Research Area: Human Disease

Covid-19 scientific leaders share expertise in new MIT class
Greta Friar | Whitehead Institute
September 9, 2020

As the Covid-19 pandemic swept across the globe, bringing everyday life to a screeching halt, researchers at MIT and its affiliates ramped down much of their lab work and stopped teaching classes in person, but refused to come to a standstill. Instead, they changed tacks and took action investigating the many unknowns of Covid-19 and the virus that causes it (SARS-CoV-2), organizing pandemic responses, and communicating with the public and each other about what they knew.

One result of this period was the advent of a new course, aimed at providing MIT students with information on the science of the pandemic. The MIT Department of Biology tapped two scientists with experience working on pandemics to spearhead a course, 7.00 (COVID-19, SARS-CoV-2 and the Pandemic), which began Sept. 1. Whitehead Institute member and MIT Professor Richard Young, who had been quick to organize Covid-19 related research efforts, and Ragon Institute Associate Director Facundo Batista, a resident expert on immunology and infectious disease, agreed to lead the course.

The class meets virtually on Tuesday mornings, and a public livestream and recordings are available for anyone who wants to watch the lectures. Students who are taking the course for credit also gain access to a weekly session led by Lena Afeyan, a teaching assistant and MIT graduate student in Young’s lab at the Whitehead Institute. The session provides relevant background information on the science before the lectures.

Getting students up to speed on what is and is not known about the pandemic is no easy task. The science is complex and, in these early days, full of unknowns. Experts in many fields must pool their knowledge; virologists, immunologists, epidemiologists, public health researchers, clinicians, and more are focused on important pieces of the puzzle. Therefore, Young and Batista reached out to the leaders in all of those fields to give lectures in the course. Students will hear from experts that include Anthony Fauci, the longtime director of the National Institute of Allergy and Infectious Diseases, as well as David Baltimore of Caltech; Kizzmekia Corbett of the National Institutes of Health; Britt Glaunsinger of the University of California at Berkeley; Akiko Iwasaki of Yale University; Eric Lander of the Broad Institute of MIT and Harvard; Michel Nussenzweig of Rockefeller University; Arlene Sharpe of Harvard Medical School and Brigham and Women’s Hospital; Bruce Walker of the Ragon Institute of Massachusetts General Hospital, MIT, and Harvard; and others at the forefront of Covid-19 efforts. The course faculty agree that the best way to get accurate information to students is to have the experts provide it directly.

Designing the course

For many of the students, Covid-19 may be their first serious encounter with a pandemic, but a number of the lecturers have worked on the AIDS pandemic or other widespread infectious diseases, which they draw on when teaching.

“I like to put the coronavirus in the context of viruses I know better, like flu and HIV and polio virus,” says David Baltimore, the Nobel laureate professor of biology and president emeritus at Caltech who was previously the first director of the Whitehead Institute and a professor at MIT. However, the scientists’ relevant backgrounds can only help so much. The new coronavirus is a unique and difficult research subject.

“It has no obvious evolutionary relationship to other viruses. It’s got a much longer RNA, many more genes, so more complexity of function, more complexity of genetics, and it’s received relatively little study up until recently,” Baltimore says. “There is a lot more work that needs to be done.”

When planning the class, Young wanted to give all of the information needed to understand what is likely the first pandemic to powerfully impact the lives of the undergraduates taking the course. His motives were pedagogical — and practical.

“If we give people knowledge of what’s known and not known about the virus, provided by experts whom they trust, they can help us come up with solutions,” Young says.

Young and Batista expect that some of their students will soon be conducting their own Covid-19 research. Batista hopes that this experience will encourage students to think even beyond the scope of the current pandemic.

“I think the U.S. and the Western world have underestimated the risk of infectious diseases because the big pandemics have been happening elsewhere. This class is about bringing people together on Covid-19, and more than that, [it is about] creating a consciousness about the threat of future infections,” Batista says.

Where to start?

The first lecture was given by Bruce Walker, director of the Ragon Institute. Walker provided an overview of the available information, including how the pandemic appears to have started, how the virus causes disease, and what the prospects are for treatment and vaccines. The level of the science is aimed at MIT undergraduates, but because the livestream audience may have different science backgrounds, Walker made sure to define basic terms and concepts as he went. The lecture was attended by 250 students, with more than 7,000 people watching the livestream.

Registered students can ask questions during a Q&A at the end of each lecture. Walker addressed students’ concerns about the U.S. response to the pandemic, the risk of reinfection, mutability of the virus, and challenges with new types of vaccines. With the aim of providing accurate information, his answers were not always reassuring. However, in spite of the many uncertainties that the scientists are grappling with, the course faculty’s message for students is an optimistic one.

“People have felt powerless in this pandemic,” Afeyan says. “A course like this can help people feel like they have the tools to do something about it. There is a plethora of problems that will stem from the pandemic, so there are lots of ways to get involved regardless of your field.”

Researchers have banded together across MIT, Whitehead Institute, Ragon Institute, and around the globe to address the pandemic. For students who want to join the research effort, the content of the lectures is paired with discussions during Afeyan’s sessions with researchers earlier in their careers, who can talk to the students about next steps should they choose to pursue one of the fields presented in the course.

As for students and audience members simply looking to understand the public health event that has so strongly impacted their world, the faculty hope that the course will provide them with the answers they need. Scientists are not the only ones dealing with lots of uncertainty these days, and there is value in learning what the experts know as they know it, straight from the source.

A computational approach to cancer

Toni-Ann Nelson transformed remote summer research into an opportunity to learn a new set of tools for analyzing tumors.

Raleigh McElvery
August 20, 2020

Toni-Ann Nelson has wanted to find a cure for cancer ever since she was nine years old and lost her grandfather to the disease. “I remember thinking there must be something that the doctors and scientists were missing,” she recalls. “It just couldn’t be that complicated.” Now one semester away from earning her degree in molecular biology, Nelson is realizing cancer is just that — complicated. After conducting cancer research during MIT’s Summer Research Program in Biology (MSRP-Bio), she understands much more about the intricacies of tumors and metastasis. But she’s also glimpsed just how many cellular puzzles remain to be solved.

Growing up in Jamaica, Nelson enjoyed all her science classes, but preferred biology because she knew it would provide the foundation to probe cancer. She graduated as the valedictorian of her high school class, and earned a scholarship to Alcorn State University in Mississippi, where she began in the spring of 2017.

Alcorn doesn’t have any cancer research facilities, so Nelson secured a position as an undergraduate researcher in Yan Meng’s plant tissue culture lab. For three years, Nelson aimed to improve viral disease resistance in sweet potatoes. Even though she wasn’t conducting clinical research, she mastered key molecular biology techniques like PCR, gel electrophoresis, and tissue culture.

