Enzymatic Transition States and Logical Inhibitor Design
Enzymes catalyze virtually all of the chemical transformations necessary for biological life. Knowledge of the transition-state structure of enzymatic reactions permits the design of powerful inhibitors. Methods have been developed in this laboratory for the determination of geometry and charge features that characterize enzymatic transition states. This information is used for the logical design of transition-state analogues. Chemical synthesis is accomplished by our chemistry collaborators at the Victoria University of Wellington, New Zealand. Our novel transition state analogues have the potential to be new drugs Specific projects include:
Human genetic deficiency of purine nucleoside phosphorylase causes a specific T-cell insufficiency. Our inhibitors of this enzyme are powerful anti T-cell agents and anti-gout candidates. Immucillin-H (Mundesine®) was approved for use in Japan for peripheral T-cell lymphoma (PTCL). DADMe-Immucillin-H (Ulodesine®) has completed phase 2 clinical trials for gout. We are characterizing the enzyme-drug interactions at the kinetic, atomic and drug-resistant levels to define their exceptional drug characteristics.
Purine salvage is essential for growth of parasitic protozoa. A family of powerful inhibitors has been prepared against two target enzymes from the malaria parasite. Promising results have been obtained in cell culture and in infected primates. Galidesivir®, first characterized here, is in clinical trials for Covid-19 and Yellow Fever in Brazil.
Human cancers are genetically unstable. The epigenetic changes make cancer cells more susceptible to agents that disrupt epigenetic control. Regulatory methylation of proteins and DNA are our epigenetic anticancer targets. S-Adenosylmethionine is the source for methyl transfer reactions, essential in cancer cells. We are targeting three enzymes in the epigenetic pathways. Our goal is to developed powerful transition state analogue inhibitors with anticancer activity.
Enzymatic transition states have lifetimes of a few femtoseconds (10-15 sec) but catalyze reactions on the millisecond time scale (10-3 sec). We make isotopically heavy enzymes to understand what happens at the fsec timescale. These studies are textbook-changing studies into the fundamentals of catalysis. We collaborate with computational quantum chemists at the University of Arizona, nice to visit in January and February.
Clostridium difficle is a growing health problem. We use transition state theory to design and synthesize antitoxins to fight this growing infectious disease threat.
Students in this laboratory can receive training in enzymology, catalysis, protein expression, inhibitor design, computer modeling, inhibitor synthesis, and in drug metabolism studies in cells and animals. We collaborate in NMR, X-ray crystallography, mass spectroscopy, synthetic organic chemistry, cancer and medicine projects. Graduates from this laboratory are well trained for future careers as university researchers, the pharmaceutical industry or biomedical research.
Selected References
Transition State Analogues Enhanced by Fragment-Based Structural Analysis: Bacterial Methylthioadenosine Nucleosidases. Zhang D, Burdette BE, Wang Z, Karn K, Li HY, Schramm VL, Tyler PC, Evans GB, Wang S. Biochemistry 59, 831-835 (2020).
Enhanced Antibiotic Discovery by PROSPECTing. Schramm VL, Meek TD.Biochemistry. 20; 58, 3475-3476 (2019).
Selective Inhibitors of Helicobacter pylori Methylthioadenosine Nucleosidase and Human Methylthioadenosine Phosphorylase. Harijan RK, Hoff O, Ducati RG, Firestone RS, Hirsch BM, Evans GB, Schramm VL, Tyler PC. J Med Chem. 62, 3286-3296 (2019). Antibacterial Strategy against H. pylori: Inhibition of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis. Joshi S, Fedoseyenko D, Mahanta N, Ducati RG, Feng M, Schramm VL, Begley TP. ACS Med Chem Lett. 10, 363-366 (2019)..
Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase. Harris LD, Harijan RK, Ducati RG, Evans GB, Hirsch BM, Schramm VL ACS Chem Biol. 13, 152-160 (2018).
Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum. Ducati RG, Namanja-Magliano HA, Harijan RK, Fajardo JE, Fiser A, Daily JP, Schramm VL. Proc Natl Acad Sci U S A 115. 2114-2119 (2018). Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels. Harijan RK, Zoi I, Antoniou D, Schwartz SD, Schramm VL. Proc Natl Acad Sci U S A. 115, 6209-6216 (2018). Transition-State Analogues of Campylobacter jejuni 5'-Methylthioadenosine Nucleosidase. Ducati RG, Harijan RK, Cameron SA, Tyler PC, Evans GB, Schramm VL. ACS Chem Biol. 13, 3173-3183 (2018).
Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase. Gebre ST, Cameron SA, Li L, Babu YS, Schramm VL. J Biol Chem. 2017 Aug 9. pii: jbc.M117.801779. doi: 10.1074/jbc.M117.801779. [Epub ahead of print]
Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase. Harijan RK, Zoi I, Antoniou D, Schwartz SD, Schramm VL. Proc Natl Acad Sci U S A. 2017 Jun 20;114(25):6456-6461.
Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase. Firestone RS, Cameron SA, Karp JM, Arcus VL, Schramm VL. ACS Chem Biol. 2017 Feb 17;12(2):464-473.
Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase. Stratton CF, Poulin MB, Du Q, Schramm VL. ACS Chem Biol. 2017 Feb 17;12(2):342-346.
Oligonucleotide transition state analogues of saporin L3. Mason JM, Yuan H, Evans GB, Tyler PC, Du Q, Schramm VL. Eur J Med Chem. 2017 Feb 15;127:793-809.
Continuous Fluorescence Assays for Reactions Involving Adenine. Firestone RS, Cameron SA, Tyler PC, Ducati RG, Spitz AZ, Schramm VL. Anal Chem. 2016 Dec 6;88(23):11860-11867.
Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase. Wang Z, Chang EP, Schramm VL. J Am Chem Soc. 2016 Nov 16;138(45):15004-15010.
Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2. Poulin MB, Schneck JL, Matico RE, Hou W, McDevitt PJ, Holbert M, Schramm VL. J Am Chem Soc. 2016 Jun 1;138(21):6699-702.
Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis. Namanja-Magliano HA, Stratton CF, Schramm VL. ACS Chem Biol. 2016 Jun 17;11(6):1669-76.
Human DNMT1 transition state structure. Du Q, Wang Z, Schramm VL. Proc Natl Acad Sci U S A. 2016 Mar 15;113(11):2916-21.
Modulating Enzyme Catalysis through Mutations Designed to Alter Rapid Protein Dynamics. Zoi I, Suarez J, Antoniou D, Cameron SA, Schramm VL, Schwartz SD. J Am Chem Soc. 2016 Mar 16;138(10):3403-9.
Transition State Structure of RNA Depurination by Saporin L3. Yuan H, Stratton CF, Schramm VL. ACS Chem Biol. 2016 May 20;11(5):1383-90.
Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36. Poulin MB, Schneck JL, Matico RE, McDevitt PJ, Huddleston MJ, Hou W, Johnson NW, Thrall SH, Meek TD, Schramm VL. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):1197-201.
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