Metalloprotein scaffolds are attractive candidates for MRI contrast agents because the interaction of paramagnetic metals with water molecules within the active site provides an easy read out for MRI. The protein scaffold itself is highly tunable and can be engineered to bind reversibly and specifically to one target in the presence of many structurally related compounds. However, the current methods developed using this strategy is limited by the inherent relaxivity of the agents. As improved relaxivity corresponds to better signal and resolution and may increase the scope of small molecule targets available for imaging, engineering protein sensors with higher relaxivity is important for realizing the full potential of this approach. Our proposal addresses this goal. Water exchange rate and innate spin of the metal both have profound effects on the relaxivity of the contrast agent and we will target both of these avenues for exploration. The cytochrome p450 BM3h platform has proved to be highly tunable and amenable to the directed evolution approach and we will construct sensors from this scaffold. Preliminary studies in the Arnold lab have identified mutation can lead to better flexibility and improved water access to the binding site. We believe that a targeted screen of specific active site residues via iterative saturation mutagenesis will lead to improvements in relaxivity of Mn(III) based sensors. As a complementary approach, we will also synthesize gadolinium(III) porphyrins for incorporation into BM3h. In both cases, directed evolution will be used to produce sensors with good thermostability, improved relaxivity, and high specificity for a neurochemical target like dopamine or norepinephrine. The sensors will be fully characterized by absorbance, mass spectrometry, MRI measurements, and other techniques previously used.
The ability to monitor neurochemical fluctuations in the brain in real time is essential to the next generation of diagnostic medicine and treatment neurological disease yet very few imaging techniques are capable of this feat. The development of MRI contrast agents that can selectively bind one molecule and relay the binding event via changes in MRI signal is a promising avenue of research toward this goal. By engineering protein-based sensors with good target selectivity and higher innate relaxivity, we will improve the signal and resolution of existing agents, which will allow us to examine small changes in concentrations of important signaling molecules like dopamine, serotonin and norepinephrine.
|Wang, Z Jane; Peck, Nicole E; Renata, Hans et al. (2014) Cytochrome P450-Catalyzed Insertion of Carbenoids into N-H Bonds. Chem Sci 5:598-601|
|Renata, Hans; Wang, Z Jane; Kitto, Rebekah Z et al. (2014) P450-catalyzed asymmetric cyclopropanation of electron-deficient olefins under aerobic conditions. Catal Sci Technol 4:3640-3643|
|Farwell, Christopher C; McIntosh, John A; Hyster, Todd K et al. (2014) Enantioselective imidation of sulfides via enzyme-catalyzed intermolecular nitrogen-atom transfer. J Am Chem Soc 136:8766-71|
|Wang, Z Jane; Renata, Hans; Peck, Nicole E et al. (2014) Improved cyclopropanation activity of histidine-ligated cytochrome?P450 enables the enantioselective formal synthesis of levomilnacipran. Angew Chem Int Ed Engl 53:6810-3|
|McIntosh, John A; Coelho, Pedro S; Farwell, Christopher C et al. (2013) Enantioselective intramolecular C-H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew Chem Int Ed Engl 52:9309-12|