Halogenated compounds, such as acutumine, can be valuable in medicinal chemistry because of their distinctive chemical appendages, which cause them to react in intriguing ways with other biomolecules, and drug designers can employ them to create compounds that perform specific roles in the body. Halogenation is used in about 20% of medicinal substances nowadays. “Unfortunately, the capacity of chemists to efficiently install halogen atoms in appropriate places for beginning compounds has been extremely limited,” Weng explains.
The majority of natural halogenated compounds are produced by microorganisms such as algae or bacteria, whereas acutumine is the only halogenated substance produced by plants. Although the reaction is time-consuming and expensive (10 mg of manufactured acutumine can cost roughly $2,000), chemists ultimately succeeded in synthesizing the molecule in 2009.
Colin Kim, a Ph.D. student at the Whitehead Institute’s Weng lab, was curious about how these plants completed this difficult process using only their own genetic material. “We reasoned, why not ask how the plants manufacture it and then scale up the reaction [to produce it more efficiently]?” Kim states.
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“By knowing how real organisms, such as the moonseed plant, accomplish chemically difficult halogenation chemistry, we may be able to create new biochemical techniques to produce novel halogenated chemicals for drug discovery,” Weng adds.
Kim was aware that for every halogenated molecule in an organism, there is a halogenate enzyme that catalyzes the reaction that causes the halogen to stick.
Halogenases are useful in the production of pharmaceuticals because a strategically inserted halogen can help fine-tune the bioactivities of diverse medications. So Weng, who is also an associate professor of biology at MIT, and Kim, who led the project, set about identifying the helper molecule responsible for the production of acutumine in moonseed plants.
Discovery for a Cancer Cure
First, the scientists collected three Menispermaceae plant kinds. Acutumine was known to be produced by two of them: common moonseed (Menispermum canadense) and Chinese moonseed (Sinomenium acutum). They also obtained a snake vine (Stephania japonica) plant from the same family, which did not yield the chemical.
They began their inquiry by utilizing mass spectrometry to hunt for acutumine in all three plants and then determine where it was found. They discovered the chemical throughout the first two chapters, as well as some additional ones in the roots of common moonseed. The third plant, snake vine, has none, as expected, and could thus be used as a reference species because it would apparently never develop the gene for the halogenase enzyme.
The researchers then began looking for the gene. They began by sequencing the RNA that was expressed in the plants (RNA acts as a messenger between genomic DNA and functional proteins) and then produced a massive database of RNA classified by tissue.
The additional acutumine in common moonseed roots came in handy at this stage. Previous studies on other halogenases in bacteria revealed that one specific kind of enzyme, known as Fe(II)/2-oxoglutarate-dependent halogenases, or 2ODHs for short, was capable of site-specifically adding a halogen in the same way that the moonseed’s mystery enzyme did.
Notwithstanding the fact that no 2ODHs had yet been discovered in plants, the researchers decided this lead was worth investigating. So, they looked for transcripts that looked like 2ODH sequences and were more abundant in the roots of common moonseed than in the leaves and stems.
Kim and Weng were confident they had found what they were seeking after analyzing the RNA transcripts: one gene in particular (dubbed McDAH, short for M. canadense dechloroacutumine halogenase) was strongly expressed in the roots of common moonseed. Then, in Chinese moonseed, they discovered SaDAH, a protein that shared 99.1 percent of McDAH’s sequence. There was no matching protein found in snake vine, indicating that this protein was most likely the desired enzyme.
To be sure, the researchers investigated the enzyme in the lab and discovered that it was the first plant 2ODH capable of attaching the chlorine molecule to the alkaloid molecule dechloroacutumine to generate acutumine. Interestingly, the enzyme was quite picky; when they gave it other alkaloids such as codeine and berberine to see if it would install a halogen on them as well, the enzyme ignored them, indicating that it was highly specific towards its preferred substrate, dechloroacutumine, the precursor of acutumine. They compared the enzyme’s activity to that of other comparable enzymes and discovered that the key to its ability was the substitution of aspartic acid for glycine in the active site.
Kim and Weng were curious about what else the enzyme responsible for the moonseed’s halogenation reactions could do now that they had identified it. They reasoned that a chemical capable of initiating such a complex reaction could be valuable for chemists attempting to manufacture other molecules.
Then they fed the enzyme dechloroacutumine and a variety of alternative anion to see if it could catalyze a reaction with any of these molecules instead of chlorine. The enzyme only catalyzed a reaction with azide, a three-nitrogen-atom compound, out of a list of anions that included bromide, azide, and nitrogen dioxide.
“That is super cool because there isn’t any other naturally occurring azidating enzyme that we know of,” Kim says. The enzyme could be used in click chemistry, a nature-inspired method to create a desired product through a series of simple, easy reactions.
In future studies, they hope to use what they’ve learned about the McDAH and SaDAH enzymes as a starting point to create enzymes that can be used as tools in drug development. They’re also interested in using the enzyme on other plant products to see what happens. “Plant natural products, even without chlorine, are pretty effective and bioactive, so it would be cool to see if you can take those plant natural products and then install chlorines to see what kind of changes and bioactivity it has, whether it develops new-to-nature functions or retains its original bioactivity with enhanced properties,” Kim says. “It expands the biocatalytic toolbox we have for natural product biosynthesis and its derivatization.”
Source: Medindia
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