I am directly involved in the development of novel fluorescence tools for the discovery of cardiac calcium pump therapeutics. The sarco/endoplasmic reticulum calcium ATPase (SERCA) is the calcium pump responsible for maintaining cellular calcium homeostasis. Diminished SERCA function has been directly linked to numerous degenerative disease states, such as heart failure. The pathological progression of heart failure is associated with an elevated level of cytosolic calcium, and impairs the function of the muscle contraction-cycle. The overarching goal of my research is to discover novel small-molecule effectors, capable of enhancing SERCA’s ability to pump and store calcium within the sarcoplasmic reticulum (SR). Drugs that increase the calcium pumping efficiency of SERCA will restore calcium homeostasis by reducing the calcium content in the cytosol, and enhance impaired cardiac function.
The process of drug discovery is a high-risk effort, and involves screening millions of small-molecules to fortuitously discover a lead compound with high-therapeutic potential. The precise placement of two fluorescent proteins at specific locations along SERCA’s cytosolic headpiece, allows for the detection of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorescent proteins. I have generated human cell lines that overexpress this fluorescent fusion protein and created a live-cell biosensor. The rate of energy transfer (FRET) is dependent on the distance between the fluorescent probes and linked to the enzymatic activity of SERCA. FRET tracks SERCA’s structural status, while it pumps calcium into the sarcoplasmic reticulum. These biosensors are grown in vast quantities, harvested, and utilized for high-throughput drug screening. The cells are dispensed into high-density microplates, where each well contains a different compound.
Through my research, I have demonstrated that FRET can be utilized for the purposes of high-throughput drug screening by using proprietary fluorescence technology, capable of recording the nanosecond fluorescence decay rate (lifetime) and the full emission spectrum. Both lifetime and spectral modes offer incredibly fast speeds, with high resolution and precision. High-throughput screening by lifetime mode offers the advantage of resolving the structural status of the FRET biosensor because the mole fraction of each structural state is assessed, and candidate compounds found during the screening process can be characterized by their structural effect on the biosensor. High-throughput screening by spectral mode increases assays precision by taking into account the shape of the fluorescence emission spectrum. The shapes of these spectra are decomposed into the contribution of known components by a novel spectral unmixing method, and further used to accurately evaluate FRET. When coupled with lifetime mode, spectral-based drug screening increases assay precision and removes artifacts from cellular autofluorescence and fluorescent compounds. The complementary advantages of coupling spectral and lifetime fluorescence measurements significantly reduce the rate of false-positives from high-throughput drug screens. The development of these technologies and FRET biosensor assays, drastically increases the probability of identifying a novel drug with great therapeutic potential.
I am currently interested in applying these novel technologies for the development of new FRET-biosensors targeting other promising therapeutic pathways. I have training and research experience in a broad range of fields (neuroscience, immunology, stem cells, molecular biology, structural biology, and optical fluorescence). My long-term career goal is to work in industry but still remain in close contact with academic research to hopefully make a lasting impact in both areas.
University of Minnesota, Minneapolis, MN Ph.D., Biochemistry, Molecular Biology, and Biophysics Advisor: David. D. Thomas, PhD
University of Minnesota, Minneapolis, MN BS,Neuroscience BS, Biochemistry
University of Wisconsin River Falls, WI
TEACHING & RESEARCH EXPERIENCE
|2018||HHMI BioInteractive Training|
|2018||Preparing Future Faculty Certificate, University of Minnesota|
|2018 Summer||BIOL 1100, Intro to Genetics (TREM Scholar at Normandale Community College)|
|2015 Summer||BIOC 4125, Laboratory in Molecular Biology and Biotechnology|
|2014 Fall||BIOC 4125, Laboratory in Molecular Biology and Biotechnology|
|2008-2009||Undergraduate Research Assistant, University of Minnesota (Mark Thomas, PhD)|
FUNDING & AWARDS
2017-Present NIH K12 Training Research Educators in Minnesota Postdoctoral Scholar
2017 NIH T32 Minnesota Muscle Postdoctoral Fellow
2016 Ross A. Gortner Award
2014-2016 NIH Chemistry-Biology Interface Training Grant
2012-2016 3M Graduate Fellowship
2013-2017 Arnold H. Johnson Graduate Fellowship
2014 Charles Carr / William Peterson Award
2008-2009 Undergraduate Research Grant (UROP)
Tory Schaaf, Kurt Peterson, Benjamin Grant, David Thomas, and Greg Gillispie. 2017. High Precision FRET Assays. U.S. Patent Application 62450188. Filed on Jan. 25, 2017, Patent Pending.
