George Lountos

George T. Lountos, Ph.D.

  • Center for Cancer Research
  • National Cancer Institute


Dr. Lountos is currently involved in a number of highly collaborative research projects within NCI at Frederick involving the structural determination of proteins engaged in cancer and infectious diseases with particular emphasis on the structure-based design of novel small molecule inhibitors. A variety of targets are currently under investigation, including protein kinases and phosphatases, viral proteases, the SUMO-conjugating enzyme, Ubc9, tyrosyl-DNA phosphodiesterase I (TDP1) and other enzymes. Dr. Lountos determined and published the first crystal structure of the Middle East respiratory syndrome coronavirus 3CL protease in 2015 (  Dr. Lountos played a pivotal role in determining high-resolution crystal structures of human checkpoint kinase 2 (Chk2) in complex with various inhibitors that have informed the rational design of potent and specific inhibitors with anti-cancer activity ( In collaboration with several NCI laboratories, Dr. Lountos has successfully applied fragment-based drug discovery to identify and structurally characterize the first small molecule scaffolds bound to the cancer drug targets, Ubc9  ( and TDP1 ( by high resolution X-ray crystallography. Current research is also focused on structural studies of the E. coli LonA protease using single particle cryo-electron microscopy. Our research has been highlighted on the covers of the journals Nucleic Acids Research, Royal Society of Chemistry Chemical Science, Royal Society of Chemistry Chemical Biology, Chemical Biology & Drug Design, and ACS Biochemistry.

Areas of Expertise

Crystallization Of Macromolecules Including Protein-ligand Complexes
X-ray Diffraction Data Collection
Structure Determination Of Macromolecules
Structure-based Drug Design
Fragment-based Drug Discovery
Protein Expression And Purification
Cryo-electron Microscopy


Selected Key Publications

Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery

*Botos, I.,* Lountos, G.T., Wu, W., Cherry, S., Ghirlando, R., Kudzhae, A.M., Rotanova, T.V., de Val, N., Tropea, J.E., Gustchina, A., and Wlodawer, A
Current Research in Structural Biology. 1: 13-20, 2019. [ Journal Article ]

Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening

Lountos, G.T., Zhao, X.Z., Kiselev, E., Tropea, J.E., Needle, D., Pommier, Y., Burke, T.R., and Waugh, D.S.
Nucleic Acids Research. 47: 10134-10150, 2019. [ Journal Article ]

Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

Lountos GT, Jobson AG, Tropea JE, Self CR, Zhang G, Pommier Y, Shoemaker RH, and Waugh DS.
J. Struct. Biol. 176: 292-301, 2011. [ Journal Article ]

Utilization of nitrophenylphosphates and oxime-based ligation for development of nanomolar affinity inhibitors of the Yersinia pestis outer protein H (YopH) phosphatase

Bahta M, Lountos GT, Dyas B, Kim SE, Ulrich RG, Waugh DS, and Burke TR Jr.
J. Med. Chem. 54: 2933-43, 2011. [ Journal Article ]

Cellular inhibition of checkpoint kinase 2 (Chk2) and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019

Jobson AG, Lountos GT, Lorenzi PL, Llamas J, Connelly J, Cerna D, Tropea JE, Onda A, Zoppoli G, Kondapaka S, Zhang G, Caplen NJ, Cardellina JH, Yoo SS, Monks A, Self C, Waugh DS, Shoemaker RH, and Pommier Y.
J. Pharmacol. Exp. Ther. 331: 816-26, 2009. [ Journal Article ]


NAR cover

Nucleic Acids Research

Published Date

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3′ end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.



See: Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening by George T. Lountos, Xue Zhi Zhao, Evgeny Kiselev, Joseph E. Tropea, Danielle Needle, Yves Pommier, Terrence R. Burke, Jr. and David S. Waugh in Nucleic Acids Research201947, 10134–10150.

