Bioorthogonal imaging of therapeutic B-Raf inhibition

B-Raf is a protein kinase that is part of the MAP kinase signal transduction cascade that controls cell proliferation and survival. Due to the incidence of a specific genetic alteration of the corresponding oncogene, mutant B-Raf is expressed having an 800-fold increased kinase activity leading to uncontrolled proliferation and growth of tumor cells. This B-Raf mutation is most frequently observed in human melanoma. About 50% of all cases of this aggressive form of skin-cancer are related to mutant B-Raf, causing a disproportionate number of deaths worldwide. Recently, several compounds have been identified as potent and selective mutant B- Raf inhibitors already leading to chemotherapeutic applications. In the course of the proposed project the development of a bioorthogonal approach for molecular imaging of the protein kinase inhibition by the most promising mutant B-Raf targeting drugs, vemurafenib and dabrafenib, is intended. Based on the structural scaffolds of these compounds, so called bioorthogonally imaging drugs will be prepared by total synthesis of strained- cycloalkene-labeled drug derivatives. These compounds can be localized in vitro and in vivo via highly selective bioorthogonal reaction with fluorogenic tetrazines leading to the intended development of imaging probes that have properties close to the parent drugs. After the preparation of several prospective bioorthogonally imaging drugs by introducing various strained cycloalkenes, a small library of these compounds will be investigated by in vitro methods and cell imaging, including co-localization studies using antibody co-staining to assess the capability for selective imaging of mutant B-Raf. A novel bioorthogonal proteomics approach will be applied for target identification studies to examine polypharmacologic properties of these compounds also leading to valuable conclusions for the parent drugs. Lead agents will then be selected for in vivo (intravital) imaging at subcellular resolution using turn-on probes and novel fluorescence microscopy techniques. Overall, these investigations should lead to the development of a reliable approach for the imaging of therapeutic B-Raf inhibition and a better understanding of drug pharmacokinetics at the single cell level, finally to get insight into the underlying mechanisms of this emerging cancer related research topic.

B-Raf is an enzyme that is part of an important signal transduction cascade that controls cell growth and survival. Due to the incidence of a specific genetic alteration of the corresponding oncogene, mutant B-Raf is produced in the cell having an 800-fold increased activity leading to uncontrolled growth of tumor cells. This B-Raf mutation is most frequently observed in human melanoma. About 50% of all cases of this aggressive form of skin-cancer are related to mutant B-Raf, causing a disproportionate number of deaths worldwide. The major goal of this project was the development of (bioorthogonal) imaging agents for the visualization of therapeutic B-Raf inhibition based on the structural scaffolds of the two FDA-approved drugs vemurafenib and dabrafenib. Modifiable analogs of these two compounds have been synthesized enabling further modification without significantly altering the binding to the target. Different fluorophores have been attached to afford imaging agents for fluorescence microscopy. A BODIPY-derivative of vemurafenib was successfully used for in vivo imaging in xenografted mice and a bioorthogonal derivative of dabrafenib could be used for imaging and target identification studies. Such a bioorthogonal analog bears a stable bioinert functional group that does not interfere with the binding of the drug to its target, but can be used to perform in vivo click chemistry after the binding event in living cells. Labeling with so called smart probes or turn-on probes (fluorogenic imaging agents that will only be fluorescent after the click reaction) can be used for imaging without washing, thus significantly shortening the time for image acquisition after adding the imaging agent and thus preventing loss of signal due to dissociation of non-permanently binding drugs. The availability of a complementary system (directly labeled vs. bioorthogonal drug derivatives) enabled studying the drug uptake in resistant melanoma cells.Furthermore, we used the bioorthogonal strategy in combination with click-activatable pro-drugs within a proof-of-concept study to show pretargeted bioorthogonal pro-drug activation for the treatment of resistant cells. Bioorthogonal elimination or click-to-release chemistry was used to design such click-activatable pro-drugs, which basically are drugs protected with a bioorthogonal moiety that is cleaved upon reaction with its bioorthogonal counterpart. Overall, we have been able to develop fluorescently and bioorthogonally labeled imaging agents to study the drug uptake in sensitive as well as resistant melanoma cells (in cells and in vivo), and to use the bioorthogonal two-step strategy for a selective activation of pro-drugs applying click-to-release chemistry. The developed tools and methods are expected to enable further insight into therapeutic B-Raf inhibition and targeted pro-drug activation potentially leading to improved strategies for diagnostics and therapy.