Episode 32: Peptide Protectors - New Defenders in the Fight Against Superbugs with Key Opinion Leader Bill Wimley

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And I'm Dr. Elaine Hamm, the Executive in Residence for the Tulane School of Medicine.

And this is Bio from the Bayou.

Welcome back to Bio from the Bayou. I'm your host today, Dr. Elaine Hamm, and I am the executive in residence at Tulane School of Medicine. Today's guest is Dr. Bill Wimley, and he is the professor of biochemistry and molecular biology at Tulane University School of Medicine, and his lab is at the cutting edge of research on antimicrobial peptides to treat drug-resistant infections. And I'm I'm really thrilled to be able to dust off my microbiology hat and talk to a fellow microbiologist. So, welcome to the show, Bill.

Well, Elayne, I'm really glad to be here.

Well, wonderful. So, as I mentioned, your lab does a lot of work concerning antimicrobial peptides, or AMPs for short. Can you explain to us what an antimicrobial peptide is exactly?

Yeah, an antimicrobial peptide is a type of antibiotic that Mother Nature uses. They're made by humans, they're made by Insects and other animals are made by other bacteria in sort of a germ warfare that bacteria use to combat each other. And unlike most conventional antibiotic drugs, antimicrobial peptides, they're a different kind of material. They're basically small fragments of proteins, and they act in a very different way from conventional antibiotics. Meaning they, they attack the bacterial membrane, which is The barrier between the inside of the bacteria and the rest of the universe. So this is a very valuable target, and it's a vulnerable target for bacterial survival, you know, in an infection where the host is trying to combat the infection. So antimicrobial peptides attack this critical barrier and are able to kill bacteria that have evolved, for instance, drug resistance to conventional antibiotics.

Oh, I remember first hearing about antimicrobial peptides Years and years ago, as it related to the Komodo dragon. I don't know. One of the things that the Komodo dragon can do in terms of killing its victims is that they ended up dying from sepsis from one bite, and they just follow their, their victim until they die. Well, what made the Komodo dragon not die from its own mouth, you know, teeming full of bacteria? And they found that it was these antimicrobial peptides that they actually Made for themselves to protect themselves against the infections that they could cause their victims. So I just remember being really fascinated by antimicrobial peptides and sort of how they— we really derived them from nature and are able to try to use them to combat nature when nature goes bad, as it were.

That's right. There's a great story that the very first antimicrobial peptide that was discovered was discovered by a guy named Michael Zaslav, and he was He was studying Xenopus oocytes. So oocytes are large egg cells, really the largest single cell available to a researcher. He was getting these oocytes from Xenopus, which is basically the African clawed frog.

Yeah, frogs, yeah.

Yeah, and Zaslav noticed that they would do surgical removal of the eggs on these frogs, open them up, take the eggs for their experiments, and sew them back up and drop them back into their dirty fish tanks, and they would use the same frogs over and over again. And he realized that these surgical wounds never became infected, the frogs were always fine.

Yeah.

So this launched him into a career of studying why these frogs were protected from infection. It turns out in the skin mucus of the African clawed frog is the first antimicrobial peptides discovered called magainin. And these frog skin antimicrobial peptides really started the whole game. And now researchers have found Antimicrobial peptides are really everywhere. Most organisms have some form of antimicrobial peptides. Humans have many antimicrobial peptides, some secreted in certain tissues and some sequestered in certain types of immune cells. And most amphibians and most reptiles, including the American alligator and the Komodo dragon and many others, have antimicrobial peptides as well. So again, how long ago was that first antimicrobial peptide That was discovered in the early '80s, maybe 1982 or 1983. Around the same time, some Japanese researchers discovered an antimicrobial peptide in silk moths called sacropins, and some researchers in California discovered some human antimicrobial peptides called defensins. And all this happened in the first half of the 1980s, and then the field really exploded from there.

Why the bigger interest right now in terms of antimicrobials, and why antimicrobial peptides versus others?

