George T. Lountos, Ph.D.
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 molecular 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 played a pivotal role in solving high-resolution crystal structures of human checkpoint kinase 2 (Chk2) in complex with various inhibitors that have enabled the rational design of potent and specific inhibitors with anti-cancer activity. Current research is also focused on structural studies of the E. coli LonA protease using single particle cryo-electron microscopy.
1) crystallization of macromolecules including protein-ligand complexes, 2) X-ray diffraction data collection, 3) structure determination of macromolecules, 4) structure-based drug design, 5) fragment-based drug discovery, 6) protein expression and purification 7) cryo-electron microscopy
Structure-Based Drug Design:
A variety of drug targets involved in cancer and infectious disease are currently being studied by X-ray crystallography in collaboration with medicinal chemists and biochemists within the CCR. Our goal is to elucidate the structural basis for inhibitor binding as a first step towards the optimization of these inhibitors by rational design. In addition to studying protein-inhibitor complexes of compounds identified from high-throughput screening, our group is leading fragment-based drug discovery efforts for drug targets by screening libraries of small drug-like fragments by crystallographic methods. X-ray crystallography is a very powerful tool that can be used to identify and confirm binding of low-affinity fragments to proteins that are not detectable by other screening methods. Structural information gleaned from these studies can be used to optimize low-affinity fragments into inhibitors with higher potency while retaining favorable drug-like properties.
Current targets under collaborative investigation using structure-based drug design methods include the SUMO-conjugating enzyme, Ubc9 (Dr. Jay Schneekloth, Chemical Biology Laboratory) and tyrosyl-DNA phosphodiesterase I (TDP1) (Dr. Yves Pommier, LMP). Very recently, we have solved the first high-resolution crystal structure of the main protease (3CLpro) from the highly pathogenic Middle East Respiratory Syndrome (MERS) virus. In collaboration with Dr. Terrence Burke (Chemical Biology Laboratory) and Dr. Robert Ulrich (USAMRIID), we have used structure-based design methods to develop highly potent and specific inhibitors of the Yersinia pestis protein tyrosine phosphatase, YopH. Our group also has played a pivotal role in the rational design of human checkpoint kinase 2 inhibitors in collaboration with Dr. Yves Pommier and Dr. Robert Shoemaker (NCI Division of Cancer Prevention). We were also the first group to report high-resolution crystal structures of the anti-cancer drug target, TDP1, in complex with small molecule inhibitors. In collaboration with the labs of Dr. Burke and Dr. Pommier, we are using structure-based design methods to design new inhibitors of TDP1.
In collaboration with Dr. Lesley-Ann Giddings (Department of Chemistry, Smith College), we are studying the crystal structures and mechanisms of enzymes involved in the biosynthesis of siderophores and metal-chelating secondary metabolites that help microorganisms thrive in iron-limited environments. These enzymes are potentially useful as biocatalysts in the synthesis of clinically-approved siderophores and could be exploited as novel drug targets to combat certain human pathogens.
The energy-dependent Lon proteases play a key role in cellular regulation by degrading short-lived regulatory proteins and misfolded proteins in the cell. Under the direction of Dr. Alexander Wlodawer, we are using single-particle cryo-electron microscopy to study the full-length structure of the E. coli LonA protease. Recently, we solved the structure of the catalytically inactive S679A mutant of E. coli LonA protease by cryo-EM at 3.5 Å resolution without a bound substrate (*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. (2019). Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery. Current Research in Structural Biology. 1:13-20.). EcLonA without a bound substrate adopts a hexameric open-spiral quaternary structure that might represent the resting state of the enzyme that is poised to bind to substrate. Our ongoing research efforts are to solve structures of LonA protease bound to various substrates to study the dynamics of structural changes upon substrate binding.
Selected Key Publications
Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery.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.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.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 .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.J. Pharmacol. Exp. Ther. 331: 816-26, 2009. [ Journal Article ]
Dr. Lountos received his undergraduate degree in chemistry with high honor (2000) from the Georgia Institute of Technology where he performed research in organic chemistry in the laboratory of Prof. Suzy Beckham. He obtained a Ph.D. in chemistry (2005) from the School of Chemistry & Biochemistry, Georgia Institute of Technology, under the direction of Prof. Allen Orville where he conducted crystallographic studies of flavin-dependent enzymes. From 2006-2011, he was a postdoctoral fellow in the Protein Engineering Section of the Macromolecular Crystallography Laboratory, under the direction of Dr. David Waugh. His postdoctoral research focused on structural studies of proteins involved in the Yersinia pestis type III secretion system and structure-based drug design of inhibitors of the human checkpoint kinase 2 and Yersinia pestis YopH,
Dr. Lountos was the recipient of a Federal Technology Transfer Award in 2008 and the NIH Fellows Award for Research Excellence (FARE) in 2009 and in 2010. In 2011, he was appointed as Scientist I with the Basic Research Program, Leidos Biomedical Research, Inc., where he works in support of the Macromolecular Crystallography Laboratory. In 2016, he was a recipient of the Special Achievement Award during the 19th Annual Frederick National Laboratory Achievement Awards Program. Dr. Lountos is an active member of the American Crystallographic Association (ACA) where he served as chair of the ACA Young Scientist Scientific Interest Group (2015). In 2017, he was appointed to the ACA Council where he served as the Young Scientist Representative to council until January 2020.