Chemical Biology Cover

Chemical Biology & Drug Design

Published Date

We have developed competitive and direct binding methods to examine small-molecule inhibitors of protein tyrosine phosphatase activity. Focusing on the Yersinia pestis outer protein H, a potent bacterial protein tyrosine phosphatase, we describe how an understanding of the kinetic interactions involving Yersinia pestis outer protein H, peptide substrates, and small-molecule inhibitors of protein tyrosine phosphatase activity can be beneficial for inhibitor screening, and we further translate these results into a microarray assay for high-throughput screening


Hogan, M., Bahta, M., Cherry, S., Lountos, G.T., Tropea, J.E., Zhao, B., Burke Jr., T.R., Waugh, D.S., and Ulrich, R.G. (2013). Biomolecular interactions of small-molecule inhibitors affecting the YopH protein tyrosine phosphatase. Chem. Biol. Drug Des. 81:323-333

Biochem Cover

Biochemistry Cover

Published Date

The crystal structure of choline oxidase provides mechanistic insights and reveals a novel FAD C4a-adduct. The solvent accessible surfaces of the homodimeric enzyme are colored to illustrate the dimer interface and the major domains within one subunit. The FAD binding (blue) and substrate binding (green) domains are partially separated by a loop (orange) that sequesters the active site within the interior of the enzyme


*Quaye, O., *Lountos, G.T., Fan, F., Orville, A.M., and Gadda, G. (2008). Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase. Biochemistry 47: 243-256. * authors contributed equally

Chemical Science Cover

Chemical Science

Published Date

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3′- and 5′-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3′-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.


Zhao, X. Z.Kiselev, E.Lountos, G. T.Wang, W.Tropea, J. E.Needle, D.Hilimire, T. A.Schneekloth, J. S.Waugh, D. S.Pommier, Y.Burke, T. R. Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sitesChem. Sci. 2021123876 DOI: 10.1039/D0SC05411A

protein structure image on journal cover

Identification of multidentate tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors that simultaneously access the DNA, protein and catalytic-binding sites by oxime diversification

Published Date

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family that can downregulate the anticancer effects of the type I topoisomerase (TOP1) inhibitors by hydrolyzing the 3'-phosphodiester bond between DNA and the TOP1 residue Y723 in the critical stalled intermediate that is the foundation of TOP1 inhibitor mechanism of action. Thus, TDP1 antagonists are attractive as potential enhancers of TOP1 inhibitors. However, the open and extended nature of the TOP1-DNA substrate-binding region has made the development of TDP1 inhibitors extremely challenging. In this study, starting from our recently identified small molecule microarray (SMM)-derived TDP1-inhibitory imidazopyridine motif, we employed a click-based oxime protocol to extend the parent platform into the DNA and TOP1 peptide substrate-binding channels. We applied one-pot Groebke-Blackburn-Bienayme multicomponent reactions (GBBRs) to prepare the needed aminooxy-containing substrates. By reacting these precursors with approximately 250 aldehydes in microtiter format, we screened a library of nearly 500 oximes for their TDP1 inhibitory potencies using an in vitro florescence-based catalytic assay. Select hits were structurally explored as their triazole- and ether-based isosteres. We obtained crystal structures of two of the resulting inhibitors bound to the TDP1 catalytic domain. The structures reveal that the inhibitors form hydrogen bonds with the catalytic His-Lys-Asn triads ("HKN" motifs: H263, K265, N283 and H493, K495, N516), while simultaneously extending into both the substrate DNA and TOP1 peptide-binding grooves. This work provides a structural model for developing multivalent TDP1 inhibitors capable of binding in a tridentate fashion with a central component situated within the catalytic pocket and extensions that project into both the DNA and TOP1 peptide substrate-binding regions.


Xue Zhi Zhao, Wenjie Wang, George T. Lountos, Evgeny Kiselev, Joseph E. Tropea, Danielle Needle, Yves Pommier and Terrence R. Burke, Jr.

RSC Chemical Biology, 20234, 334-343