For Zhilei Chen, PhD, associate professor in the Texas A&M College of Medicine, protein engineering is the "hammer" she uses to tackle "nails" like Zika virus and C. diff. Join us for a discussion about her recent research on neutralizing toxins produced by C. diff bacteria.
Christina Sumners: Welcome to Science Sound Off. I’m Christina Sumners.
Tim Schnettler: And I’m her co-host, Tim Schnettler.
Christina Sumners: And we’re here today with Dr. Zhilei Chen. She is an associate professor from the Department Of Microbial Pathogenesis & Immunology at the Texas A&M College of Medicine. Welcome to the show.
Zhilei Chen: Thank you.
Christina Sumners: We’re thrilled to have you here today. So your research is on protein engineering. Is that a fair statement?
Zhilei Chen: That’s right, yeah.
Christina Sumners: Okay, so what is protein engineering?
Zhilei Chen: Okay. So protein engineering, basically we try to engineer proteins for different applications so it can be used in laundry detergent and it can be used in medicine. So that’s what we are interested in. And it can be used in materials to build coatings for implants and also can be used for making materials to grow cells in it. So kind of a big field, but we are most interested in engineering proteins for therapeutic application.
Christina Sumners: These are drugs that are different from the normal pharmaceuticals that we take. How are they different from just the drugs you usually get in the pharmacy?
Zhilei Chen: Yeah, so I would say in the 19th century, until now, all the medicines are made of small chemicals. They are made by chemists and they are engineered by medicinal chemists who make these molecules. You can think of different small atoms, putting them together to make different drugs. So, with the development of the recombinant technology in your common DNA technology in the 20th century, it opens the door to a new set of tools people can use to generate proteins.
That’s what we are doing. We try to make medicines out of proteins because in nature, so proteins are our natural defense mechanism. Our body uses protein to fight off infections, various kinds of diseases. We tried to borrow this page from nature and tried to try to use that to develop medicines to treat different diseases. The protein medicine everybody knows about is the antibodies, right?
Christina Sumners: Oh okay.
Zhilei Chen: Antibodies are proteins and they are basically engineered by the body to fight various infections. Throughout your lifetime you get different infection, you have different ones over a different time and your body is constantly engineering different antibodies to fight off this infection over time. What we try to do is try to do this, help in case someone who is not able to fight off the infection themself, then we try to give them a boost like when you get sick, you take a med. And some people will get over the sickness just by themself, but sometimes you need a little help with these medicines, so we try to engineer proteins that can help people get better quicker.
Christina Sumners: That makes sense. Okay. And so it really is taking the body’s own natural ability and just boosting it, like you said, but with recombinant DNA you can build these building blocks to do almost anything. Is that right?
Zhilei Chen: I wouldn’t say about anything but a lot of things because proteins are very diverse and the protein can have a lot of functions. Usually, I would say most proteins, they cannot handle high temperature, very harsh conditions, but some proteins do. Just like most organism cannot live at very high temperatures, but some organisms do and they evolve ways to do that and usually by having their proteins with different properties, like the most stable, or stable against these different chemicals. Yeah, so you can engineer a protein to have many different properties, almost unlimited amount, a variety of properties proteins can have.
Tim Schnettler: You mentioned fighting off the infection and some people not being able to fight off the infection. Is that something maybe that this has been developed for toward someone who’s already sick with something like cancer, or something like that, to help them fight off other things that happen when they’re dealing with this?
Zhilei Chen: Well, yes and no. Say just like a medicine, right? For example, somebody gets a cold, most people get over a flu pretty quickly, some people don’t, so we literally just try to use protein to mimic these small molecules to have these different properties. I want to say a biggest advantage of protein versus a small molecule is when you take a drug, the common thing the doctors say will be a side effect. What would be the side effect?
Christina Sumners: Sure.
Tim Schnettler: Right.
Zhilei Chen: The side effect usually, I would say almost all of the time, it comes from the small molecule, because it’s so small, it targets your target, but it also targets something it’s not intended to because it does not have the specificity that it can be so specific to your target protein, or a receptor in your body, but protein is much bigger. The biggest advantage is it can generate to bind the two with much higher specificity and affinity. As a consequence of that you will have much fewer side effects, so side effects is much, much reduced compared to conventional small molecule drug.
Christina Sumners: Because it’s just not affecting all the things you don’t want it to, that you’re touching.
