Transition State Analogues of Purine Nucleoside Phosphorylase: the Work of Vernon L. Schramm

Abstract

Transition State Analogue Inhibitors of Purine Nucleoside Phosphorylase from Plasmodium falciparum

(Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219–3225)

Purine-less Death in Plasmodium falciparum Induced by Immucillin-H, a Transition State Analogue of Purine Nucleoside Phosphorylase

(Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226–3231)

Achieving the Ultimate Physiological Goal in Transition State Analogue Inhibitors for Purine Nucleoside Phosphorylase

(Lewandowicz, A., Tyler, P. C., Evans, G. B., Furneaux, R. H., and Schramm, V. L. (2003) J. Biol. Chem. 278, 31465–31468)

Vernon L. Schramm grew up in South Dakota. In high school he excelled in science and, as a result, enrolled in the South Dakota State College of Agricultural and Mechanical Arts as a chemistry major. To pay his way through college, he worked in the school's chemistry stockroom preparing reagents and standards for the chemistry laboratories. However, after taking an elective class in bacteriology in his junior year, Schramm was offered a paid undergraduate research post by departmental chairman Edward C. Berry, on the condition he switch majors for his final year. He did this and spent his time isolating, resolving, and quantitating the organic acids from fermented silage.

Vernon L. Schramm

After graduating, Schramm received a fellowship for the master of science program in the department of nutrition at Harvard University. He took two biochemistry courses, one taught by Journal of Biological Chemistry (JBC) Classic author Konrad E. Bloch (1) and the other by Gene Brown and Vernon Ingram, both of which greatly influenced his career direction.

As his 2-year master's program was drawing to a close, Schramm noticed an ad in the Harvard Medical School Library for international research scholars to study biochemistry at the Australian National University in Canberra. He applied, was accepted, and set sail with his wife and daughter for an 18-day boat trip from Long Beach to Sydney. In Australia, Schramm joined the laboratory of John F. Morrison, who was interested in applying steady-state kinetic theory to allosteric enzymes. He graduated with a thesis titled “Kinetic Studies of Allosteric Phosphotransferases” and then spent the next 2 years at the Life Sciences Division of the NASA Ames Research Center in Mountain View, CA, working with Lawrence Hochstein. The lab focused on enzymes from halophilic bacteria isolated from salt pans, and Schramm's project was on N-ribosyltransferases with AMP nucleosidase.

In 1971, Schramm was offered a position as assistant professor of biochemistry at the Temple University School of Medicine in Philadelphia. He brought a preparation of the allosteric AMP nucleosidase with him and began working on the characterization of its kinetic, binding, and inhibition properties. He discovered that the natural product formycin became a transition state analogue when it was phosphorylated to the 5′-phosphate (2), which led to his interest in transition state analogues. Schramm reported his results at an American Society for Biochemistry and Molecular Biology meeting in Atlanta, after which JBC Classic author W. Wallace Cleland (3) suggested that he use the system for the application of kinetic isotope effects. Curious, Schramm attended a Steenbock Symposium on isotope effects in enzymology and realized that it was possible to achieve a complete description of an enzymatic transition state using a combination of synthetic isotope chemistry, quantitative analysis, and computational chemistry.

Over the next several years, Schramm and his technician Hazel (Barner) Leung assembled the precursors and enzymes to convert glucose with ³H or ¹⁴C labels in specific positions to ATPs containing specific ³H or ¹⁴C labels (4). His postdoctoral fellow, David W. Parkin, measured their first complete set of isotope effects, which was published in the JBC in 1984 (5). Three years later, Schramm, Parkin, and postdoctoral fellow Frank Mentch published a paper showing agreement between experimental kinetic isotope effects and a quantum chemical model of an enzymatic transition state for AMP nucleosidase (6).

In 1987, Schramm left Temple University to join the faculty of the Albert Einstein College of Medicine of Yeshiva University in New York, where he decided to focus on enzymatic transition state analysis and using transition state information as a blueprint to design transition state analogue inhibitors. In theory, these inhibitors would bind tighter to the enzyme than substrate by the factor of the enzymatic rate enhancement, which meant the inhibitors could be useful in drug design.

Schramm's postdoc, Benjamin A. Horenstein, modeled molecular electrostatic potential maps to serve as inhibitor design blueprints for the transition state structure for nucleoside hydrolase (7), several of which were synthesized and proved to be powerful inhibitors (8). Wanting to synthesize more inhibitors, Schramm formed a collaboration with Richard H. Furneaux and Peter C. Tyler, leaders in the carbohydrate chemistry group at Industrial Research Ltd. in New Zealand, whom he met through a faculty member at Einstein.

The first inhibitor they designed was Immucillin-H. It matched an early, dissociative N-ribosidic bond loss transition state structure, typified by bovine purine nucleoside phosphorylase (PNP). It turned out to be a 23 pm inhibitor of bovine PNP, binding almost 1 million times tighter than the normal substrates (9), and a 56 pm inhibitor of human PNP.

PNPs also play an important role in malaria. Plasmodium falciparum, the parasite responsible for the majority of deaths due to malaria, must salvage purines from its mammalian host because it cannot synthesize them itself. Hypoxanthine is the major purine precursor for purine salvage, and the sole pathway of hypoxanthine production in P. falciparum and in human erythrocytes is through the phosphorolysis of inosine to hypoxanthine, which is catalyzed by PNP. As reported in the first JBC Classic reprinted here, Schramm and his colleagues cloned, expressed, and isolated large quantities of pure P. falciparum PNP, which they then characterized and used to define substrate specificity and transition state inhibitors. The scientists also determined that P. falciparum PNP has a reduced affinity for Immucillin-H and several of its derivatives, as compared to human PNP.

In the second Classic, published back to back with the first, Schramm and his colleagues show that both erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. They found that Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes. Growth inhibition by Immucillin-H was reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. These results suggested that PNP could be a viable target for anti-malarial therapy.

The final Classic deals with another medical application of PNP inhibition. Human genetic deficiency of PNP causes the onset of T-cell immune deficiency; thus, inhibitors of the enzyme could be beneficial in cases of T-cell cancers and T-cell autoimmune disorders. Schramm and his colleagues discovered that the transition state structure of human PNP was more dissociated than bovine PNP; it had greater separation between the ribosyl group and the departing purine ring. As detailed in the Classic, they designed a second generation inhibitor, 4′-deaza-1′-aza-2′-deoxy-1′,9-methylene (DADMe)-Immucillin-H, which was a 9 pm inhibitor of human PNP, binding over 4 million times tighter than substrates. This molecule has cleared a Phase IIa clinical trial for autoimmune disorders, and yet another member of this family is undergoing antimalarial trials in primates.

Schramm eventually became the Ruth Merns Chair of the department of biochemistry at Albert Einstein College of Medicine, a position he still holds today. He was elected to the National Academy of Sciences in 2007 and also was the recipient of the 2006 Repligen Award from the Biological Chemistry Division of the American Chemical Society, the Harry Eagle Award for Outstanding Basic Science Teaching from Albert Einstein College of Medicine, the Rudi Lemberg Award from the Australian Academy of Science, and the George A. Sowell Award for Excellence in Teaching from Temple University School of Medicine.