Research in the Tonge laboratory is focused on three major areas that explore the role of Time in biology: (i) the mechanism of drug action, (ii) photoreceptor biophysics and biology, and (iii) PET imaging. We design and synthesize inhibitors of enzyme drug targets involved in diseases such as cancer and infection, and use techniques such as pharmacokinetic/pharmacodynamic (PK/PD) modeling, mass spectrometry and positron emission tomography (PET) to explore the role of drug-target binding kinetics in drug activity at the cellular and whole organism level. We also use biophysical methods such as ultrafast spectroscopy coupled with site-specific protein modification to understand the mechanism of photoreceptor activation as a prelude to the development of optogenetic devices. In addition to exploring the role of drug action using PET imaging, we also develop radiotracers to detect and localize bacterial infection in humans.
The Mechanism of Drug Action
Time-dependent enzyme inhibitors are of particular interest in drug discovery programs since the rate of complex dissociation (koff) can be slower than the time scale of in vivo drug elimination, leading to sustained target occupancy at low drug concentration, enabling dosing frequency and exposure to be reduced and thus increasing the therapeutic window.1, 2 We have developed mechanistic PK/PD models that enable the kinetic parameters for time-dependent enzyme inhibition to be used to predict in vivo drug efficacy.3-5 To inform and interrogate the PK/PD model, we are developing compounds with altered residence times on their targets and quantifying the molecular factors that modulate the coupling of time-dependent enzyme inhibition to prolonged drug activity following compound washout. This analysis provides direct insight into target vulnerability. In addition, we are developing positron emission tomography radiotracers to quantify in vivo target occupancy and assist in translating our discoveries into humans. The ability to accurately quantify target engagement as a function of time and drug concentration is expected to dramatically improve the prediction of in vivo drug activity across all therapeutic areas. Targets currently being studied include kinases involve in cancer and inflammation, and enzymes that synthesize essential components of bacterial cell membranes.
Photoreceptor Biophysics and Biology
Organisms sense and respond to light using photoactive proteins that contain an embedded chromophore. The focus of our program is on the blue-light using FAD (BLUF) and the light oxygen voltage (LOV) domain photoreceptors, and photoswitchable fluorescent protein such as Dronpa. Both photoswitchable fluorescent proteins and photoreceptors have unique and critical applications in dissecting biology and in rationally controlling biological function. Photochromic fluorescent proteins can be reversibly switched between bright (on) and dark (off) states using light and play a fundamental role in live cell imaging, while photoreceptors control a wide-variety of biological processes by converting light into protein structural change, and have applications in optogenetics. In each case, elucidating the underlying mechanism of operation remains a central challenge to drive the development of novel optogenetic tools and for their deployment in applications such as super-resolution microscopy. We are using transient infrared (IR) fs to ms time resolution spectroscopy (TRMPS),6 coupled with the site-specific incorporation of unnatural amino acid reporters to determine how ultrafast structural changes on the sub-ps time scale induced by light absorption, lead to macromolecular reorganization and photoprotein activation on the ms-s time scale: i.e. over more than 10 decades of time. The goal of this project is define the reaction coordinate for photoreceptor function in both time and space, and identify specific residues that can be altered to manipulate photoreceptor output, a key challenge for optogenetic applications. Recent publications on BLUF,7, 8 and LOV domain photoreceptors,9, 10 and Dronpa,11 are included below.
Imaging Bacterial Pathogens In Vivo
Bacterial infections such as those of prosthetic joints, bones (osteomyelitis) and heart valves (infective endocarditis) are difficult to difficult to diagnose and treat, and are a major cause of mortality, morbidity and health care costs. We are developing positron emission tomography (PET) radiotracers that can be used for non-invasive PET imaging to detect and localize bacterial pathogens in humans. Such radiotracers will distinguish between different pathogen populations, serve as non-invasive diagnostics, and inform on bacterial load during chemotherapy, thereby identifying and improving treatment of patients with infectious diseases. Our 1st generation novel PET tracer, [18F]F-PABA, can distinguish Staphylococcus aureus infection from inflammation,12 unlike FDG which is currently used as a diagnostic tool. Second generation tracers are under development that show improved uptake and sensitivity.