Zhilei Chen: Exactly, yeah.
Christina Sumners: Okay.
Zhilei Chen: That’s with the body. We all have so many different antibodies, we don’t have side effects.
Christina Sumners: Usually yeah. You recently had an article in the journal PLOS Biology and you were looking at C. diff, the bacteria we know C. difficile. Tell us a little bit about that research and what y’all found?
Zhilei Chen: Oh okay. This is actually a project initially started with the College Of Medicine seed grant, so the CSTR Foundation gave us a seed grant to allow us to generate initial results for this study. So C. diff is an infection that affects, I will say, millions of people each year. It’s actually the number one hospital acquired bacterial infection, which is leading to death in patients. So C. diff is a bacteria that’s everywhere in the environment, but its specific affect patient in hospital because when patient take antibiotics it’s believed to be the major reason why people will acquire the C. diff infection. The problem with C. diff infection is it recurs, so people can have two, three, four, five times infection and it kills, something like maybe 30% of people would die in 30 days after the initial infection.
Christina Sumners: Oh wow.
Zhilei Chen: It’s actually a really big problem, so CDC consider that to be the top one urgent problem we need to deal with in this country. So C. diff is a bacteria. It’s a spore that’s floating around in the environment. Everybody can breathe in, eat, it gets into your stomach. And unfortunately usually the stomach acid will kill most of the bacteria. In this case, the stomach acid, actually activates the spore to allow the bacteria to germinate. And then it germinates in your stomach and it goes into your gut and it basically just proliferates in your gut.
And then once these bacteria grow they secretes toxins. People don’t really know why the bacteria secretes toxin because they don’t need to secrete toxins.
Christina Sumners: You think they wouldn’t want to kill the host?
Zhilei Chen: Exactly. They don’t need to, but they do secrete a toxin, these toxins enters the epithelium, so that the cell that lining your gut, and kills these cells. As a result of that, you develop this colitis, which is like a canker sore in your mouth, but it’s in your gut and it can be really painful and causes bloody diarrhea. So tell, tell sign for CDF infection is a bloody diarrhea and people can have recurrent infection. Normally, the way we treat the C. diff infection is by using more antibiotics, try to kill all the bacteria, so it usually works, I will say, pretty well, but in the recent years due to the development of antibiotic-resistant bacteria, the cure rate has been going down. And then you have to do it again, give them more antibiotics if they get the infection again, the next infection, so that’s the problem.
What we want to do is try to develop a protein, specifically something that can be a drink, somebody can just drink it and it will enter the gut and neutralize these toxins, so we don’t have to affect the bacteria growth directly, but we can prevent the damage that are caused by these bacteria.
These protein we engineered, the scaffold that we chose is called a DARPin, it’s a Design Anchoring Repeat Protein, so it’s a scaffold. We choose this scaffold because it was found to be really stable. It’s a protein that can endure very high temperature and then fold very stably, so with that we engineered these protein to bind to the toxins. Specifically one of these toxin is called a TCDB. The people in my lab, they worked really hard on the project. We generate a library, so the technique we use is called directed evolution. It actually got Nobel Prize last year. Frances Arnold is my academic grandmother, so she is the founder of this directed evolution principle and that’s what we use for protein engineering.
Christina Sumners: Okay.
Zhilei Chen: The concept is rather simple. Just like natural evolution where it’s a process where mutations happens randomly and that is coupled with a selection pressure that allows the nature to select for organisms who is more fitted the properties. For example, we have many different dog varieties, right?
Christina Sumners: Sure yeah.
Zhilei Chen: They all come from wolf, but because of this random mutation coupled with selection, you are able to get dogs, they almost look like two different species. When you compare a chihuahua with a Great Dane, right?
Tim Schnettler: Right.
Zhilei Chen: In protein engineering we do something very similar, but we do that in a test tube, so we have a gene which is a DARPin, so it does not bind to our target toxin to start to neutralize it, but we introduce mutations into this gene. In this case we create a library of these DARPins, about 10 to the ninth, so that’s a trillion. Is it a trillion?
We have tension line, different DARPins, we express these DARPins phase, which is a bacteria virus. And then we select for these phase, so they can bind to our target toxin, so from there we further screen the library we have. Just because they bind to a toxin does not mean they can neutralize the toxin, so from all those binders we did a secondary functional screening. We look for those ones that can neutralize a toxin and that’s how we derive a first panel of 12 different DARPins that have a very strong neutralization activity.
