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Chemical Biology, Enzymology and Spectroscopy
Research in the Tonge Group is focused on understanding how proteins control and modulate the properties of small molecule ligands. We are interested in understanding the fundamental aspects of enzyme catalysis and in determining how enzymes cause and stabilize charge rearrangement. Some of the enzymes we study are drug targets in pathogens such as Mycobacterium tuberculosis, Francisella tularensis, Burkholderia pseudomallei and methicillin-resistant Staphylococcus aureus (MRSA). We use mechanistic information to design and synthesize high affinity enzyme inhibitors that have long residence times on their targets based on the knowledge that drug-target residence time is a critcal factor for in vivo antibacterial activity. The long residence time inhibitors are also being used to image bacterial populations in humans using positron emission tomography. In addition to enzymes, we are also interested in understanding how fluorescent proteins, such as green fluorescent protein (GFP), control the formation and fluorescence of the embedded chromophore. Studies on GFP are now being extended to other light activated proteins such as AppA, a BLUF domain antirepressor from the photosynthetic bacterium Rhodobacter sphaeroides.
Microbial Enzyme Drug Targets: Residence Time and
In Vivo Activity
An area of research that has grown out of an interest in enzyme mechanisms involves our efforts to develop inhibitors of known or putative drug targets. These studies are focused on pathogens such as Mycobacterium tuberculosis (MTB), Francisella tularensis and S. aureus and include enzymes from pathways involved in bacterial fatty acid biosynthesis as well as the biosynthesis of the electron carrier menaquinone.
Fatty Acid Biosynthesis
Bacterial fatty acid biosynthesis is a validated target for drug discovery and our primary focus is on the enoyl reductase enzyme from this pathway (FabI). We hypothesize that high affinity inhibition of the FabI enzyme class is coupled to ordering of a loop of amino acids close to the active site slow onset inhibitors bind to the enzyme. Using structure-based approaches we developed a series of diphenyl ether inhibitors of InhA, the FabI enzyme from MTB. The most potent first generation compound has a Ki value of 1 nM for InhA and MIC90 values of 1-2 μg/mL against sensitive and INH resistant strains of MTB (Sullivan et al. (2006) ACS. Chem. Biol. 1, 43-53). Selected
diphenyl ethers are also slow-onset nM inhibitors of the FabI enzyme from Francisella tularensis (ftuFabI), inhibit bacterial growth with MIC90 values of ~0.1 μg/ml
and have antibacterial activity in an animal model of tularemia (Lu et al.
(2009) ACS Chem. Biol. 4, 221-231).
Importantly, the in vivo antibacterial
activity of the compounds correlates with their residence time on the target
rather than with their thermodynamic affinity for ftuFabI.
Future Directions: We are now expanding the chemical diversity of our compound libraries to improve the ADME properties of the inhibitors. This project requires close collaboration between compound design and synthesis, and studies involving pharmacokinetics, pharmacodynamics and compound evaluation in animal models of TB infection. In addition, we are focusing our efforts to probe interactions within the cell that are critical for enzyme and inhibitor activity. This will involve mass spectrometry and the dissection of protein-protein interactions using chemical tools. We are also expanding our inhibitor discovery efforts to other enzymes and we plan to screen focused chemical libraries using transferred NOE NMR spectroscopy to obtain inter-ligand NOEs .
β-Ketoacyl-ACP Synthases
We are extending our
antibacterial discovery efforts to other targets in the FAS-II pathway
including the β-ketoacyl-ACP synthases that catalyze the Claisen condensation
of malonyl-ACP with the growing fatty acid. We have demonstrated that the natural product
thiolactomycin (TLM) is a slow-onset inhibitor of KasA, the β-ketoacyl-AcpM
synthase in the M. tuberculosis FAS-II
pathway. TLM binds more tightly to the
acyl-enzyme intermediate formed during the Claisen condensation catalyzed by
KasA. We have determined X-ray
structures of KasA-TLM inhibitor complexes and have elucidated the structural
basis for the very long chain substrate specificity of the enzyme. This information is being used to rationally
design novel TLM analogues with improved antibacterial activity. We are also using interligand NOE NMR to
drive the fragment-based assembly of novel inhibitors.
Menaquinone Biosynthesis
Menaquinone (MK) is the sole quinone in the
mycobacterial electron transport chain.
Enzymes involved in MK biosynthesis are promising drug targets since the
pathway is absent in humans and also because compounds that affect respiration
may be active against latent MTB populations.
We have initiated a coordinated series of activities to investigate the
importance of this pathway including cloning, expressing and characterizing the
putative TB men enzymes, as well as studying MK biosynthesis using mass
spectrometry to follow the incorporation of isotopically labeled precursors
into MK.
Light Activated Proteins
Interests in utilizing spectroscopic methods to elucidate the precisedetails of enzyme catalyzed reactions have expanded in several directions. The ability of enzymes to promote catalysis through noncovalent interactions has important parallels with the control of photophysical properties exerted by the green fluorescent protein on the embedded chromophore. Since optical and structural events in GFP occur on a very fast time scale following light absorption, our steady state vibrational methods have been supplemented with ultrafast time resolved infrared spectroscopy (TRIR). A highlight of this work was the use of TRIR to obtain direct proof that the excited state proton transfer (ESPT) reaction in GFP results in the protonation of a glutamate close to the chromophore (Stoner-Ma et al. (2005) J. Am. Chem. Soc., 127, 2864-5).
Time resolved studies on GFP are now being expanded to other light activated molecules including the BLUF (Blue Light Receptor Using FAD) protein AppA, an antirepressor from Rhodobacter sphaeroides. Light absorption by the AppA flavin chromophore causes subtle changes in chromophore-protein interactions that result in dissociation of AppA from the transcriptional repressor PpsR and the subsequent down regulation of photosystem biosynthesis. Using TRIR we are studying how formation of the FAD excitation causes structural changes to the protein matrix that are thought to include rotation of a glutamine side chain that is hydrogen bonded to the chromophore (Stelling et al. (2007) J. Am. Chem. Soc., 129,
15556-64).
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