“Fundamental research is important because many times finding a cure requires starting with the basics, and understanding what’s going on inside the cell,” she says.

When Nelson was accepted into MSRP-Bio as a Gould Fellow and assigned to work in Tyler Jacks’ lab, she was elated to get her first hands-on cancer research experience. But in April 2020 — two months before the program was slated to begin — MIT’s campus temporarily shut down due to the COVID-19 pandemic, and MSRP-Bio 2020 became a remote learning experience.

As a result, Nelson and her MSRP-Bio cohort conducted their research from home. She took on a computationally-intensive project that was conducive to remote work and required taking an online quantitative methods class. In a manner of weeks, she learned an entirely new set of skills, including programming languages like Python.

“I always thought that I wouldn’t need those types of computational tools as part of my cancer research,” she explains. “But working at MIT was enlightening, because it showed me that they are key to understanding disease. I can definitely see myself using them on my own projects in the future.”

Pink and purple histology image
Light micrograph of a lung adenocarcinoma. Credit: Vasilena Gocheva/Jacks Lab, Koch Institute

The Jacks lab studies the genetic events that contribute to cancer, and Nelson’s project centered on lung adenocarcinoma. The predominant form of non-small cell lung cancer, it begins in alveolar type II (AT2) cells. Past studies showed that, as the tumor progresses, AT2 cells change state and lose their original identity. Nelson wanted to determine which genes and proteins underlie this evolution. Her analyses showed that genetic markers characteristic of AT2 cells tend to decrease over time, while markers denoting faster-growing “high grade” tumors become more prevalent.

“The kinetics of these gene expression changes that are happening early on are still poorly understood,” she explains. “It just goes to show how complicated this pathology is, which I find even more fascinating.”

Once researchers can pinpoint the genes and proteins that drive changes in cancer cell state, they’ll be better equipped to design drugs that target and prevent metastatic processes.

Although Nelson couldn’t visit the lab in person, as on-campus research slowly began ramping up again, her graduate student mentor Amanda Cruz would show her around during their video conference calls. Cruz also helped Nelson explore the scientific literature, choose studies for the lab’s journal club, and perform computational analyses.

Given the unprecedented circumstances, Nelson says having a solid support system was key to her success. Nelson and her MSRP cohort also relied on one another for encouragement, and were each assigned a graduate student “pal” for guidance outside of lab.

“The program catered to our every need, and it’s structured to ensure that someone will always check up on you if you’re feeling alone,” Nelson says. “I never expected to get so much from this experience, especially because I’m not physically on campus. But what I learned this summer was so much more than I could ever have anticipated.”

Her time in the Jacks lab has solidified her fervor for cancer research, and she intends to apply to cancer biology PhD programs in order to continue this line of inquiry. “I’ve realized there’s still so much more to learn,” she says, “but we’re getting there.”

Top image courtesy of Toni-Ann Nelson
Posted: 8.19.20
SMART research enhances dengue vaccination in mice
Singapore-MIT Alliance for Research and Technology
August 13, 2020

Researchers from the Singapore-MIT Alliance for Research and Technology (SMART), MIT’s research enterprise in Singapore, have found a practical way to induce a strong and broad immunity to the dengue virus based on proof-of-concept studies in mice. Dengue is a mosquito-borne viral disease with an estimated 100 million symptomatic infections every year. It is endemic in over 100 countries in the world, from the United States to Africa and wide swathes of Asia. In Singapore, over 1,700 dengue new cases were reported recently.

The study is reported in a paper titled “Sequential immunization induces strong and broad immunity against all four dengue virus serotypes,” published in NPJ Vaccines. It is jointly published by SMART researchers Jue Hou, Shubham Shrivastava, Hooi Linn Loo, Lan Hiong Wong, Eng Eong Ooi, and Jianzhu Chen from SMART’s Infectious Diseases and Antimicrobial Resistance (AMR) interdisciplinary research groups (IRGs).

The dengue virus (DENV) consists of four antigenically distinct serotypes and there is no lasting immunity following infection with any of the DENV serotypes, meaning someone can be infected again by any of the remaining three variants of DENVs.

Today, Dengvaxia is the only vaccine available to combat dengue. It consists of four variant dengue antigens, one for each of the four serotypes of dengue, expressed from attenuated yellow-fever virus. The current three doses of immunization with the tetravalent vaccine induce only suboptimal protection against DENV1 and DENV2. Furthermore, in people who have not been infected by dengue, the vaccine induces a more severe dengue infection in the future. Therefore, in most of the world, the vaccination is only given to those who have been previously infected.

To help overcome these issues, SMART researchers tested on mice whether sequential immunization (or one serotype per dose) induces stronger and broader immunity against four DENV serotypes than tetravalent-formulated immunization — and found that sequential immunization induced significantly higher levels of virus-specific T cell responses than tetravalent immunization. Moreover, sequential immunization induced higher levels of neutralizing antibodies to all four DENV serotypes than tetravalent vaccination.

“The principle of sequential immunization generally aligns with the reality for individuals living in dengue-endemic areas, whose immune responses may become protective after multiple heterotypic exposures,” says Professor Eng Eong Ooi, SMART AMR principal investigator and senior author of the study. “We were able to find a similar effect based on the use of sequential immunization, which will pave the way for a safe and effective use of the vaccine and to combat the virus.”

Upon these promising results, the investigators will aim to test the sequential immunization in humans in the near future.

The work was supported by the National Research Foundation (NRF) Singapore through the SMART Infectious Disease Research Program and AMR IRG. SMART was established by MIT in partnership with the NRF Singapore in 2007. SMART is the first entity in the Campus for Research Excellence and Technological Enterprise (CREATE) developed by NRF.  SMART serves as an intellectual and innovation hub for research interactions between MIT and Singapore, performing cutting-edge research of interest to both Singapore and MIT. SMART currently comprises an Innovation Centre and five IRGs: AMR, Critical Analytics for Manufacturing Personalized-Medicine, Disruptive and Sustainable Technologies for Agricultural Precision, Future Urban Mobility, and Low Energy Electronic Systems. SMART research is funded by the NRF Singapore under the CREATE program.

The AMR IRG is a translational research and entrepreneurship program that tackles the growing threat of antimicrobial resistance. By leveraging talent and convergent technologies across Singapore and MIT, they aim to tackle AMR head-on by developing multiple innovative and disruptive approaches to identify, respond to, and treat drug-resistant microbial infections. Through strong scientific and clinical collaborations, they provide transformative, holistic solutions for Singapore and the world.

