochemistry has lost a great scientist and scholar. Norm was witness to the early development of lipid biochemistry as a discipline and set many of the scientific and academic standards for the field. His contributions to the chemical characterization, enzymology, and pharmacology of cerebroside metabolism have left an indelible mark for which the lipid research community should be immensely grateful. Finally, his novel concept of synthesis inhibition therapy as a strategy for the treatment of sphingolipidoses has been clinically established and promises to improve the lives of thousands of patients with Gaucher disease and potentially those with other sphingolipidoses for many years to come. 8

9 References 1. Kishimoto, Y., and N. S. Radin Isolation and determination methods for brain cerebrosides, hydroxy fatty acids, and unsaturated and saturated fatty acids. Journal of Lipid Research 1: Kishimoto, Y., and N. S. Radin Composition of cerebroside acids as a function of age. Journal of Lipid Research 1: Radin, N. S., D. Rittenberg, and D. Shemin The role of acetic acid in the biosynthesis of heme. J Biol Chem 184: Radin, N. S., D. Rittenberg, and D. Shemin The role of glycine in the biosynthesis of heme. J Biol Chem 184: Radin, N. S., J. R. Brown, and F. B. Lavin The preparative isolation of cerebrosides. J Biol Chem 219: Hajra, A. K., and N. S. Radin Biosynthesis of odd and even numbered cerebroside fatty acids: evidence for two routes. Biochim Biophys Acta 70: Kopaczyk, K. C., and N. S. Radin In Vivo Conversions of Cerebroside and Ceramide in Rat Brain. J Lipid Res 6: Morell, P., and N. S. Radin Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid. Biochemistry 8: Ullman, M. D., and N. S. Radin Enzymatic formation of hydroxy ceramides and comparison with enzymes forming nonhydroxy ceramides. Arch Biochem Biophys 152: Vunnam, R. R., and N. S. Radin Analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain. Chem Phys Lipids 26: Inokuchi, J., and N. S. Radin Preparation of the active isomer of 1 phenyl 2 decanoylamino 3 morpholino 1 propanol, inhibitor of murine glucocerebroside synthetase. J Lipid Res 28: Shayman, J. A Eliglustat tartrate: Glucosylceramide Synthase Inhibitor Treatment of Type 1 Gaucher Disease. Drugs of the future 35: