department of pharmacology

Chris Dealwis, Ph.D.

Dealwis

 
Associate Professor

Phone: (216) 368-1652, 368-3337
Fax: (216) 368-1300
E-mail: chris.dealwis@case.edu
W333

Research:

The Dealwis lab has been investigating how the Life's essential enzyme ribonucleotide reductase (RR) functions by catalyzing the rate limiting step of DNA synthesis and is responsible for maintaining a balanced nucleotide pool in the cell. The lab is interested in developing novel anticancer agents that target RR. In a second front, neutron diffraction and ultrahigh resolution x-ray diffraction is used to investigate transition states of catalytic mechanisms, solvent structure and dynamics. Our lab uses biochemical, biophysical and structural tools such as x-ray diffraction, neutron diffraction, electron microscopy, isothermal titration calorimetry and spectroscopy to unravel the mysteries of life. Fragment-based drug design, molecular modeling and in silico screening techniques are used in drug discovery.

Ribonucleotide Reductase

We study the structure-function and regulation of the anti-cancer target ribonucleotide reductase (RR). RR converts nucleotides to deoxynucleotides (dNTPs), the rate-limiting step in de novo DNA synthesis. Control of the dNTP pool is essential; an excess of deoxynucleotides causes mutations, while scarcity can lead to cell death due to improper cell division. RR is highly regulated transcriptionally, allosterically, by subunit compartmentalization and, in S. cerevisiae, by its protein inhibitor sml1. In 2006, we solved the structure of the first eukaryotic RR1 from S. cerevisiae (see Figure below). Our structures provide a molecular basis for how RR selects for specific nucleotide diphosphate substrates based on exquisite loop rearrangements induced by effector binding at a selectivity site, and balance cellular nucleotide pools (Xu et al., 2006).

Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation.

Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4022-7. Md. Faiz Ahmad., et al., J Mol Biol. 2012

Allosteric regulation by the activator ATP and inactivator dATP based on oligomerization

Oligomerization of proteins as a regulatory mechanism is becoming generally accepted. For many years the consensus was that RR was a multi-subunit enzyme consisting of the large subunit RR1, containing the catalytic subunit, which associates with RR2 housing the essential free radical required for catalysis, together assembling into a heterotetramer. This view was challenged in 2000 where mammalian enzymes were shown to form hexamers. Subsequently several labs including our own have shown the existence of dimers and hexamers in eukaryotic and mammalian RRs (Fairman et al., 2011). In 2011 we reported the first hexamer x-ray structure induced by dATP binding at the allosteric site called the activity site (see figure below). In the same publication, we reported an electron microscopy structure of the first eukaryotic holo complex, demonstrating how a dimer of RR2 binds inside the hexameric RR1 ring (see figure below). New data from other labs show that the dATP-induced hexamer is stabilized by a protein IRBIT to assist in dATP's allosteric inhibition of the enzyme. Moreover, chemotherapeutic agents such as clofarabine and cladarabine have been reported to form persistent hexamers while binding at the catalytic site and the activity site. Therefore the pursuit to understand the role of oligomerization of RR has just begun.

Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization. Nat. Struct. Mol. Biol. 2011 Mar;18(3):316-22. PMID: 21336276; PMCID: PMC3101628.

Md. Faiz Ahmad and Chris Dealwis. The structural basis for the allosteric regulation of ribonucleotide reductase. Prog Mol Biol Transl Sci. 2013;117:389-410

Developing a new class of anticancer agents targeting RR

Several of the antimetabolite class of drugs used in the clinic target RR. These include nucleoside base drugs such as gemcitabine commonly used in treating breast, lung and pancreatic cancer as well as clofarabine, fludarabine and cladarabine. Unfortunately, these nucleoside drugs lead to chain termination and their lack of specificity causes unwanted side effects. Therefore we have pursued developing a novel class of anticancer agents that are non-nucleoside reversible inhibitors with improved target specificity to reduce the toxic side effects. We were immensely aided by the structure of the human enzyme which we determine in 2011 as a starting model for in silico screening. A detailed description of the method used for discovering the first non-nucleoside reversible inhibitors is found in (J Med Chem 2015 by Md. Faiz Ahmad et al., and see figure on drug design cycle). In order to achieve this, the PI has assembled a group of experts in medicinal chemistry, enzymology, cell biology, radio- biology and mouse co-facility at the Case Comprehensive Cancer Center.

Md. Faiz Ahmad et al., Identification of Non-nucleoside Human Ribonucleotide Reductase ModulatorsJ. Med. Chem. 2015 Dec 24;58(24):9498-509.

The small protein inhibitor of S.cerevisiae RR Sml1

Sml1 is a protein inhibitor of RR1 under the cell cycle control involving the Mec1/Rad53 pathway. Interestingly, sml1 is an intrinsically disordered protein that is stowed as a dimer (Gupta et al., 2004). During the pre-S-phase of the cell cycle sml1 binds RR1 in a 1:1 complex and upon entering the S-phase or during radiation damage it is phosphorylated by Dun1 kinase (Uchiki et al., 2004) leading to ubiquitination and clearance from the cell. While there is no crystal structure for sml1 or complex with RR, we have deduced the kinetic mechanism using steady-state enzymology demonstrating that the mode of inhibition is determine by nucleotide binding (Misko et al., 2016). Structural work is ongoing.

Misko TA, Wijerathna SR, Radivoyevitch T, Berdis AJ, Md. Faiz Ahmad,  Harris ME, Dealwis CG. Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme. FEBS.Lett. (2016), 590:1704-12

Gupta V,et al., Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p. Biochemistry. 2004 Jul 6;43(26):8568-78.

Uchiki Tet al., Identification of phosphorylation sites on the yeast ribonucleotide reductase inhibitor Sml1. J Biol Chem. 2004 Mar 19;279(12):11293-303.

Neutron diffraction and ultrahigh resolution x-ray diffraction

We use neutron and ultra-high resolution x-ray diffraction to solve controversial mechanistic questions. The contribution of hydrogen atoms in noncovalent interactions and enzymatic reactions underlies all aspects of biology at the molecular level, yet their “visualization” is quite difficult.  Neutron diffraction (ND) is well suited to such studies, as it is able to resolve the positions of hydrogen atoms in macromolecules. ND is coming of age with the advent of the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory. Our lab is one of the first to use Spallation neutrons to solve macromolecular structure. Using DHFR as our test enzyme, we were able to use 0.3mm3 non-perdeuterated crystals to obtain 2.2Å resolution data at the Protein Crystallography station (PCS) at Los Alamos National Lab. The neutron crystallography cycle is depicted pictorially below. Our lab continues to work on other signaling molecules to unravel the mechanisms of proton-coupled signal transduction.

Wan Q, Bennett BC, Wilson MA, Kovalevsky A, Langan P, Howell EE, Dealwis C.
Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. Proc. Natl. Acad. Sci. USA (PNAS). 2014 Dec 23;111(51):18225-30.

Bennett B, Langan P, Coates L, Mustyakimov M, Schoenborn B, Howell EE, Dealwis C. Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate. Proc Natl Acad Sci U S A. 2006 Dec5;103(49):18493-8.

Other projects:

Amyloids

Soluble proteins and peptides can sometimes aggregate into insoluble, self-assembled filamentous aggregates including amyloid and amyloid-like structures. Current interest in amyloid fibrils and related aggregates arises from their involvement in diseases such as Alzheimer's disease (AD), type 2 diabetes, prion diseases, and other protein misfolding disorders. Since aggregates of the amyloid-beta (Aβ) peptide have been implicated in the molecular mechanism of Alzheimer's disease (AD), reversing or preventing Aβ aggregation is an important prospective approach to AD therapy. We have solved the first three-dimensional x-ray structure of the immunodominant N-terminus of Aβ in complex with the Fab fragment of the mAb PFA1 (see Figure right). Our structures provide a molecular basis for Fab-Aβ(N-terminus) interactions (Gardberg et al PNA S 2008). The crucial interactions are made between the WWDDD motif from CDRH2 with an EFRH motif of Aβ. We have also shown that the N-terminus directed mAbs have the potential to cross-react with EFRH-like sequences that are found within proteins found in the brain. Our structures provide a basis for improving selectivity and potency towards Aβ.

Gardberg AS, Dice LT, Ou S, Rich RL, Helmbrecht E, Ko J, Wetzel R, Myszka DG, Patterson PH, Dealwis C. Molecular basis for passive immunotherapy of Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15659-64

Lab Photo Gallery »

Selected Publications:

  • Knappenberger AJ, Ahmad Md. Faiz, Viswanathan R, Dealwis CG, Harris ME. Nucleotide analog triphosphate allosterically regulate human ribonucleotide reductase and identify chemical determinants that drive substrate specificity. Biochemistry, 2016, 55 (41), pp 5884–5896
  • Misko TA, Wijerathna SR, Radivoyevitch T, Berdis AJ, Ahmad Md. Faiz, Harris ME, Dealwis CG. Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme. FEBS.Lett. (2016), 590:1704-12.
  • Md. Faiz Ahmad, Huff SE, Pink J, Alam I, Zhang A, Perry K, Harris ME, Misko T, Porwal SK, Oleinick NL, Miyagi M, Viswanathan R, Dealwis CG. Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators.  J. Med. Chem. 2015 Dec 24;58(24):9498-509.
  • Zhang Y,  Desai A,  Yang S Y,  Bae Ki Beom,  Monika I. Antczak,  Stephen P. Fink,  Shruti Tiwari, Joseph E. Willis,  Noelle S. Williams,  Dawn M. Dawson, David Wald,  Wei-Dong Chen,  Zhenghe Wang,  Lakshmi Kasturi,  Gretchen A. Larusch,  Lucy He,  Fabio Cominelli,  Luca Di Martino,  Zora Djuric,  Ginger L. Milne,  Mark Chance,  Juan Sanabria, Chris Dealwis,  Debra Mikkola,  Jacinth Naidoo,  Shuguang Wei, Hsin-Hsiung Tai,  Stanton L. Gerson,  Joseph Ready, Bruce Posner, James K. V. Wilson,  Sanford D. Markowitz .  Inhibition of the Prostaglandin Degrading Enzyme 15-PGDH Potentiates Tissue Regeneration. Science 348 (2015) aaa2340-1.
  • Wan Q, Bennett BC, Wilson MA, Kovalevsky A, Langan P, Howell EE, Dealwis C. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. Proc. Natl. Acad. Sci. USA (PNAS). 2014 Dec 23;111(51):18225-30.
  • Wan Q, Kovalevsky AY, Wilson MA, Bennett BC, Langan P, Dealwis C. Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP+. Acta Crystallogr F Struct Biol Commun. 2014 Jun;70(Pt 6):814-8.
  • Md. Faiz Ahmad and Dealwis CG. The structural basis for the allosteric regulation of ribonucleotide reductase. Prog Mol Biol Transl Sci. 2013;117:389-410.
    Md. Faiz Ahmad, Wan Q, Jha S, Motea E, Berdis A, Dealwis C. Evaluating the therapeutic potential of a non-natural nucleotide that inhibits human ribonucleotide reductase. Mol Cancer Ther. 2012 Oct;11(10):2077-86.
    Md. Faiz Ahmad, Kaushal PS, Wan Q, Wijerathna SR, An X, Huang M, Dealwis CG. Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase. J Mol Biol. 2012 Jun 22;419(5):315-29.
  • Langan, P., Fisher, Z., Kovalevsky, A., Mustyakimov, A., Sutcliffe, V., Afonine, AV., Bennett, B., Dealwis, C and Schoenborn, B. Protein structures by Spallation neutron crystallography. (2008), J. Synch. Rad, 15, 215-218.
    Bennett, B.C., Gardberg, A. S., Blair, M and Dealwis, C.  On the determinants of amide backbone exchange in proteins: a neutron crystallographic comparative study. (2008), Acta. Cryst. D. 64, 764-83.
  • Xu, H., Fairman, J.W., Wijerathna, Kreischer, N.R., LaMacchia, J., Helmbrecht, E., Cooperman, B.S and Dealwis, C. The Structural Basis for Peptidomimetic Inhibition of Eukaryotic Ribonucleotide Reductase: a Conformationally Flexible Pharmacophore. (2008), J. Med Chem. 51, 5653-9.
  • Brad C. Bennett, Qun Wan, Md. Faiz Ahmad, Paul Langan and Chris G. Dealwis.  X-ray structure of the ternary MTX•NADPH complex of the anthrax dihydrofolate reductase: a pharmacophore for dual-site inhibitor design. (2009), J. Struct. Biol. 166,162-171
  • Anna Gardberg, Lezlee Dice, Kathleen PridgenJan Ko, Paul Patterson, Susan Ou, Ronald Wetzel, Chris Dealwis. Structures of Aβ-related peptide-monoclonal antibody complexes. Biochemistry. (2009), 48, 5210-7.
  • Dianqing Sun, Hai Xu, Sanath, R. Wijerathna, Chris Dealwis*, and Richard E. Lee*. Chem. Med. Chem. (2009), Structure-based Design, Synthesis, and Evaluation of 2'-(2-Hydroxyethyl)-2'-Deoxyadenosine and Its 5'-Diphosphate as Novel Ribonucleotide Reductase Inhibitors, ChemMedChem, 4, 1649-56 (*joint corresponding authors)
  • Jaskiran Kaur, Shalini Jha, Chris Dealwis and Barry S. Cooperman. (2009), Design, synthesis and structure of peptidomimetic inhibitors of eukaryotic ribonucleotide reductase: A target for cancer chemotherapy. Proceedings of the 21st American Peptide Society, Pages 80-88
  • Minami, S.S., Sidahmed, E., Aid, S., Shimoji, M., Niikura, T., Mocchetti, I., Rebeck, G.W., Prendergast, J.S., Dealwis, C., Wetzel, R., et al. Therapeutic versus neuroinflammatory effects of passive immunization is dependent on Abeta/amyloid burden in a transgenic mouse model of Alzheimer's disease. J Neuroinflammation 7, 57.
  • Miyagi, M., Wan, Q., Ahmad, M F., Gokulrangan, G., Tomechko, S. E., Bennett, B. & Dealwis, C. Histidine hydrogen‐deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase. (2011) PLoS One 6, e17055.
  • Fairman, J. W., Wijerathna, S. R., Ahmad, M. F., Xu, H., Nakano, R., Jha, S., Prendergast, J., Welin, R. M., Flodin, S., Roos, A., Nordlund, P., Li, Z., Walz, T. & Dealwis, CG. Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide‐induced oligomerization. (2011) Nat Struct Mol Biol 18, 31622.
  • Wan, Q., Md.Faiz Ahmad., Fairman, J., Gorzelle, B., de la Fuente, M., Dealwis, C. & Maguire, M. E. X‐Ray Crystallography and Isothermal Titration Calorimetry Studies of the Salmonella Zinc Transporter ZntB. (2011). Structure 19, 70010.
  • Anna S. Gardberg, Lezlee T. Dice, Elizabeth Helmbrecht, Jan Ko, Susan Ou, Paul H. Patterson, Rebecca Rich, David Myszka, Ronald Wetzel, and Chris Dealwis. (2007) Proc. Natl. Acad. Sci. USA, 104(40): 15659-64. Molecular basis for passive immunotherapy of Alzheimer's disease.
  • Brad Bennett, Hai Xu, Richard F. Simmerman, Richard E. Lee and Chris G. Dealwis. Crystal structure of the anthrax drug target. Bacillus anthracis dihydrofolate reductase. (2007)J. Med Chem, 18: 4374-81.
  • Brad Bennett, Paul Langan, Leighton Coates, Marat Mustyakimov, Benno Schoenborn, Elizabeth Howell, and Chris Dealwis.  Neutron diffraction studies of E. coli DHFR in complex with Methotrexate. (2006). Proc. Natl. Acad. Sci. USA103: 18493-18496.
  • Hai Xu, Catherine Faber, Tomoaki Uchiki, James W. Fairman, Joseph Racca, and Chris Dealwis. Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation (2006)Proc. Natl. Acad. Sci. USA 103: 4022-4027.
  • Hai Xu, Catherine Faber, Tomoaki Uchiki, Joseph Racca, and Chris Dealwis. Structures of eukaryotic ribonucleotide reductase I define gemcitabine diphosphate binding and subunit assembly (2006). Proc. Natl. Acad. Sci. USA 103: 4028-4033.
  • Uchiki T, Dice LT, Hettich RL, and Chris Dealwis. Identification of phosphorylation sites on the yeast ribonucleotide reductase inhibitor Sml1. (2004)J. Biol. Chem., 279: 11293-303.
  • Gupta V, Peterson CB, Dice LT, Uchiki T, Racca J, Guo JT, Xu Y, Hettich R, Zhao X, Rothstein R, and Chris Dealwis. Sml1p Is a Dimer in Solution: Characterization of Denaturation and Renaturation of Recombinant Sml1p. (2004)Biochemistry43: 8568-8578.
  • Wall, JS, Gupta V, Wilkerson M, Schell M, Loris R, Adams P, Solomon A, Stevens F, and Chris Dealwis. Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four Vλ6 proteins. (2004). J. Mol. Recognit. 17: 323-31.
  • Chris Dealwis and Jon Wall. Towards understanding the structure-function relationship of human amyloid disease. (2004)Curr. Drug Targets 5: 159-71.
  • Uchiki T, Hettich R, Gupta V, and Chris Dealwis Characterization of monomeric and dimeric forms of recombinant Sml1p-histag protein by electrospray mass spectrometry. (2002)Anal. Biochem., 301: 35-48.

Lab Members:

  • Faiz Ahmad Mohammed (research associate)
  • Tessianna Misko (graduate student)
  • Sarah Huff (graduate student)
  • Andrew. J Knappenberger (visiting graduate student)

Past members:

  • Brad Bennett (former graduate student. Currently assistant professor Stamford University)
  • Tomoaki Uchiki (former graduate student. Currently in Vivo Science Inc. Kawasaki, Kanagawa, Japan )
  • James Fairman (former graduate student. Currently X-ray crystallographer and Informatics at Roche pharmaceuticals)
  • Sanath Wijerathna (former graduate student. Lecturer Green Bay medical school)
  • Matthew Wilkerson (former master student)
  • Leslie Dice (former master student. Research assistant Oak Ridge National Labs)
  • Charlene Jha (former master student. Graduate student at UNC)
  • Vibha Gupta (former postdoc. Currently Assistant Professor at Jaypee Institute of Information Technology in New Delhi, India)
  • Anna S Gardberg (former postdoc. Currently Senior Scientist in Structural Biology at Constellation Pharmaceuticals in Boston)
  • Catherine Farber (former postdoc.)
  • Hai Zu (former postdoc. Currently Senior Scientist at Trevigen Inc. in Washington DC)
  • Qun Wan (former postdoc. Currently Professor at Nanjing Agricultural University in China)
  • Prem Kaushal (former postdoc. Currently postdoctoral fellow at Briggs laboratory Wandsworth Center, New York)
  • Intekhab Alarm (former postdoc. Currently postdoc at University of Michigan)
  • Jenny Lee Keiser (former postdoc. Currently RA at Case Western Reserve University)