Proteins and labs come together to prevent Rett syndrome
Greta Friar | Whitehead Institute
July 22, 2020

New discoveries about the disruption of condensates in the neurodevelopmental disorder Rett syndrome provide insights into how cells compartmentalize chromosomes as well as new potential paths for therapies.

Scientists have, for many years, conceptualized the cell as a relatively free-flowing space, where–apart from the organization provided by specific cellular structures–molecules float freely, somehow ultimately ending up in the right place at the right time. In recent years, however, scientists have discovered that cells have much more spatial organization than previously thought thanks to a mechanism called phase separation, which occurs in cells when certain molecules form large droplet-like structures that separate what’s inside of the droplet from the rest of the cell. The droplets, called condensates, help sequester and concentrate molecules in specific locations, and appear to increase the efficiency of certain cellular functions.

Whitehead Institute Member Richard Young, also a professor of biology at Massachusetts Institute of Technology (MIT), has been exploring the previously unknown role that condensates play in gathering the molecules needed for gene transcription–the process by which DNA is read into RNA. In order to better understand when and how cells use phase separation, Charles Li, a graduate student in Young’s lab, set out to identify more proteins that can form condensates. That search led him to MeCP2, a protein associated with the severe neurodevelopmental disorder Rett syndrome, studied by Young’s colleague at Whitehead Institute, Founding Member Rudolf Jaenisch, who is also a professor of biology at MIT. No cure for Rett syndrome currently exists, and Jaenisch’s lab has been investigating the biology of the disorder in the hopes of discovering a medical therapy that can rescue neurons affected by Rett syndrome.

With the discovery of MeCP2’s condensate forming ability, Young and Jaenisch saw the opportunity for a promising collaboration between their labs. Led by co-first authors Li and Eliot Coffey, another graduate student in Young’s lab, the two labs investigated MeCP2 and whether the disruption of its condensate-forming ability contributes to Rett syndrome. During these investigations, the researchers also uncovered how cells may use condensates to help organize the active and inactive parts of chromosomes. Their findings, published in the journal Nature on June 22, report on these insights and suggest new paths for developing therapies for Rett syndrome.

PHASE SEPARATION AND RETT SYNDROME

Proteins that form condensates often contain intrinsically disordered regions (IDRs), long spaghetti-like strands that transiently stick together to form a dynamic mesh. Research has historically focused on the structured regions of proteins, which bind very specifically to other molecules, while IDRs have largely been overlooked. In this case, MeCP2’s large IDRs were exactly what drew Li to it.

“What was striking to me was that this protein has been studied for decades, and so much function has been ascribed to the protein as a whole, yet it only has one structured domain with a recognized function, the DNA binding domain. Beyond that, the entire protein is disordered, and how its parts function was largely unknown,” Li says.

The researchers found that MeCP2 used its IDRs to glom together and form condensates. Then they tested many of the mutations in the MECP2 gene that are associated with Rett syndrome and found that they all disrupt MeCP2’s ability to form condensates. Their findings suggest that therapies targeting condensates associated with the protein, rather than the protein itself, may be promising in the hunt for a Rett syndrome treatment.

“MeCP2 and Rett syndrome have been studied intensely for many years in many labs and yet not a single therapy has been developed. When the project began, I was immediately fascinated by the idea that we might find a new disease mechanism that could help us finally understand how Rett syndrome arises and how it could be treated,” Coffey says.

“Rick [Young] has shown that condensates play key roles in maintaining normal cellular function, and our latest collaboration illuminates how their disruption may drive diseases such as Rett syndrome,” Jaenisch says. “I hope the insights we have gained will prove useful both in our continued search for a treatment for Rett syndrome and more broadly in research on condensates and disease.”

COMPARTMENTALIZING CHROMOSOMES

The researchers’ investigation into MeCP2’s condensate forming behavior also shed light on how chromosomes are organized into regions of active and inactive genes. When MeCP2 is functioning normally, it helps to maintain heterochromatin, the roughly half of our chromosomes where genes are “turned off,” unable to be read into RNA or further processed to make proteins. MeCP2 binds to sequences of DNA marked with a certain type of regulatory tag that is typically found in heterochromatin. This helps crowd MeCP2 to the threshold concentration needed to form heterochromatin condensates. These condensates, in turn, help to sequester the molecules needed to maintain it apart from euchromatin, the half of our chromosomes filled with active genes. Different proteins form condensates near euchromatin, concentrating the molecular machinery needed to transcribe active genes there.

Since condensates form when proteins with large spaghetti-like IDRs stick together, one might expect that any protein containing IDRs could interact with any other IDR-containing protein to form droplets, and that is what the researchers have often seen. However, what they observed with MeCP2, which is associated with heterochromatin, is that key condensate-forming proteins associated with euchromatin refused to mix.

It’s important for the health of the cell that the genes in heterochromatin not be inadvertently turned on. The researchers reason that discrete euchromatin and heterochromatin condensates may play a key role in ensuring that transcriptional machinery localizes to euchromatin only, while repressive machinery–like MeCP2–localizes to heterochromatin. The researchers are excited to turn their attention to how proteins are able to join condensates selectively, and when and where else in the cell they do so.

“There’s a chemical grammar waiting to be deciphered that explains this difference in the ability of some proteins to move into one condensate versus another,” Young says. “Discovering that grammar can help us understand how cells maintain the crucial balance between the active and silent halves of our genome, and it could help us understand how to treat disorders such as Rett syndrome.”

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Written by Greta Friar

Richard Young’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at the Massachusetts Institute of Technology.

Rudolf Jaenisch’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.

Li, C.H., Coffey, E., et al. (2020). MeCP2 links heterochromatin condensates and neurodevelopmental disease. Nature. DOI: 10.1038/s41586-020-2574-4

Discovery of how cancer drugs find their targets could lead to a new toolset for drug development
Whitehead Institute
June 17, 2020

In the watery inside of a cell, complex processes take place in tiny functional compartments called organelles. Energy-producing mitochondria are organelles, as is the frilly golgi apparatus, which helps to transport cellular materials. Both of these compartments are bound by thin membranes.

But in the past few years, research at Whitehead Institute and elsewhere has shown that there are other cellular organelles held together without a membrane. These organelles, called condensates, are tiny droplets which keep certain proteins close together amidst the chaos of the cell, allowing complex functions to take place within. “We know of about 20 types of condensate in the cell so far,” says Isaac Klein, a postdoc in Richard Young’s lab at Whitehead Institute and oncologist at the Dana-Farber Cancer Institute.