References for the Research Section
1. Tonge, P. J. (2018) Drug-Target Kinetics in Drug Discovery, ACS Chem Neurosci 9, 29-39.
2. Lu, H., Iuliano, J. N., and Tonge, P. J. (2018) Structure-kinetic relationships that control the residence time of drug-target complexes: insights from molecular structure and dynamics, Curr Opin Chem Biol 44, 101-109.
3. Walkup, G. K., You, Z., Ross, P. L., Allen, E. K., Daryaee, F., Hale, M. R., O’Donnell, J., Ehmann, D. E., Schuck, V. J., Buurman, E. T., Choy, A. L., Hajec, L., Murphy-Benenato, K., Marone, V., Patey, S. A., Grosser, L. A., Johnstone, M., Walker, S. G., Tonge, P. J., and Fisher, S. L. (2015) Translating slow-binding inhibition kinetics into cellular and in vivo effects, Nat Chem Biol 11, 416-423.
4. Daryaee, F., Chang, A., Schiebel, J., Lu, Y., Zhang, Z., Kapilashrami, K., Walker, S. G., Kisker, C., Sotriffer, C. A., Fisher, S. L., and Tonge, P. J. (2016) Correlating Drug-Target Kinetics and In vivo Pharmacodynamics: Long Residence Time Inhibitors of the FabI Enoyl-ACP Reductase, Chem Sci 7, 5945-5954.
5. Daryaee, F., Zhang, Z., Gogarty, K. R., Li, Y., Merino, J., Fisher, S. L., and Tonge, P. J. (2017) A quantitative mechanistic PK/PD model directly connects Btk target engagement and in vivo efficacy, Chem Sci 8, 3434-3443.
6. Brust, R., Lukacs, A., Haigney, A., Addison, K., Gil, A., Towrie, M., Clark, I. P., Greetham, G. M., Tonge, P. J., and Meech, S. R. (2013) Proteins in action: femtosecond to millisecond structural dynamics of a photoactive flavoprotein, J Am Chem Soc 135, 16168-16174.
7. Gil, A. A., Haigney, A., Laptenok, S. P., Brust, R., Lukacs, A., Iuliano, J. N., Jeng, J., Melief, E. H., Zhao, R. K., Yoon, E., Clark, I. P., Towrie, M., Greetham, G. M., Ng, A., Truglio, J. J., French, J. B., Meech, S. R., and Tonge, P. J. (2016) Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa on the Forward and Reverse Ground-State Reactions, J Am Chem Soc 138, 926-935.
8. Gil, A. A., Laptenok, S. P., Iuliano, J. N., Lukacs, A., Verma, A., Hall, C. R., Yoon, G. E., Brust, R., Greetham, G. M., Towrie, M., French, J. B., Meech, S. R., and Tonge, P. J. (2017) Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues, J Am Chem Soc 139, 14638-14648.
9. Gil, A. A., Laptenok, S. P., French, J. B., Iuliano, J. N., Lukacs, A., Hall, C. R., Sazanovich, I. V., Greetham, G. M., Bacher, A., Illarionov, B., Fischer, M., Tonge, P. J., and Meech, S. R. (2017) Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain, J Phys Chem B 121, 1010-1019.
10. Iuliano, J. N., Gil, A. A., Laptenok, S. P., Hall, C. R., Tolentino Collado, J., Lukacs, A., Hag Ahmed, S. A., Abyad, J., Daryaee, T., Greetham, G. M., Sazanovich, I. V., Illarionov, B., Bacher, A., Fischer, M., Towrie, M., French, J. B., Meech, S. R., and Tonge, P. J. (2018) Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy, Biochemistry 57, 620-630.
11. Laptenok, S. P., Gil, A. A., Hall, C. R., Lukacs, A., Iuliano, J. N., Jones, G. A., Greetham, G. M., Donaldson, P., Miyawaki, A., Tonge, P. J., and Meech, S. R. (2018) Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa, Nat Chem 10, 845-852.
12. Zhang, Z., Ordonez, A. A., Wang, H., Li, Y., Gogarty, K. R., Weinstein, E. A., Daryaee, F., Merino, J., Yoon, G. E., Kalinda, A. S., Mease, R. C., Iuliano, J. N., Smith-Jones, P. M., Jain, S. K., and Tonge, P. J. (2018) Positron Emission Tomography Imaging with 2-[(18)F]F- p-Aminobenzoic Acid Detects Staphylococcus aureus Infections and Monitors Drug Response, ACS Infect Dis 4, 1635-1644.