Well, I mean, as you indicated earlier, drug-resistant bacterial infections are on the rise, and the fraction of clinical isolates of bacteria taken from patients in the hospital that are resistant to drugs and resistant to many conventional drugs is continuing to increase. And for some species of bacteria, like Pseudomonas aeruginosa and Staph aureus, essentially 100% of the isolates taken from patients in hospitals are drug-resistant. So Drug resistance is growing very rapidly and becoming a widespread problem. And the number of patient deaths from sepsis and other bacterial infections is also growing. So this is a tremendous problem that has led some media outlets and even some scientists to sort of proclaim that the end of the antibiotic era is near.

I'm probably one of those scientists. I definitely am more worried about antimicrobial resistance As the next new pandemic versus the avian flu, which is, you know, the hot topic these days. But antimicrobial resistance is always one of the bigger challenges that are really looming over us, whether we realize it or not. And I really think that, having worked on traditional antibiotic programs myself, it is really, really challenging to develop a new antibiotic, a traditional antibiotic where we think of it as a small molecule. And I really don't think— That I'm gonna call them sort of antimicrobial alternatives in the sense of like, they're not small molecules. But I think that we really need to start thinking about alternatives to the traditional small molecules, 'cause those are the ones that are the more easy to get resistance to, whether it's the bacteria flushing them out through efflux pumps or, you know, those types of defenses that they've developed over the years. I feel like antimicrobial peptides and things like phage too have really not been looked at and not really been given their due in terms of the commercial applicability. Would you kind of expand on that, that idea of like, why haven't AMPs been more at the forefront of this massive need for a new drug?

Well, I mean, first, the conventional antibiotics seemed for decades since the discovery of penicillin, they seem so easy. Small molecules that are very cheap to synthesize and you could produce literally tons of penicillin or a similar conventional antibiotic, and they solved all the problems. Everything was great, you know, until, of course, very shortly after the introduction of each new conventional antibiotic, resistance developed. So classical antibiotics typically have a single biochemical target, a single protein, a single biochemical process. And when you have a single target, then it becomes relatively easy to develop resistance by essentially by testing mutations in that single target. And as you mentioned, things like efflux pumps, which are generic drug flushing mechanisms, become very effective at getting rid of all molecules that are similar to the conventional antibiotics. And conventional antibiotics, of course, these small molecules, they're constrained by their Chemistry, so they all have very similar physical properties in terms of how they get across membranes. And so the efflux pumps use this to their advantage and can pump them out essentially indiscriminately. So the industry and the science of antibiotic development is slowly coming around to realize that the miracle drugs have weaknesses, and we need to start thinking about drugs that use different mechanisms of action. So Phage drugs, for instance, they work pretty well. And they've been— there's been a lot of research over a lot of decades, and phage drugs are pretty effective. Antimicrobial peptides are really pretty effective. They work pretty well, and they work against a lot of drug-resistant bacteria. But these approaches are— they're much more complex. The molecules are more difficult to make. They're more expensive to make. The phage are more expensive to produce. You know, peptides are relatively much more expensive than making a small molecule in terms of synthesis and purification. And so, you know, there's been a lot of resistance to apply these new sort of modalities to antibiotic development. But the world is coming around as more and more bacteria are becoming completely resistant to all conventional antibiotics. These new approaches are gaining traction. There's more attention, there's more research interest focused on them. And, and, you know, the critical thing— that is needed is that the drug development industry has to become interested. And so there is growing interest in the industry as well. So I think we're, we're really seeing a change in thinking, a change in emphasis on the types of drugs that, that the biotech industry is thinking about. Antimicrobial peptides are basically coming into their own as we speak.