From there we did a secondary screening, so we try to combine these 12 DARPins in a random fashion, see if they can have something that work even better. That allows us to get a second generation DARPin, which is a dimer, that it turn out to neutralize a toxin at picomolar EC50, so that’s a really, really strong potency for these DARPins.
And then with a collaborator, Professor Junjie Zhang, so he helped us to solve the cryo-EM structure of the toxin in complex with our DARPins to help us understand how do these DARPin work, the mechanism of action and he generates some really beautiful EM structure images for us. And another co-author on the paper is Professor Hanping Feng. He is from University of Maryland and then he help us do the in vivo study to see whether these DARPins can protect from these toxin challenge.
Christina Sumners: You’ve basically created these proteins and shown that they seem to work in animal models. What’s next? Are you going to move them forward?
Zhilei Chen: Yeah, so that’s our goal. What we are doing right now, we have submitted a patent application through the A&M. And in the second generation we want to basically increase their stability further because in the gut we have all these digestive enzymes, so for something to work really well in the gut, they also need to be stable against these proteases. We have some data that haven’t been published. We were able to do, I would say, maybe third generation engineering to get them to be very protease stable, so in the next experiment we’ll launch a test in additional animal models. Basically, it’s in a model where we can challenge the animal with the spores of these bacteria and see if our DARPins can protect them from the toxicity of these toxins.
Tim Schnettler: I want to back up a little bit. You mention that this is a common thing in hospitals and that the CDC has identified as one of their top priorities. Why is it such a common thing in hospitals? Why do you see this infection in hospitals?
Zhilei Chen: Oh, it’s just because in hospitals people are taking antibiotics, right? Especially in hospitalized patient, so the doctor usually gives them all the antibiotics when they are hospitalized and because of these antibiotics, that allows these bacteria, an opportunistic infection, allow them to take residence in these patients. And then, even once they get discharged and when they go home, they realize, “Uh oh, I’m having bloody diarrhea and I’ll have to come back to the hospital.” It’s a major reason for people to come back and be hospitalized again, and then again, because of the recurrent infection.
Christina Sumners: It’s ironic that a bacteria infection can be caused by antibiotics. I don’t think most people really realize that that happens.
Zhilei Chen: Well, considering antibiotic resistance is a global problem, so the more antibiotic we use, the more we have to use it to kill the bacteria so we don’t have a good solution to it. I think that’s why the NIH is looking for way to treat this bacterial infection without fostering more antibiotic resistance development.
Christina Sumners: So even when we create new antibiotics and those bacteria become resistant to those, and it’s a continuous cycle. But your approach would bypass that all together.
Zhilei Chen: Hopefully, that’s our goal, so hopefully. One of the way to treat infection is try to leave the organism alone and then deal with the effect of this infection, right?
Tim Schnettler: Mm-hmm (affirmative).
Zhilei Chen: In this case, C, diff alone, they’re not a pathogenic. In fact, people have done a trial where they just feed the people with these strains of C. diff that does not secrete a toxin. It turn out to actually have benefits. Patient just reduced their infection rates because of the non-pathogenic strain can compete with the pathogenic strain to reduce the toxin load. We really think if you can just neutralize the toxin, you can probably just leave the bacteria alone. Then you don’t have to worry about fostering more antibiotic resistance.
Christina Sumners: And eventually the body will fight it off or not, and either way it’s not really doing any harm.
Zhilei Chen: Yes. Eventually your body, any other bacteria, just takes time for all the other bacteria to take residence in your gut.
Christina Sumners: A microbiome to reassert itself.
Zhilei Chen: That’s right, that’s right. Yeah, it’s just this bacteria, they grow really fast, so they first come and take over. And they cause a lot of damage to your gut, so your gut is not able to recover, but if you allow the gut to recover without these damages then other bacteria can restore the balance in your gut.
Tim Schnettler: Let’s talk about how you got involved in this research and what made you decide, “This is where I wanna go; this is what I wanna study.”.
Zhilei Chen: We are very interested in protein engineering. I’m fascinated with this ability, like the rest evolution, ever since my grad school, the ability to almost play God in a way that we can create the proteins. These have been long time consider, the forbidden thing, something produced by nature, proteins, people cannot change them. But now, with these genetic tools, we can really make many wonderful things that in the past only God can make, right?