Now, in a paper published in Science on June 19, Klein and Ann Boija, another postdoc in Young’s lab, show the mechanism by which small molecules, including cancer drugs, are concentrated in these cellular droplets — a finding that could have implications for the development of new cancer therapeutics. If researchers could tailor a chemical to seek out and concentrate in one kind of droplet in particular, it might have a positive effect on the delivery efficiency of the drug. “We thought, maybe that’s an avenue by which we can improve cancer treatments and discover new ones,” says Klein.

“This [research] is part of a revolutionary new way of looking at the organization within cells,” says Phillip Sharp, a professor at the Massachusetts Institute of Technology’s Koch Institute for Integrative Cancer Research and a co-author on the study. “Cells are not little pools of soup, all mixed together. They are actually highly organized, compartmentalized units, and that organization is important in their function and in their diseases. We’ve just started to understand that, and this new paper is a really important step, using that insight, to understand how to potentially treat diseases differently.”

CONDENSATES AND DRUG DELIVERY

To explore how different properties of condensates inside the cell’s nucleus affected the delivery of cancer drugs, Boija and Klein selected a few example condensates to study. These included splicing speckles, which store cellular materials needed for RNA splicing, nucleoli, where ribosomes are formed, and a new kind of droplet Young’s lab discovered in 2018 called a transcriptional condensate. These new condensates bring together all the different proteins needed to successfully transcribe a gene.

The researchers created their own suite of four different fluorescently-labeled condensates by adding glowing tags to marker proteins specific to each kind of droplet. For example, transcriptional condensates are marked by the droplet-forming protein MED1, splicing speckles by a protein called SRSF2, and nucleoli by FIB1 and NPM1.

Now that they could tell individual droplets apart by their cellular purpose, the team, along with the help of Nathanael Gray, a chemical biologist at Harvard University and the Dana-Farber Cancer Institute, created fluorescent versions of clinically important drugs. The tested drugs included cisplatin and mitoxantrone, two anti-tumor medicines commonly used in chemotherapy. These therapeutics were the perfect test subjects, because they both target proteins that lie within nuclear condensates.

The researchers added the cancer drugs to a mixture containing various droplets (and only droplets, none of the actual drug targets), and found that the drugs sorted themselves into specific condensates. Mitoxantrone concentrated in condensates marked by MED1, FIB1 and NPM1, selectively avoiding the others. Cisplatin, too, showed a particular affinity for droplets held together by MED1.

“The big discovery with these in vitro studies is that a drug can concentrate within transcriptional condensate independent of its target,” Boija says. “We used to think that drugs come to the right place because their targets are there, but in our in vitro system, the target is not there. That’s really informative — it shows the drug is actually being concentrated in a different way than we thought.”

To understand why some drugs were drawn into transcriptional condensates, they screened a panel of chemically-modified dyes and found that the important part of many drugs — the part that led them to concentrate in transcriptional condensates  —  is the molecules’ aromatic ring structure. Aromatic rings are stable, ring-shaped groupings of carbon atoms. The aromatic ring in some drugs are thought to stack with rings in MED1’s amino acids, leading the drug to concentrate in transcriptional condensates.

Being able to tailor a drug to enter a certain condensate is a powerful tool for drug developers. “We found that if we add an aromatic group to a molecule, it becomes concentrated within the transcriptional condensate,” Boija says. “It’s that type of interaction that is important when we design new drugs to enter transcriptional condensates — and maybe we can improve existing drugs by modifying their structure. This will be very exciting to look into.”

WHERE DRUGS CONCENTRATE AFFECTS HOW WELL THEY FIGHT CANCER

In order for this tool to be practically useful in drug development, the researchers had to make sure that concentration in specific droplets would actually impact the drugs’ performance. Boija and Klein decided to test this using cisplatin, which is drawn to transcriptional condensates by MED1 and works to fight cancer by adding clunky platinum molecules to DNA strands. This damages tumor cells’ genetic material. When the researchers administered cisplatin to a mixture of different condensates, both in the test tube and in cells, the drug preferentially altered DNA that lay within transcriptional condensates.

This could explain why cisplatin and other platinum drugs are effective against so many diverse cancers, says Young, who is also a professor of biology at MIT; cancer-causing genes often carry regions of DNA called super enhancers, which are extremely active in transcription, leading to very large transcriptional condensates. “We now think the reason that drugs like cisplatin can work well in patients with diverse cancers is because they’re becoming selectively concentrated at the cancer-causing genes, where these large transcriptional condensates occur,” he said. “The effect is to have the drug home in on the gene that’s causing each cancer to be so deadly.”

A DRUG RESISTANCE MYSTERY, SOLVED

The new insights in condensate behavior also provided some answers to another question in cancer research: why people become immune to the breast cancer drug tamoxifen.Tamoxifen works by attaching itself to estrogen receptors in the cancer cells, preventing them from getting the hormones they need to grow and eventually slowing or stopping the formation of new cancer cells altogether. The drug is one of the most effective treatments for the disease, reducing recurrence rates for ER+ breast cancers by around 50%.

Unfortunately, many patients quickly develop a resistance to tamoxifen — sometimes as soon as a few months after they start taking it. This happens in a variety of ways — for example, sometimes the cancer cells will mutate to be able to kick the tamoxifen out of the cells, or simply produce fewer estrogen receptors for the drug to bind. One form of resistance was associated with an overproduction of the protein MED1, but scientists didn’t know why.

With their newfound knowledge of how a drug’s activity is affected by where it concentrates, Boija and Klein had a hypothesis: the extra MED1 might increase the size of the droplets, effectively diluting the concentration of tamoxifen and making it more difficult for the drug to bind its targets. When they tested this in the laboratory, the team found that more MED1 did indeed cause larger droplets, leading to lower concentrations of tamoxifen.

A NEW TOOLSET FOR DRUG DESIGNERS

The ability to better understand the behavior of drugs in cancer cells — how they concentrate, and why the cancer could become resistant to them — may provide drug developers with a new arsenal of tools to craft efficient therapeutics. “This study suggests that we should be exploring whether we can design or isolate drugs that are concentrated in a given condensate, and to understand how existing drugs are concentrated in the cell,” says Phil Sharp. “I think this is really important for drug development — and I think [figuring it out] is going to be fun.”

Making medicine runs in the family
Greta Friar | Whitehead Institute
May 5, 2020

What do the painkillers morphine and codeine, the cancer chemotherapy drug vinblastine, the popular brain health supplement salidroside, and a plethora of other important medicines have in common? They are all produced in plants through processes that rely on the same family of enzymes, the aromatic amino acid decarboxylases (AAADs). Plants, which have limited ability to physically react to their environments, have instead evolved to produce a stunning array of chemicals that allow them to do things like deter pests, attract pollinators, and adapt to changing environmental conditions. A lot of these molecules have also turned out to be useful in medicine—but it’s unusual for one family of enzymes to be responsible for so many different molecules of importance to both plants and humans. New research from Whitehead Institute Member Jing-Ke Weng, who is also an associate professor of biology at the Massachusetts Institute of Technology, and postdoctoral researcher Michael Torrens-Spence delves into the science behind the AAADs’ unusual generative capacity.