Yeah. And I think that, I mean, sort of one of the challenges with antimicrobials versus As you mentioned, the small molecules, I mean, they're just, at the time, many years ago, that they were just much more expensive to make. And so therefore they weren't as commercially of interest because of that limitation. So one of the challenges as it comes to sort of, you know, creating antimicrobial peptides is obviously designing antimicrobial peptides and manufacturing antimicrobial peptides. So what has your lab specifically done in terms of You know, engineering and sort of optimizing the AMPs themselves.

We did some studies a while ago that we had developed several very good antimicrobial peptides, that is antimicrobial peptides that were very effective in the Petri dish, very effective in the test tube against all kinds of human pathogens. We thought this was great, and when we tested these peptides in animal models of infection, they were essentially completely ineffective. And so this started us, and the same sort of story has been observed in several human clinical trials of antimicrobial peptides, where they work very, very well in the laboratory, but don't work as well in the clinic. So we launched into a long series of experiments where we tried to really understand what are the impediments to the activity of antimicrobial peptides in the body versus, you know, in the test tube. And essentially, we We quantitated these things, we learned what's important, and what we found is what's most important, or one of the major impediments, is that antimicrobial peptides bind at least weakly to host cells. They bind at least weakly to host tissue. When you compare them side by side, antimicrobial peptides bind very well to bacteria, they bind very weakly to host cells. Everyone thought this was okay, you know, a little bit of background binding to the host cells, But it turns out, even in a very serious infection, you know, the host tissue, the host cells outnumber the bacteria, you know, 1,000 to 1 or 1 million to 1. And so even very weak binding to host cells was enough to completely prevent activity because host cells were so abundant. There was that host cell binding, and we also discovered that, you know, proteolytic degradation was important, an important impediment to the activity of antimicrobial peptides, and The lack of high solubility was an impediment. And so what we did is we, once we learned and quantitated what these impediments were, we carried out what we call synthetic molecular evolution, which really just means we made a library of potential antimicrobial peptides based on some peptides that we had already discovered. And we screened this library under conditions that included highly concentrated host cells. We used red blood— human red blood cells as the host.

Wow.

So we screened this library under conditions where we could select for peptides that had very high solubility, select for peptides which retained their activity in the presence of concentrated host cells. And it turns out, as we already knew, you know, the presence of concentrated human red blood cells eliminated the activity of 99.9% of the members of this peptide library. But That 0.1%, that 1 in 1,000 peptides that did not lose activity in the presence of concentrated red blood cells, you know, they were our winners. So we were able to identify those by screening under these conditions that included these important impediments. And so we identified a family of peptides that retained extremely potent antimicrobial activity in the presence of concentrated host cells. This was the start of The lineage of peptides that we have continued to evolve over the last few years, basically for their broad spectrum antimicrobial activity that is retained in vivo, in the body.

Yeah, I can tell you that I was part of a company where protein binding absolutely killed our company. And we were even working on a small molecule, but, you know, drugs will bind to things within the human body, but whether it's at a good high percentage or a bad high percentage has always remained to be seen. And you definitely have to take that into consideration when you, when you put something in a, work Amazingly well in a test tube, but the moment that you put it in an animal, it may just completely crash. And one of those reasons is, yeah, protein or cell binding as it relates to AMPs. So can you talk a little bit about your engineered peptide Dkanga and its subsequent generations, and how effective is it in treating bacterial infections, and sort of what types of bacterial infections have you looked at?

