BY ROHAN BADLANI
Targeting molecules with fluorescent probes enables scientists to investigate a wide variety of phenomena in the human body at the molecular level. This important area of bioorganic chemistry is currently being investigated by Dr. James Wilson, an assistant professor in the chemistry department at the University of Miami. Wilson’s projects focus on developing fluorescent analogs of biomolecules along with probes to target DNA, transmembrane proteins, receptors and enzymes. Through the use of combinatorial synthesis, a technique allowing an investigator to essentially cast a wide net and test a number of molecules for their compatibility with target structures, he is able to create dyes that target specific macromolecules.
Of note is his research on the norepinephrine transporter (NET), which is a sodium-neurotransmitter symporter that binds to norepinephrine and other substrates. The results of his study were published in his paper Fluorescent stilbazolium dyes as probes of the norepinephrine transporter: structural insights into substrate binding. The NET is the target of ligands which include recreational drugs such as cocaine along with inhibitors such as desipramine. In this study, the investigative team tested various sterically demanding dyes in order to gain more insight into the binding of substrates to the norepinephrine transporter. A series of bulky stilbazolium dyes, including six newly synthesized compounds, were evaluated to determine the effect of extending the molecular probes’ heads or tails.
To design a probe, Wilson and his team used known substrates for the transporter and assigned a family of related dyes to probe the functional limits of the NET. They then executed timed experiments to evaluate probe binding and displacement of both desipramine and norepinephrine as competitive inhibitors. In the process, the team exposed cells to 1 µM solutions of the stilbazolium dyes. Some factors that were taken into consideration were the structural elements of each dye along with whether the molecule had a “head-first” or “tail-first” orientation.
It was shown through the evaluation of binding and displacement kinetics that the NET is able to bind to dyes that possess significant expansion. However, this was only the case for probes that were expanded along the long axis. Extensions of the N-alkyl pyridinium tails was well tolerated, while expansion of the aryl head, an electron donor, reduces the attachment of the substrate to its binding site. Through the investigation of dimers, it was suggested that a headfirst orientation alone triggered the turn-on emission characteristic of stilbazolium dyes.
Creating biomolecular analogs is no easy task. “Humans are really terrible at designing molecules,” Wilson explains. “You can try to rationally design something … and it just doesn’t work.” To overcome this obstacle, Wilson utilizes the combinatorial strategy of testing a large number of fluorophores with the protein to see which molecules are effective. Interestingly enough, molecules that behave in a manner similar to target molecules may actually look quite different. This was the case with norepinephrine in his study of the norepinephrine transporter.
This type of research has a number of different applications. By creating probes for different molecules, Wilson is able to explore the efficacy of different forms of therapy, such as chemotherapy for cancer patients. Using these probes in marking certain drugs, he is able to see how the medication interacts with the target cells and if it is effective in treatment.
What fuels Wilson’s drive to find the answers to pressing questions is his interest in the subject. He advises, “If you’re going to do well, you have to be obsessed with it. If you’re just looking for all these grant opportunities, you’re not going to do well. It’s about trying to take the stuff you love and do something useful.” However, Rome was not built in a day. Wilson experienced a great change in his outlook from starting out as a graduate student to becoming a postdoc. He elaborates, “Your perspective goes from being task oriented and trying to do what your boss told you, to trying to answer the big questions.”