Tim Schnettler: Yeah.
Zhilei Chen: And I’m really fascinated by this way to mimick these natural evolution in the test tubes to create new fascinating proteins with new properties. Yeah, so I’m interest in therapeutic proteins because I see a lot of potentials. For us, particularly, I want to make proteins that can be used to fight infectious disease, so that’s one of my passion.
And part of the reason is that most of the pharmaceutical companies in this country, also in around the world, people are mostly focused on engineering antibodies for cancer therapy. And antibodies are really expensive to make and then antibody therapy are really expensive. For cancer, we can handle it. For cancer it’s rare, in a general sense, among the population, so we can handle it, but for infectious disease it affects millions and millions more people, so antibody is just not a solution to treat infectious disease, in my opinion. So that’s why there’s interest in this Design Anchoring Repeat Protein because these proteins can be produced very cheaply, almost at a fraction of the price as an antibody just because antibody requires a mammalian cell culture to produce, which is just naturally very expensive. While the protein we are interested in, the DARPin, it can be produced literally a bucket amount in bacteria, so E. coli can very efficiently produce these DARPins. We use this, really a protein that can be very cheaply produced. If we can engineer them to mimic the function of antibody, it maybe has a potential to treat infectious disease for a much greater population. That’s how I got started with engineering DARPin, using DARPin as a scaffold to engineer therapeutic proteins.
In the lab we also engineer DARPins against other infectious disease. One of them is it’s called EHEC, so it’s a bacteria that secretes a different toxin called the Shiga toxin and its effect causes diarrhea. And in that case it causes kidney damage, especially in children, so we think by engineering DARPins that can neutralize these toxins we can help a lot of these patients affected by these infection.
Christina Sumners: So you mentioned bacteria. Can these techniques be used against viruses as well?
Zhilei Chen: Yes, definitely. So, as I said, proteins are our tools and we can use it to treat various diseases. So another area we’re very interested in is viral infections, so bacteria infection and viral infections. Specifically, we are interested in the Zika virus infection and influenza virus infection. We chose these two viruses in part because these are viruses that can potentially affect huge populations and then we don’t have a really effective way of treating these infections.
In a case of influenza, a lot of experts I talk to, they always say it’s not whether we going to have a next big outbreak, it’s when we’re going to have a next outbreak, right?
Christina Sumners: Mm-hmm. Yup.
Zhilei Chen: If the next outbreak happens, are we ready for it? Well, the answer is probably not.
Christina Sumners: Uh-oh.
Zhilei Chen: With the medicines we have, there’s no way we can handle another outbreak, so that’s why I see our DARPin has a potential because if we can engineer these DARPins that can be rapidly produced that is effective, then you can potentially distribute it to millions of people in a short period of time rather than antibody takes a long lifecycle to produce. Even though we do have antibody that’s effective against these influenza infection, there’s just no way we can make enough in a timely manner to make an impact…
Christina Sumners: To stop a pandemic before it can-
Zhilei Chen: That’s right, to stop the pandemic.
Christina Sumners: Wow, okay. And you mentioned Zika as well? I know that’s been in the news in the last few years, does that have a similar approach?
Zhilei Chen: Yeah, so the Zika is similar approach. In that case we are working on engineering proteins that can prevent the disseminated infection of a Zika because the Zika is a mosquito. The infection starts with a mosquito bite, so the goal is, say try to engineer these DARPins that it can prevent the spread of the virus from a local bite to other organs in the body, so definitely prevent it from getting into the fetus.
Christina Sumners: And that’s where the big problem is, of course, with Zika is-
Zhilei Chen: The big problem, yes. That’s right.
Christina Sumners: Is when it gets to the fetus and causes problems, microcephaly and such. That would be very cool if you could just keep it in the arm or wherever you got the mosquito bite.
Zhilei Chen: That’s right, so that’s the goal.
Christina Sumners: Fantastic. Well, we’ll have to get you back on the podcast at some point to talk about how all these projects are coming.
Zhilei Chen: Yes. I’ll be happy to.
Christina Sumners: Well, thank you so much for coming to talk to us today.
Zhilei Chen: All right. Okay, thank you.
Christina Sumners: And thank you all so much for listening, and we’ll see you next time.