Sure. The peptide Dkanga essentially was a rational variant of the peptides that were selected in this screen I just mentioned. And now we've gone beyond and we have a couple additional gain-of-function peptides related to Dkanga. But essentially, Dkanga was a consensus sequence that we derived from this high-throughput screen, and it really has all of the properties you would like an antimicrobial peptide to have. It has broad spectrum, sterilizing activity against a large set of the human bacterial pathogens, including the pathogens we typically find infecting wounds. So this is a good place for an antimicrobial peptide to be. This is a good target. An infected wound is a good target for antimicrobial peptides. We find that D. conga has very good activity against many of the bacteria we would find in a wound. D. conga also has several other very Important activities, things that we did not screen for specifically, but that it has as part of its properties. And one is D. conga is extremely effective at killing bacterial biofilms. So biofilms, especially in wounds and in medical devices like PICC lines and catheters, etc., are an extremely important problem because bacteria in a biofilm are essentially already protected from antibiotics. They're metabolically somewhat quiescent, so antibiotics targeting protein synthesis or DNA synthesis, for instance, are not very effective there. Bacteria in biofilms are physically protected from the environment, so many antibiotics don't even have good access to them. And so even bacteria that are susceptible to certain conventional antibiotics become resistant by virtue of being— of participating in a biofilm. So bacterial biofilms are a very important problem. When we tested Dikanga and its successors against bacterial biofilms formed by gram-negative bacteria, formed by gram-positive bacteria, we found very good killing activity against the bacteria in these biofilms. So this is a very important activity, and after the fact, we can understand what's going on here, that these peptides attack the bacterial membrane, and so the bacteria don't have to be metabolically highly active. The membrane is vulnerable Even if they're somewhat quiescent in a biofilm. And so these antimicrobial peptides, Dkanga especially, is very good at killing bacteria that are protected within a bacterial biofilm. We showed this effect even that Dkanga has very strong antibiofilm effects even in an infected wound in an animal model. The second thing we found that is attracting a lot of our attention these days in the lab is that Dkanga does not induce resistance. So we took a bacterium called Pseudomonas aeruginosa, one of the very common drug-resistant bacteria that cause infections, and we passed the Pseudomonas against a set of conventional antibiotics. And as we all— as many labs have shown, Pseudomonas gains resistance to these drugs very quickly. When we passaged Pseudomonas aeruginosa against D. conga and several related peptides, we found no acquisition of resistance, even over many passages.

Wow.

These are overnight passages, so that each passage is many generations of bacterial replication. We find no increase in resistance, so no change in susceptibility of Pseudomonas aeruginosa to these drugs over these many passages. This is a very important observation because Pseudomonas, for instance, it has mechanisms to defend itself against antimicrobial peptides. It can make— physical changes to the state of its membranes. And Pseudomonas, for instance, can quickly become resistant to colistin, for instance, which is an FDA-approved antimicrobial peptide. Colistin has some problems, such as it causes a lot of kidney toxicity, so it's generally used as a drug of last resort. But Pseudomonas, for instance, quickly becomes resistant to colistin under the same conditions where it does not become resistant to Dkanga. So again, we're extremely interested in the mechanism of what we call resistance avoidance. And at this point, I don't want to speculate on the mechanism, but we're, we're working hard to understand this because this is a critical property that needs to be present in any future antibiotics that we use. If bacteria can rapidly become resistant to a new antibiotic, it's only going to be useful for a short period of time. If we can develop antibiotics which avoid the rapid development of resistance, then, you know, we've got a drug that's Potentially useful for a much longer period of time.

Yeah, that's really key. I mean, and that's one of the things that the FDA is now on the checklist of understanding mutation, you know, frequency and the ability to get resistance to the new drug as well. And I kind of want to pivot away from sort of thinking through like human health. I mean, when we think about antibiotics, we tend to just sort of immediately think of, well, someone that has an infection, but, you know, antimicrobials are in a lot of supply chains throughout a lot of different areas that impact our lives. And when we think about animal feed supplies and pet food and even cosmetics having antimicrobials in them as well, I mean, there's a lot of different things that touch our lives are impacted by antimicrobials. So, so just sort of thinking with that lens, like what are the broader implications of your drug beyond just sort of human drug that we, you know, beyond just sort of the standard thing that we would put into a prescription bottle?

