Abstract: In the traditional paradigm of pharmacology, diseases arising from excess protein function are treated through potent and selective inhibitors. In contrast, diseases originating from lack of protein function are refractory to this approach and present a tremendous unmet medical need. In the Burke lab, we propose that small molecules can imperfectly mimic missing proteins, acting as a prosthesis on the molecular scale. We have identified hinokitiol, a tropolone natural product capable of replacing protein iron transporters. In order to understand hinokitiol’s activity and enable the development of a mobilization-optimized therapeutic, we seek to understand the atomistic underpinnings that govern its transport activity through establishment of structure activity relationships (SAR). We synthesized an array of tropolones with alkyl substituents with different length, branching patterns, and isotopes to systematically analyze the impact of both lipophilicity and substituent constitution. We have also shown that other iron chelators can be engineered for iron mobilization, following the same rules we uncovered for tropolones. Using our synthetic route, we have synthesized site-selective 13C-tropolones, which will serve as mechanistic probes in solid-state NMR paramagnetic relaxation enhancement (PRE) experiments. The understanding gained from our SAR studies will allow for rational design of a hinokitiol-inspired derivative with optimal iron mobilization.
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Congratulations on such fantastic research! This was a great talk and so much impressive work done. I am curious about the binding of the iron to hinokitiol… you show in your slides that the iron is bound by the carbonyl and the alcohol. Do you plan to make any derivatives at those two positions to prove that the iron is binding there and to see if derivatives have increased/decreased activity?
Hi Shelby, thank you for your question!
We have proven that iron binds to the carbonyl and alcohol oxygens by obtaining a crystal structure of the hinokitiol-iron complex. In addition, we have previously synthesized a derivative of hinokitiol with the alcohol group replaced by a proton, and this derivative showed no iron mobilization activity.
Nice talk Ruby! I have a few questions about your liposome assay.
1. How did you quantify the concentration of iron outside of the liposome?
2. Did you verify that the liposome was intact? (ie, how do we know that the iron you’re detecting outside of the liposome isn’t just from liposomes that ruptured?)
3. Why do you think higher concentrations of the small molecule ultimately lead to a dropoff in iron transport? I would expect the iron transport to simply level off at higher concentrations rather than dropping. Do you think the small molecules are potentially aggregating in solution at higher concentrations?
4. Assuming I’m reading your data properly, there doesn’t appear to be very much iron transported out of the liposomes (15 mM inside to 0.3 mM outside). Is your assay performed under dilute conditions, or is only a small amount of iron being transported? And do you think that’s enough to be relevant in a biological system?
Again, really nice talk!
Hi Philip, thank you so much for these questions!
1. The concentration of iron was quantified using a dye ferrozine that binds to iron.
2. Liposomes are typically very stable. Also as you mentioned in your 4th question, the transported amount of iron is rather small compared to the total amount we put into the liposome. If the liposomes are ruptured, I would expect the amount of iron outside of the liposome to be more significant. Moreover, we know that liposomes can be broken by detergent. If the iron we see outside of the liposome results from the lipid being ruptured, I would expect the longer chain derivatives (more detergent-like) to have higher activity, which contradicts our observation. For these reasons I believe we are seeing actual iron mobilization and not liposome rupturing.
3. Yes, just like you mentioned, we currently think that aggregation of the small molecules lead to lower activity, and even the toxicity. We have obtained some preliminary results visualizing the Hino-iron aggregates using TEM that shows larger aggregates form at higher concentrations of the small molecules.
4. I believe this result indicates a rather small amount of iron is being transported, but I think it is relevant in a biological system. The iron transported will be uptaken by other parts of the cell that need the iron, which will drive more iron transport to reach equilibrium of the iron concentration on both sides of the membrane.
You have synthesized many compounds using a variety of synthetic reactions. What were the yields of your reactions (a range is fine or the yields of the most relevant reaction steps)
Also, what methods did you use to determine the identity of your synthetic products. Any special methods used for the isotopically labeled compounds (Deuterated and 13C labeled)
Hi Anton, thank you very much for the questions!
The yield to get from cyclohexadienes to the promo-tropolone building blocks, over 4 steps, is around 10%, and the yields for the coupling reactions followed by methyl group deprotections are around the range of 10%-30%, depending on the alkyl substituent we are attaching.
All the synthetic products have been characterized with 1H and 13C NMR, in addition to high and low resolution mass spectrometry.
For the 13C labeled compounds, we simply run 13C NMR on the sample, and the isotopic label can be identified as an enhanced peak in the spectrum as compared to other signals (natural abundance of 13C is only 1.1%). The identities of the intermediates in which a proton is directly attached to the 13C carbon were also confirmed through 1H NMR, where 13C-1H coupling can be observed. All the intermediates and products have also been characterized with mass spectrometry.
On the other hand, deuterated derivatives required more special characterization. To identify the deuterium peaks, we took deuterium NMR spectra for these compounds. In their 13C NMR spectra, the 13C peaks are split by the couplings with deuteriums, and the structure assignments can also be corresponded with these splitting patterns. In addition, to better resolve these 13C peaks, we were able to characterize all deuterated compounds with simultaneously proton and deuteron decoupled 13C NMR with the help of Dr. Zhu in the NMR lab.
Hope this answers your questions!