Stroik D, S.Yuen, K Janicek, T Schaaf, J Li, D Ceholski, R Hajjar, R Cornea, and D Thomas. Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells. Nature Scientific Reports. Accepted.
Lo, CH, N. Vunnam, AK Lewis, TL Chiu, BE Brummel, TM Schaaf, BD Grant, P Bawaskar, DD Thomas, and JN Sachs. An innovative high-throughput screening approach for discovery of small molecules that inhibit TNF Receptors. SLAS Discovery, Published online May 22, 2017.
Schaaf, TM, KC Peterson, BD Grant, P Bawaskar, S Yuen, J Li, JM Muretta, GD Gillispie, and DD Thomas. 2017. High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA. SLAS Discovery 22:262-273. PMC5323330.
Schaaf, TM, KC Peterson, BD Grant, DD Thomas, and GD Gillispie. 2017. Spectral unmixing plate reader: high-throughput, high-precision FRET assays in living cells. SLAS Discovery 22:250-261. PMC5323330.
Gruber, SJ, RL Cornea, J Li, KC Peterson, TM Schaaf, GD Gillispie, R Dahl, KM Zsebo, SL Robia, and DD Thomas. 2014. Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells. J Biomol Screening 19:215-222 (cover article). PMC4013825.
Filareto, A, S Parker, R Darabi, L Borges, M Iacovino, T Schaaf, T Mayerhofer, J Chamberlain, J Ervasti, R McIvor, M Kyba, and R Perlingeiro. 2013. An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells. Nature Communications 4: 1549. PMC3595133.
Tory M. Schaaf, KC Peterson, BD Grant, J Li, P. Bawaskar, S Yuen, GD Gillispie, and DD Thomas. 2017. Spectral unmixing plate reader: High-throughput FRET-based screening in living cells to identify small-molecule effectors of SERCA. Biophysical Society 61th Annual Meeting, February 14, New Orleans, Louisiana.
Tory M. Schaaf, Ji Li, Samantha L. Yuen, Prachi Bawaskar, Benjamin D. Grant, Kurt C. Peterson, David D. Thomas, Gregory D. Gillispie. 2016. SUPR: Spectral unmixing plate reader for live-cell FRET biosensor drug screening. Biophysical Society 60th Annual Meeting, March 2, Los Angeles, California.
Tory M. Schaaf, Ji Li, Rocio Foncea, Simon J Gruber, Kurt C Peterson, Karl J Petersen, David A Bernlohr, Gregory D. Gillispie, and David D. Thomas. 2015. A two-color non-muscle SERCA FRET sensor for diabetes drug discovery using fluorescence lifetime detection. Chemistry-Biology Interface Career Development Workshop. August 10, Vanderbilt University, Tennessee.
Tory M. Schaaf, Ji Li, Rocio Foncea, Simon J Gruber, Kurt C Peterson, Karl J Petersen, David A Bernlohr, Gregory D. Gillispie, and David D. Thomas. 2015. A two-color non-muscle SERCA FRET sensor for diabetes drug discovery using fluorescence lifetime detection. Biophysical Society 59th Annual Meeting, February 10, Baltimore, Maryland.
Tory M. Schaaf, Ang Li, Benjamin D. Grant, Prachi Bawaskar, Evan Kleinboehl, Ji Li, Gregory D. Gillispie and David D. Thomas. Red-shifted FRET biosensors for high-throughput lifetime screening. Institutional Research and Academic Career Development Awards (IRACDA) 2018 NIH K12 Conference. July 16, 2018, Atlanta, Georgia. (Outstanding Poster Award).
Tory M. Schaaf, Ang Li, Benjamin D. Grant, Prachi Bawaskar, Evan Kleinboehl, Ji Li, Gregory D. Gillispie and David D. Thomas. Red-shifted fluorescent proteins improve FRET biosensors for high-throughput fluorescence lifetime screening. Biophysical Society 62th Annual Meeting, Feb 19, 2018, San Francisco, California.
Tory M. Schaaf, Kurt C. Peterson, Benjamin D. Grant, David D. Thomas, and Gregory D. Gillispie 2016. Spectral unmixing plate reader for high-precision FRET assays in living cells. Society of Laboratory Automation and Screening 5th Annual Meeting, January 23rd-27th. San Diego, California.
Tory M. Schaaf, Simon J Gruber, Kurt C Peterson, Karl J Petersen, Rocio Foncea, David A Bernlohr, Gregory D. Gillispie, and David D. Thomas. 2014. A two-color non-muscle SERCA FRET sensor for diabetes drug discovery using fluorescence lifetime detection. Biophysical Society 58th Annual Meeting, February 15th-19th, San Francisco, California.