Well, yes, it's exactly as you said. The broader implications go, you know, beyond treatment in the hospital. They go into everyday products we use that we would like to be free from bacterial contamination. I mean, we have a collaboration ongoing that's just begun with the FDA, and they're trying to develop Dkanga and similar peptides into products that could be used for protection of the blood supply, for instance, for killing bacteria that could potentially contaminate donated blood, for instance. And so we're interested in protecting the blood supply. And these peptides, there's some potential for them to be active under those circumstances. And places where you'd want a sterile environment would be things like, as I mentioned earlier, the surfaces of medical implants like catheters, which is biofilms forming on the surfaces of such devices are a very common problem and a starting point for a lot of more serious bacterial infections. So antimicrobial peptides have the potential to be able to protect surfaces, for instance. And there's a lot of research in the field on coating surfaces with antimicrobial peptides, for instance, and that gives them antibiotic properties at the surface level, which is important. And we think about things like contact lenses and those sorts of devices where bacterial contamination is a common minor problem and occasionally a very serious problem. So, Antibacterial compounds are useful in all of these applications. And, you know, it's worth noting that the antibacterial compounds that we currently find in soap and other cosmetic products are really very, very poor antibiotics. So they're more effective as a selling point than as an antibacterial compound.

So yeah, I mean, that's because there's other applications for antimicrobial peptides beyond drugs. I mean, one of the key aspects would be, would this be shelf-stable? You know, as you talk about D. conga specifically, is this shelf-stable and easy to make?

Peptides are not easy to make, at least relative to conventional small molecule antibiotics. But once you make them and purify them, they are highly stable. They have a very long shelf life, and they can be stored in powdered form. So they have a very long shelf life, even at room temperature. So, you know, the cost of Something like a peptide synthesis to be used as a drug or as an antibacterial compound, of course, goes down with scale. So once the industry has decided that one of these molecules is needed and could be useful in a large-scale application, the cost of production, you know, will go down by orders of magnitude compared to the very small-scale production that we do for the laboratory, for instance. So, you know, the scale improves the cost dramatically.

Right.

Peptides will never be as cheap to make as a small molecule antibiotic, but as we've discussed today, they have significant advantages as antibiotics. And so I think that cost-benefit analysis will eventually turn in the favor of antimicrobial peptides.

Yeah, you can have a really cheap antibiotic, but if it's just going to have resistance develop to it, then there's no point. So having something that has longevity, it might be a little bit more expensive, Would be worth it. Well, sort of talking along industry lines, how are you aiming to progress this research going forward, and are you looking for partnerships?

Yes, we're definitely looking for partnerships. We're interested in moving our most recent generation of peptides, you know, into the clinic. We're openly, uh, searching for partnerships in, in biotech, and, and, you know, we've had some discussions. We're still waiting to move forward in, in that direction. But what we are doing as a small academic lab is we are developing these peptides, developing the techniques for identifying such peptides. And, you know, as an inventor and a member of the Tulane community, you know, we have the intellectual property on these peptides. And so we are essentially waiting for, searching for partners to help us move this into the next step, which is a more significant investment of funds and a more significant sort of advance that will need to be carried out to show that the peptides we've developed by synthetic molecular evolution will be useful in the clinic. And I, I believe they will be.

Absolutely. And, you know, I think that having worked with you for many years now, I can say you're, you're incredibly collaborative and that you've done a variety of different types of collaborations with large companies, including some as simple as just an MTA and testing the drug in their own labs. And so And I think that that's another selling point is having good collaborators that really just want to move a project forward. And in this case, a really important piece of getting help to patients in some form or another. And so, well, I really want to thank you for sharing your expertise with us, and we'll be sure to keep an eye on your research as it progresses.

Well, thank you, Elaine. I was really glad to talk about my favorite subject today, and thanks for asking good questions.

I love to be able to talk about microbiology and dust off my old, you know, PhD every now and then. So it was my pleasure as well. So, well, as always, we will have Dr. Whimley's contact information and informational links posted in our show notes. Make sure you check them out to learn more. Thank you once again for joining us for Bio from the Bayou. Thanks for joining us for Bio from the Bayou, and we hope you'll join us again.

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