Plants create their useful molecules through biochemical pathways made up of chains of enzymes. Each enzyme acts as an assembly worker, taking in a molecule—starting with a basic building block like an amino acid—and performing biochemical modifications in sequence. The altered molecules get passed down the line until the last enzyme creates the final natural product. Once the pathway enzymes for a molecule of interest have been identified, researchers can copy their corresponding genes into organisms like yeast and bacteria that are capable of producing the molecules at scale more easily than the original plants. The AAAD family of enzymes function as gatekeepers to plants’ specialized molecule production because they operate at the beginning of many of the enzyme assembly lines; they take various amino acids, molecules that are widely available in nature, and direct them into different enzymatic pathways that produce unique molecules that only exist in plants. When an AAAD evolves to perform a new function, as has occurred frequently in their evolutionary history, this change high up in the assembly lines can cascade into the development of new biochemical pathways that create new natural products—leading to the diversity of medicines that stem from AAAD-gated pathways.

Due to the AAADs’ prominent role in the production of medically important molecules, Weng and Torrens-Spence decided to investigate how the AAADs came to be so prolific. In research published in the journal PNAS on May 5, the researchers illuminate the structural and functional underpinnings of the AAADs’ diversity. They also demonstrate how their detailed knowledge of the enzymes can be used to engineer novel enzymatic pathways to produce important molecules of interest from plants.

“We characterized these enzymes very thoroughly, which is a great starting place for manipulating the system and engineering it to do something new. That’s particularly exciting when you’re dealing with enzymes at the interface between primary and specialized plant metabolism; it can apply to a lot of downstream drugs,” Torrens-Spence says.

The AAAD family evolved from one ancestral enzyme into a diverse set of related enzymes over a relatively short period of time. This sort of diversification occurs when an enzyme gets accidentally duplicated, after which one copy has evolutionary pressure on it to maintain the same function, but the other copy suddenly has free range to evolve. If the superfluous enzyme mutates to do something new that is useful to the organism, from then on both enzymes, with their distinct roles, are likely to be maintained. In the case of the AAADs, this process occurred many times, leading to a large number of enzymes that appear almost exactly alike, yet can do very different things.

In order to explain the AAADs’ successful rate of diversification, the researchers took a close look at four enzymes in the AAAD family with different roles, and discovered the composition and three-dimensional shape—the crystal structure—of each. The crystal structure allowed the researchers to see how these molecular machines hold and modify specific molecules; this meant that they could understand why some AAADs initiate certain specialized-molecule production lines while other AAADs initiate alternative production lines. The researchers next used genetics and biochemistry to pinpoint the differences between the enzymes and how small genetic variations enact very major changes to the enzyme’s underlying machinery. This detailed analysis explained, among others things, how a subset of enzymes that evolved out of the AAADs, the aromatic acetaldehyde synthases (AASs), came to perform a completely different action on molecules while still being so similar to true AAADs that the two types of enzymes are often mistaken for each other.

After the researchers developed this thorough understanding of the AAAD family of enzymes, as well as knowledge of the AAAD-containing pathways that create useful medicinal molecules, they applied this knowledge by engineering an entirely new pathway to create a molecule of interest, (S)-norcoclaurine, a precursor molecule for morphine and other poppy-based painkillers. Torrens-Spence combined enzymes from pathways in different species to invent a novel chain of enzyme reactions that can produce (S)-norcoclaurine in fewer steps than is seen in nature. This experiment was a proof of concept that Torrens-Spence says shows the potential for such biosynthetic engineering, for example as a method to produce plant-based drugs more easily.

“Often with these molecules of interest, you figure out the pathway in plants and copy-paste it into a more scalable system, like yeast, that will produce larger quantities of the molecule,” Torrens-Spence says. “Here we’re applying engineering principles to biology, so that we can innovate and build something new.”

Written by Greta Friar

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Jing-Ke Weng’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an associate professor of biology at Massachusetts Institute of Technology.

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Citation:

“Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins”

PNAS, May 5, 2020

DOI: https://doi.org/10.1073/pnas.1920097117

Michael P. Torrens-Spence (1), Ying-Chih Chiang (2†), Tyler Smith (1,3), Maria A. Vicent (1,4), Yi Wang (2), and Jing-Ke Weng (1,3)

1 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

2 Department of Physics, the Chinese University of Hong Kong, Shatin, N.T., Hong Kong.

3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

4 Department of Biology, Williams College, Williamstown, Massachusetts 01267, USA.

† Present address: School of Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.

3 Questions: Michael Yaffe on treating Covid-19 patients with acute respiratory distress

MIT professor and intensivist/trauma surgeon explains the new challenges that Covid-19 brings to treating patients in acute respiratory distress.

Bendta Schroeder | Koch Institute
April 30, 2020

During the Covid-19 pandemic, frontline health care workers have had to adapt rapidly to treating patients with lung failure, not only because of shortages of equipment such as ventilators often used to treat severe cases, but also because such approaches are not always effective due to the unique and still imperfectly understood pathology of Covid-19 infections.

Michael Yaffe, the David H. Koch Professor in Science, normally divides his time among his roles as a researcher and professor of biology and biological engineering at MIT, an intensivist/trauma surgeon at Beth Israel Deaconess Medical Center (BIDMC), and a colonel in the U.S. Army Reserve Medical Corps. Currently, he is developing treatments for Covid-19 infections in his laboratory at the Koch Institute for Integrative Cancer Research at MIT. Additionally, he runs one of the Covid-19 Intensive Care Units at BIDMC and serves as co-director of the acute care and ICU section of Boston Hope, the 500-bed pop-up hospital organized by the City of Boston, Massachusetts in the Boston Convention and Exposition Center. Yaffe shares how he is working to improve outcomes for Covid-19 patients and offers his perspective on how emergency care for acute respiratory distress will need to evolve during this crisis and beyond.

Q: What are the special considerations for Covid-19 patients receiving treatment for respiratory failure?

A: We have known about acute respiratory distress syndrome (ARDS) for decades. It was first recognized in battlefield casualties during the Vietnam War, and was initially called “Da-Nang Lung,” but later was understood to be the result of many different diseases. In ARDS, fluid builds up in the tiny air sacs, or alveoli, preventing the lungs from filling up with enough air, and in severe cases is treated by putting patients on ventilators or other devices that support breathing.

