NMR in Drug Discovery: Uncovering Nature’s Potential Pharmaceuticals

Nature is a rich source of diverse plants and fungi, each possessing unique abilities and properties. One of the foremost applications of these plants is in the field of pharmaceuticals. NMR spectroscopy combined with techniques as HPLC can help today’s scientists discover the structure of these natural substances and isolate the most effective of them to be used for development of pharmaceuticals. Dan Stærk is a scientist travelling the world to find unique plants in nature and uncover how they can cure some of the diseases ravaging the world.

Dan Stærk obtained his degree in molecular biology and biotechnology at the University of Southern Denmark. After completing his thesis, he earned a PhD in the chemistry of natural substances at the Danish University of Pharmaceutical Sciences.

Dan soon after joined the Faculty of Health and Medical Sciences at UCPH as an assistant professor and quickly advanced to the position of associate professor. In 2008, he got his first professorship at the Faculty of Life Sciences (LIFE). In 2012, Dan returned to the Faculty of Health and Medical Sciences as professor and was recently appointed head of the Department of Drug Design and Pharmacology.

Unfortunately there is also downsides of leading departments as Dan explains.
As head of a department I have less than 20% academic time, and therefore spend less time on research than I would like to.

Dan’s research focuses on the biologically active and especially pharmacologically active substances in complex extracts of plants and microorganisms. He is searching for substances that binds and act through a receptor or an enzyme in the body and therefore have the capability to potentially cure diseases.

There are several different ways of finding new substances in the chemical landscape, but there is no place with such a diverse chemical and pharmacological abundance as nature. This is the reason why Dan has his eyes set on nature. If you see many of the substances isolated from nature, they are so complex that a synthetic chemist would never be able to synthesize them.

If you look at the chemical space of natural substances, it is the result of millions of years of evolution optimizing the substances to fit the particular receptors and enzymes that they target. This makes Nature the place to go, if you want to discover novel pharmacologically active substances.

In Dan’s group they are mostly interested in substances that can become either new antibiotics, future cancer pharmaceuticals, or drugs to manage type 2 diabetes.

People often discuss how we prepare for the next pandemic after corona, but we are already in the middle of it: the development of antimicrobial resistance (AMR). This AMR pandemic is not as fast as Covid-19, but fighting antimicrobial resistance is much more complex and if we don’t act now, we will run out of antibiotics to treat even simple bacterial infections.” Dan emphazises.

One of the unique aspects of how Dan’s group uses NMR is their focus on analyzing complex mixtures. One way in which Dan and his group use a NMR-based instrumental platform, where they combine NMR with HPLC and pharmacological characterization techniques.

From this instrumental platform, they obtain what they call bioactivity-mapped chemical space. Thus, when data from the above three analytical technologies are combined, it forms a 3D bioactivity-mapped chemical space. The first axis represents spectral data (as for example NMR spectra), the second axis represents the pharmacological data, and the axis represents the chromatographic separation of the substances. So with one analysis, they can see the structure, the pharmacological data and how the compound are separated from each other, all at once.

The above-mentioned chromatographic separation is done by analytical-scale HPLC, a powerful technique used to separate components in mixtures. Typically, when employing HPLC, researchers utilize a detection method like UV-light or photodiode array detection (PDA).

Instead of using conventional detection methods, we opt for high-resolution mass spectrometry combined with a well-established liquid chromatography method (LCHRMS). Coupling NMR spectroscopy to this technique is relatively rare since the sensitivity of NMR is much lower than MS and UV.

However, NMR provides highly valuable structural information that MS or UV does not, and since they are investigating nature for its potential pharmaceuticals with active substances as complex mixtures, the structure is a vital piece of information. Therefore, it makes sense to use NMR instead of other techniques.

In the complex extracts, that Dan’s group studies, there is a huge number of different substances, which is why they need to separate the individual compounds and couple them to an array of different analytical techniques. First, they fraction the substances using HPLC and then they directly record and analyze UV, MS, and NMR spectra of all of them at once. This technique is called HPLC-PDA-HRMS-SPE-NMR. This is coupled with a technique called high-resolution inhibition profiling, where all the fractions from HPLC-based micro fractionation are screened for biological activity, creating a bio chromatogram which can pinpoint the active compounds ensuring that they only investigate those substances that have the desired activity.

In a cup of coffee for example, there are hundreds of different molecules, but only a few of these are biologically active and by using this combined method we can separate the biological active from the non-active components.” Dan adds.

Dan has been using these techniques on plants from all over the world including the Nordic countries, South America, Africa, and Asia, but one of his recent collaborations has been with a group in Australia.

When working in other countries, they study plants that traditionally have been used for medicinal purposes in these countries, so they have a starting point to work from. One example is a plant family consisting of more than 200 species, which are only found in Australia. These plants contain a specific kind of chemical substances found almost exclusively in these plants, making it a source of potential new medicine and an attractive area for research collaboration.

Fungi are another source of pharmaceuticals that Dan and his group has focused on. They isolate the fungi from plants and then grow them in the laboratory, investigating what bioactive substances the fungi can produce.

The fungi can then be used as cell factories to produce medical compounds sustainably, effectively, and in a smaller space compared to agriculture or greenhouses.

Another part of his work involves looking at already established pharmaceuticals and do a quality control by using metabolomics. In this way they investigate if the drugs on the market can be trusted and if they are what they are supposed to be.
It turns out that many pharmaceuticals often do not contain what is promised, which shows why our research is so important.” Dan explains.

We have also investigated areas in the world, where there is no access to Western medicine. An example is one of his PhD students, who has been to Iran where they are struggling with the disease called leishmaniasis.” Dan adds.

Leishmaniasis is like malaria with the key difference being that it is not mosquitoes but sand flies that sting the victims. One of the variations of leishmaniasis creates huge sores on the body and face.
It is normally not lethal, but people get incapacitated and cast out from society because of the disease. The only available pharmaceutical is so toxic that there is a high risk that it kills the patient rather than the parasite, which shows that there is a huge unmet need for better drugs.

When Dan’s PhD student travelled to Iran, he interviewed the local medicine men who have found a plant with potential for curing the disease. The method they use involves grinding the plant, mixing it with wax, and then wrapping it around the wounds with a band.

Wax is a very oily substance. They investigated the plant and found that there were some substances in it that could kill the leishmania parasite. These substances dissolved well in the oily wax, supporting the used methodology of the medicine men. Unfortunately, since the leishmania parasite is not an issue in the western countries, there was no interest from investors to develop a pharmaceutical, even though a better cure was obviously lacking.

One of the great strengths of Danish NMR is the information exchange between the different groups using NMR in the various ways. The Danish NMR groups are complementary in their research interest, ranging from protein structure determination, solid-state NMR, and metabolomics in food to working with small molecules in complex mixtures.” Dan explains

Those who are NMR managers are also good at helping each other when problems arise. They can exchange knowledge on how to solve problems that others have already experienced or are struggling with now. They can also exchange of hardware if someone has an item someone else needs for their instrument.” Dan emphazises.

It is also important regarding larger initiatives to stand together; all the larger facilities only exist because Danish NMR do it together. In our group, we don’t use the larger center facilities, so it is important to have decentralized facilities as well, since the way we use our equipment here doesn’t always harmonize with the way the equipment is set up in larger facilities.” Dan ends.

Written by: Jonatan Emil Svendsen

NMR in Drug Discovery: Uncovering Nature’s Potential Pharmaceuticals

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