A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales?including those involved in cancer progression and migration?requires new imaging tools. The long-term goal of our work is to develop general strategies for macroscopic, multi-cell tracking in living organisms. The objective of this application is to engineer novel bioluminescent tools for sensitive, multi-cellular imaging in vivo. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging use the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. Over the previous granting period, we demonstrated that the substrate-binding interface of firefly luciferase can be re- engineered to generate panels of mutant enzymes that accept chemically distinct luciferins. When mutants and analogs are mixed together, light emission is produced only when complementary enzyme-substrate partners interact. Several pairs of orthogonal enzymes and substrates were identified, but they remain weak emitters and not suitable for sensitive imaging in vivo. Our central hypothesis is that improved orthogonal imaging tools can be generated using a combination of rational design and screening. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Identify the molecular determinants of orthogonality for lead pair optimization; 2) Generate orthogonal probes with improved tissue penetrance; and 3) Image tumor heterogeneity with expanded orthogonal toolsets. Under the first aim, we will use crystallography and deep-sequencing analyses to examine enzyme-substrate interactions responsible for orthogonality. These insights will be used to optimize existing orthogonal luciferase-luciferin pairs. In the second aim, we will prepare bioluminescent tools with red-shifted emission spectra. These tools will provide more sensitive imaging in vivo. In the third aim, the enzyme-substrate pairs will be used to address the roles of distinct cellular subsets in heterogeneous tumor models. Methods to rapidly differentiate the orthogonal probes in vivo will also be developed. Our approach is highly innovative, as it combines a unique blend of chemical, biological, and computational techniques to fill a long-standing void in imaging capabilities. The proposed research is significant, as the bioluminescent tools will enable the direct interrogation of cellular networks not currently possible with existing toolsets. Such studies will provide some of the first macroscopic images of tumor heterogeneity and may fundamentally change existing views on cancer progression. Additionally, similar to other imaging technologies, the bioluminescent probes will likely inspire new discoveries in a broad spectrum of fields.

Public Health Relevance

The proposed research is relevant to public health because the development of new macroscopic imaging probes is expected to increase understanding of cellular networks in vivo and their changes in cancer progression. Such tools could guide the development of new classes of therapeutics and diagnostics. Thus, the proposed research is relevant to the NIH's mission that pertains to gaining fundamental knowledge about the nature of living systems and reducing the burdens of disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM107630-07
Application #
9670120
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fabian, Miles
Project Start
2013-07-01
Project End
2022-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
7
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92617
Zhang, Brendan S; Jones, Krysten A; McCutcheon, David C et al. (2018) Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging. Chembiochem 19:470-477
Liu, Mira D; Warner, Elliot A; Morrissey, Charlotte E et al. (2018) Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases. Biochemistry 57:663-671
Yao, Zi; Zhang, Brendan S; Prescher, Jennifer A (2018) Advances in bioluminescence imaging: new probes from old recipes. Curr Opin Chem Biol 45:148-156
Jones, Krysten A; Porterfield, William B; Rathbun, Colin M et al. (2017) Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging. J Am Chem Soc 139:2351-2358
Rathbun, Colin M; Porterfield, William B; Jones, Krysten A et al. (2017) Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools. ACS Cent Sci 3:1254-1261
Steinhardt, Rachel C; Rathbun, Colin M; Krull, Brandon T et al. (2017) Brominated Luciferins Are Versatile Bioluminescent Probes. Chembiochem 18:96-100
Rathbun, Colin M; Prescher, Jennifer A (2017) Bioluminescent Probes for Imaging Biology beyond the Culture Dish. Biochemistry 56:5178-5184
Steinhardt, Rachel C; O'Neill, Jessica M; Rathbun, Colin M et al. (2016) Design and Synthesis of an Alkynyl Luciferin Analogue for Bioluminescence Imaging. Chemistry 22:3671-5
McCutcheon, David C; Porterfield, William B; Prescher, Jennifer A (2015) Rapid and scalable assembly of firefly luciferase substrates. Org Biomol Chem 13:2117-21
Evans, Melanie S; Chaurette, Joanna P; Adams Jr, Spencer T et al. (2014) A synthetic luciferin improves bioluminescence imaging in live mice. Nat Methods 11:393-5

Showing the most recent 10 out of 11 publications