The type of lung injury we are seeing in Covid-19 patients behaves very differently from the traditional type of ARDS, and seems to involve early damage to the cells that line the lungs, followed by intense inflammation. The inflammation leads to a massive increase in blood clotting that affects all of the blood vessels in the body, but particularly the blood vessels in the lungs. As a consequence, even if we can force air into the lungs, it does not get delivered very efficiently into the bloodstream.

In ICUs in Boston, New York, and Colorado, we have started a clinical trial using a clot-busting drug called tPA that we think will help rescue patients whose lungs are failing despite maximal support with a mechanical ventilator. This approach has gathered a lot of attention from other hospitals, both nationally and internationally, who are also trying this approach. The work has now led to FDA approval for this drug as an Investigational New Drug, meaning that it is now approved for use in Covid-19 ARDS in the setting of clinical trials.

Q: How has your wide-ranging expertise equipped you to address new challenges that you face in the ICU?

A: I have been very fortunate to be well-prepared to help out in this crisis. First, my training as an intensive care physician and trauma surgeon makes me comfortable in a crisis situation. The clinical problems that we are dealing with here  — ARDS, kidney failure, etc. — are exactly within the scope of my regular clinical practice. Second, my Army deployment experience as a surgeon and critical care doctor in Afghanistan and in Central America has made me very comfortable having to make decisions in resource-limited situations. Finally, it has been incredibly fortuitous that much of my lab’s work has been in the area of cell injury, particularly cancer treatment-related cell injury, but also in the setting of a condition called systemic inflammatory response syndrome, which is essentially exactly what Covid-19 is. In this area, my lab has been studying the link between inflammation and blood clotting for over a decade, and the basic science insights from that work have now become central to our understanding of Covid-19 lung failure, which no one could have foreseen when we first started that research.

Q: What implications do you think the Covid-19 pandemic will have for emergency care after it is over?

A: I think the implications of Covid-19 for the future are immense. First, I hope the lessons learned from this pandemic lead to a complete re-thinking of our national public health policy (or lack of one, really) and a re-engagement with World Health Organization officials for monitoring the outbreak of emerging diseases.

Second, I think that this crisis may fuel additional research funding in the area of critical care medicine. Before the Covid-19 crisis, very few people had heard of ARDS, or even critical care as a field of medicine, since it does not have the glamour of conditions like cancer medicine or cardiovascular disease. Historically, research in this area has been underfunded, but now that ARDS has taken the spotlight in the news, I am hopeful that the recognition that some patients with Covid-19 are dying because of critical illness and lung failure will lead to new efforts to better understand the link between inflammation, lung function, and innate immunity, including blood coagulation. The Covid-19 crisis will not end when this first wave subsides, but will re-visit us again in the fall. Additionally, other coronavirus diseases as well as viral epidemics are likely to continue to plague us in the future.

One final lesson we are learning from this terrible pandemic is how important it is to treat all of the different parts of the body as a complex interacting unit, and to apply what we know from systems biology and other fields of study to understand how those parts are integrated into one coherent system. The lung failure, kidney failure, and inflammation of the heart that are the hallmarks of Covid-19 critical illness directly reflect how different inflammatory molecules in the blood alter the function of each of these different organ systems. Our traditional medical approach of having separate specialists in infectious disease, pulmonary medicine, renal medicine, and hematology does not work well when all the organ systems are cross-talking to each other. The job of the intensive care physician is to integrate all of the relevant basic biology and pathology of these organs into a comprehensive holistic treatment approach for the patient. Covid-19 has made that need to think across multiple disciplines and connect basic science to clinical care even more apparent.

From bedside to bench, and back again
Eva Frederick | Whitehead Institute
April 22, 2020

In 2018, a 31-year-old woman checked into Massachusetts General Hospital (MGH) in Boston with a respiratory infection so bad she had to be placed on oxygen. A trip to the hospital for lung trouble was nothing new for her — several times in the past, recurrent infections required her to stay under a doctor’s supervision for days until they blew over. Now, however, it seemed that she would not be leaving the hospital until she received two entirely new lungs.

The woman had had respiratory issues since she was a baby. Her flare-ups usually presented like pneumonia — a nasty, phlegm-y cough accompanied by a fever. After years of this the pathways between the trachea and the alveoli, called bronchi, were swollen and inflamed. Her physicians suspected that these frequent respiratory bouts had something to do with the mucus produced in her airways.

Mucus is the body’s first line of defense against the dirt and pathogens we inhale when we breathe. The sticky substance, composed mostly of water, salts, and sugar-laden proteins called mucins, traps the incoming material on its sticky surface. From there, cilia — tiny finger-like protrusions from cells that can look like small eyelashes — push the mucus up through the airways where it is eventually swallowed or coughed out.

Conditions such as cystic fibrosis can cause the mucus that lines the lung pathways to become so thick that the cilia can’t push it out, leading to bronchiectasis — the swelling of the bronchi. When physicians tested the woman for such likely causes, however, the results came back negative. Her case was a total mystery.

CRACKING THE CASE STUDY

As she awaited her double lung transplant, the woman met Dr. Raghu Chivukula, at the time a pulmonary and critical care medicine fellow at MGH interested in rare and unusual lung diseases as a consequence of his PhD training in human genetics. During his time spent working with these often critically ill patients, “it became clear that there were lots of unanswered questions in lung biology and the basis of lung diseases,” he said. Chivukula soon realized that the woman’s condition was one of these unanswered questions.

Often, when doctors are unable to come to a diagnosis, they end up referring a patient to another hospital or to see a specialist. MGH, with its reputation as one of the top hospitals in America, sees quite a lot of these mysterious cases. They saw so many, in fact, that in 2016 the hospital created a program called the Pathways Consult Service, where scientists could evaluate these unusual patients to see whether their maladies might be something entirely new to science.  The program helps connect physicians with researchers in the Boston area to help come up with the technology and resources to dive deep into the biology of the patients’ undiagnosed conditions.

After his initial conversations with the woman with the lung condition, Chivukula reached out to the Pathways program to see whether they could help him further investigate her disease.

“We were so excited when Raghu, who is an incredible physician and scientist, came to us with this opportunity to learn about biology from this patient that he was seeing,” says Dr. Katrina Armstrong, the Physician-in-Chief of the Department of Medicine at MGH who works with the Pathways program.

As the woman waited for her lung transplant, Chivukula interviewed her about her medical history. He also talked to two of her siblings, who were in town to help their sister in the run-up to her operation. Talking to the three of them offered Chivukula a clue: respiratory infections ran in the woman’s family. Her two siblings showed similar, if milder, symptoms.

This finding led Chivukula, with help from the Pathways program, to send the genetic material of the woman, her parents, and her two siblings to Fowzan S. Alkuraya, a geneticist at King Faisal Specialist Hospital and Research Centre (KFSHRC), in Riyadh, Saudi Arabia. When the results came in, Alkuraya sifted through the data looking for mutations that could be playing a role in the family’s lung issues. Across all three genomes, one common difference stood out: a mutation in a gene called NEK10. “I wrote back to Raghu to tell him how excited I was for having identified this novel gene,” Alkuraya says.

Scientists weren’t sure what this gene did, although they knew it coded for a kinase — a type of protein involved in signalling by modifying other proteins with a phosphate group. Previous studies suggested the NEK10 protein might play a role in how cancer cells respond to DNA damage in humans and the formation of the nervous system in certain kinds of fish, but no research had ever linked its activity to any kind of human disease, or to the respiratory system.

Once he realized the woman’s mutation was affecting a kinase, Chivukula decided to take on the project as part of his postdoctoral research in David Sabatini’s lab at Whitehead Institute. Chivukula had initially begun working with Sabatini on a project about the role of lysosomes in the development of pulmonary fibrosis. Since Sabatini’s previous research has included a focus on understanding important protein kinases in cells, the new mutation seemed like a perfect additional project. “I was hopeful that the combination of my own interests in lung biology with David’s lab’s world-class cell biology expertise and specialized toolkit would allow us to figure out this disease,” Chivukula says.

THE MYSTERY MUTATION

To determine whether this mutation could be to blame for the woman’s condition, Chivukula and Sabatini took a closer look at the mutation itself; the changes in the woman’s DNA sequence didn’t make her cells express less NEK10, they found. Instead, the alteration caused the insertion of 7 additional amino acids in the NEK10 protein, which Chivukula hypothesized might render the protein unstable and not able to perform some key job in the woman’s lung cells.

Still, she was only one patient, and it was possible this specific mutation that appeared in the DNA of her and her siblings was unrelated to her condition. Was this just a fluke, the scientists wondered, or could NEK10 mutations be to blame in other cases of unexplained respiratory problems?

Chivukula started sending out feelers to other hospitals and research centers around the world. He hoped to find other patients with unexplained lung conditions that shared the mutations the woman and her siblings had in their NEK10 genes. Slowly, other accounts trickled in. Other hospitals had registered similar changes in patients’ DNA coding for the NEK10 protein, but didn’t have enough evidence to tie the gene to their conditions.

Chivukula’s search eventually turned up six additional patients. All of them — including several under the age of 25 — had different mutations in the NEK10 gene, but overall the effects were the same: changes in the amino acid sequence of the NEK10 protein, and a condition similar to the woman’s, marked by pneumonia-like flares and swollen, enlarged airways. Whatever NEK10 was doing, the scientists could now assume it was associated with keeping the pathways to and from the lungs healthy.

Armed with the evidence that this mutation was associated with these patients’ conditions, Chivukula went back to the lab to find out what exactly NEK10 was doing in cells. First, he needed to find where it was being used. To do this, he turned to mRNA, or messenger RNA, the intermediate step between DNA and proteins. When a cell needs to express a certain gene, it creates an mRNA transcript. That transcript carries the genetic information to the ribosomes, where it is made into a protein.

Chivukula and his colleagues obtained airway tissue from the woman — her transplant meant they had good access to tissues to study — as well as from a few from people with normal lungs. They used a kind of genetic testing that allowed them to see what RNA was being expressed in the cells, offering a clue to where the protein was used: there were large quantities of NEK10 mRNA in specialized airway tissue, but hardly any in undifferentiated lung stem cells.

To see if they could induce these undifferentiated cells to produce NEK10, the researchers cultured them in the laboratory, using a trick to mimic the lining of a human airway. By allowing the stem cells to grow on a thin film where liquid medium meets the air, the researchers coaxed the cells to slowly mature and differentiate into airway cells in the lab. When the researchers looked at this lab-grown tissue carefully, they found much higher expression of NEK10 mRNA. This meant that whatever the protein was doing, it was most active in the cells that lined the airways.

Next they wondered whether the protein might be functioning specifically within one type of airway cell, of which there are many varieties with different roles. To test this, they used a fluorescent protein to mark the cells expressing NEK10, making these cells glow green. When they allowed the cells to differentiate, the brightest glowing cells were those that were covered in cilia. This suggested to the researchers that the woman’s condition was a kind of ciliopathy, or disorder associated with cilia. Nearly all vertebrate cells have some kind of cilia, and mutations that affect their structures can have consequences such as polycystic kidney disease, retinal disease, and conditions such as obesity and cerebral anomalies.

In the lungs, cilia move mucus by wiggling back and forth in tandem with their neighbors. Moreover, previous studies had found that disruption of airway cilia could cause a disease akin to that seen in NEK10 patients. When Chivukula took a closer look at the woman’s airway cilia, he found that they still wiggled at the same speed, but something was off; while normal cilia could transport polystyrene beads on a slithery wave of mucus, her mutated cilia could barely move mucus at all.

Under a microscope, the cilia were strangely clumpy and underdeveloped. The mutation, it turned out, had caused the cilia to be too short to effectively move mucus, leading to a build-up in her airways. This mucus build-up increased her likelihood of respiratory infections and, with each infection, her bronchi grew more enlarged and swollen until she could barely breathe on her own.

A NEW DISEASE

Chivukula, Sabatini, and coauthors published their findings on the new disease in Nature Medicine in February. From what they’ve observed in the seven patients they studied, the condition follows an autosomal recessive inheritance pattern — the gene must be knocked out in both copies for airway cilia to be affected — much like cystic fibrosis and most forms of ciliopathy that affect the lungs or other tissues.

Further research will determine how variable the condition can be depending on the type of mutation in the NEK10 gene. “It’s entirely possible that there are milder or subtler variants of this gene that are not, on their own, causing this sort of end-stage lung disease,” Chivukula says.

That might mean a mutation in the NEK10 gene that led to a protein that was deformed rather than completely unstable, he says, although at this point it is impossible to know for sure. “What we do know is that this double knockout of the gene sort of phenotype is quite rare,” he says. “But like for many genetic diseases, once you understand the severe ones, you can use that information to really dig into the more common forms.”

As for the woman who received the double lung transplant, “She’s doing pretty well,” says Chivukula. “She doesn’t need oxygen and can finally walk around without becoming short of breath. Being sick for 20 years takes its toll like it would for anyone, but she’s in a much better state than she was before her transplant.”

Dr. Armstrong and others at MGH are excited by the potential applications of Chivukula’s findings. “It’s pretty unusual [for a Pathways case] to have quite as beautiful a story as Raghu was able to put together that quickly,” she says.

Maybe in the future, Chivukula says, other patients in the woman’s position will be able to be treated before their condition becomes severe enough to need a transplant in the first place. Although much research remains to be done before the condition could be cured, Chivukula believes the potential is there. Cilia, he points out, have been shown to change slightly due to external causes. For example, smokers can have cilia that are a tiny bit shorter than those of non-smokers.

“We’ve shown that delivering extra active NEK10 protein actually causes cilia function to be improved, so that does suggest that this condition could be druggable in the future,” he says. “We just need to understand the biology a little bit better.”

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By Eva Frederick

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Chivukula, R. et al. A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance. Nature Medicine. 2020 Feb. doi: 10.1038/s41591-019-0730-x.

Harnessing the moonseed plant’s chemical know-how
Eva Frederick | Whitehead Institute
April 20, 2020

In overgrown areas from Canada to China, a lush, woody vine with crescent-shaped seeds holds the secret to making a cancer-fighting chemical. Now, Whitehead Institute researchers in Member Jing-Ke Weng’s lab have discovered how the plants do it.

Plants in the family Menispermaceae, from the Greek words “mene” meaning “crescent moon,” and “sperma,” or seed, have been used in the past for a variety of medicinal purposes. Native Americans used the plants to treat skin diseases, and would ingest them as a laxative. Moonseed was also used as an ingredient in curare, a muscle relaxant used on the tips of poison arrows.

But the plants also may have a use in modern-day medicine: a compound called acutumine shown to have anti-cancer properties (although not tested specifically against cancer cells, the chemical has been shown to kill human T-cells, an important quality for leukemia and lymphoma treatments). Acutumine is a halogenated product, which means the molecule is capped on one end by a halogen atom — a group that includes fluorine, chlorine and iodine, among others. In this case, the halogen is chlorine.

Halogenated compounds like acutumine can be useful in medicinal chemistry — their unusual chemical appendages mean they react in interesting ways with other biomolecules, and drug designers can put them to use in creating compounds to complete specific tasks in the body. Today, 20% of pharmaceutical compounds are halogenated. “However, chemists’ ability to efficiently install halogen atoms to desirable positions of starting compounds has been quite limited,” Weng says.

Most natural halogenated products come from microorganisms such as algae or bacteria, and acutumine is one of the only halogenated products made by plants. Chemists finally succeeded in synthesizing the compound in 2009, although the reaction is time-consuming and expensive (10 mg of synthesized acutumine can cost around $2,000).

Colin Kim, a graduate student in the Weng lab at Whitehead Institute, wanted to know how these plants were completing this tricky reaction using only their own genetic material. “We thought, why don’t we ask how the plants make it and then upscale the reaction [to produce it more efficiently]?” Kim says.

“By understanding how living organisms such as the moonseed plant perform chemically challenging halogenation chemistry, we could devise new biochemical approaches to produce novel halogenated compounds for drug discovery,” Weng says.

Kim knew that for every halogenated molecule in an organism, there is an enzyme called a halogenase that catalyzes the reaction that sticks on that halogen. Halogenases are useful in creating pharmaceuticals – a well-placed halogen can help fine-tune the bioactivities of various drugs. So Weng, who is also an associate professor of biology at Massachusetts Institute of Technology, and Kim, who spearheaded the project, began working to identify the helper molecule responsible for creating acutumine in moonseed plants.

First, the scientists obtained three species of Menispermaceae plants. Two of them, common moonseed (Menispermum canadense) and Chinese moonseed (Sinomenium acutum), were known to produce acutumine. They also procured one plant in the same family called snake vine (Stephania japonica) which did not produce the compound.

They began their investigation by using mass spectrometry to look for acutumine in all three plants, and then find out exactly where in the plants it was located. They found the chemical all throughout the first two — and some extra in the roots of common moonseed. As expected, the third plant, snake vine, had none, and could therefore be used as a reference species, since presumably it would not ever express the gene for the halogenase enzyme that could stick on the chlorine molecule.

Next, the researchers started searching for the gene. They began by sequencing the RNA that was being expressed in the plants (RNA serves as a messenger between genomic DNA and functional proteins), and created a huge database of RNA sorted by what tissue it had been identified in.

At this point, the extra acutumine in the roots of common moonseed came in handy. The researchers had some idea of what the enzyme might look like – past research on other halogenases in bacteria suggested that one specific family of enzyme, called 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. Although no 2ODHs had yet been found in plants, the researchers thought this lead was worth a look. So they searched specifically for transcripts similar to 2ODH sequences that were more highly expressed in the roots of common moonseed than in the leaves and stems.

After analyzing the RNA transcripts, Kim and Weng were pretty sure they had found what they were looking for: one gene in particular (which they named McDAH, short for M. canadense dechloroacutumine halogenase) was highly expressed in the roots of common moonseed. Then, in Chinese moonseed, they identified another protein that shared 99.1 percent of McDAH’s sequence, called SaDAH. No similar protein was found in snakevine, suggesting that this protein was likely the enzyme they wanted.

To be sure, the researchers tested the enzyme in the lab, and found that it was indeed the first-ever plant 2ODH, able to stick on the chlorine molecule to the alkaloid molecule dechloroacutumine to form acutumine. Interestingly, the enzyme was pretty picky; when they gave it other alkaloids like codeine and berberine to see if it would install a halogen on those as well, the enzyme ignored them, suggesting it was highly specific toward its preferred substrate, dechloroacutumine, the precursor of acutumine. They compared the enzyme’s activity to other similar enzymes, and found the key to its ability lay in the substitution of one specific amino acid in the active site– aspartic acid — for a glycine.

Now that they had identified the enzyme responsible for the moonseed’s halogenation reactions, Kim and Weng wanted to see what else it could do. A chemical capable of catalyzing such a complex reaction might be useful for chemists trying to synthesize other compounds, they hypothesized.

So they presented the enzyme with some dechloroacutumine and a whole buffet of alternative anions to see whether it might catalyze a reaction with any of these molecules in lieu of chlorine. Of the selection of anions, including bromide, azide, and nitrogen dioxide, the enzyme catalyzed a reaction only with azide, a construct of 3 nitrogen atoms.

“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, Weng and Kim 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 chlorines, 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 retain its original bioactivity with enhanced properties,” Kim says. “It expands the biocatalytic toolbox we have for natural product biosynthesis and its derivatization.”

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Written by Eva Frederick

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Citation: Kim, Colin Y. et al. The chloroalkaloid (−)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants. Nature Communications. April 20, 2020. DOI: 10.1038/s41467-020-15777-w