GryphonCast Episode 5 Transcript

Speakers: Michael Lim and Kelly Boddington

Michael Lim

Since its legalization in Canada back in October 2018, a variety of cannabis products have made their way onto the market. Where your mind may immediately jump to smoking marijuana, when you first heard about cannabis. Many other products focus on the use of delta nine tetrahydrocannabinols or THC and other cannabinoids or CBDs which are known to help with a variety of ailments such as anxiety, insomnia, and chronic pain. THCs and CBDs have found new uses in a variety of products such as lotions and candles. But, have you ever wondered about how these different cannabis products were first identified and produced? Open your ears and mind? Let's chat about that.

Welcome to Gyphon CAST, a podcast where we casually chat about science coming out the College of Biological Science at the University of Guelph and how their work can affect lives around the world. I'm your host, Michael Lim. With me today is special guest and post doc, Dr. Kelly Boddington, and we chatted about some of the work she's done and Dr. [Tariq] Akhtar's lab to identify and synthesize a new cannabis compound known as bibenzyls, and we'll discuss what her work might mean for the expanding natural product market. Welcome Kelly.

Kelly Boddington

Thanks so much for having me.

Michael Lim

We're very glad to have you. So, if you had to speak to some random person on the street, or maybe you walked into some random undergrad class, how would you describe the work you do? Or I guess in general, the work done in Dr. Akhtar's lab.

Kelly Boddington

That was always a fun question to ask researchers ... can they phrase it in a way just anyone on the street can understand? In general, what we do in lab, I would use the term "part prospecting". So, in Dr. Akhtar's lab we're trying to figure out the enzymatic pathways that are involved in making a variety of different compounds. Generally, ones that show some promises, therapeutics or for other applications so that we can then produce these molecules enzymatically and study them further. We have a lot of different collaborations in the department with different professors who are interested in what our molecules can do in their different research models. We're really focused on learning how to make it generally in the way that a plant or an organism does.

Michael Lim

So, that’s kind of like “nature knows best”. So, you want to do what nature does or more that you're trying to. It's hard to make a compound from scratch, you don't know exactly what to do.

Kelly Boddington

Yes, sort of "nature knows best" - nature doesn't always know best. I'll be very honest. So, some of the different reactions can be more easily done, like chemically or whatnot, but in certain cases nature really does the best. So oftentimes, you have a molecule that you're trying to build if you're trying to put a group in a very specific spot on that molecule. So, hydroxylate one specific spot, as opposed to another. If you start doing things chemically, it just turns into a disaster because any group that can be hydroxylated gets hydroxylated and then you're like "Oh no, I didn't mean to do the whole thing at once, I only wanted a little bit here and there". And that's where enzymes generally become very useful is when you want to do one specific thing and not necessarily other things. So, nature has these little tiny machines, "enzymes" that can add your group to one specific spot. If you imagine like a little factory where they've (I never have been in a factory, full disclosure) .... I'm picturing conveyor belts, right? I'm picturing cartoon style factory where you know you have a starting product and you change it a little bit. And then it goes down conveyor belt and you change it again. And then it keeps going on that conveyor belt onto another thing. That's pretty much exactly what we're doing and all of these enzymes are the little workers of each spot along that conveyor belt, like "Oh, we'll take this here and I'll add this here". And that's what we're putting together.

Michael Lim

So, considering the wide variety compounds you can work with, and I guess just in the field in general in terms of working with enzymes and creating products. Why did you decide to research this particular topic in terms of extracting or creating bibenzyls from cannabis? Did you always want to do this kind of work like have you been tailored since you first started doing the research to working with cannabis?

Kelly Boddington

Not really. So, I'll be very honest. There are very few scientific questions that are not interesting to me. And I think a lot of researchers can relate to that. And maybe something that I found that even when you get into academia, so like, awesome science is great. Now pick one really nice thing and study it. You're like, but what about over there? What about that? So no, I didn't expect this at all. I actually started my PhD in Dr. Steffen Graether's lab studying intrinsically disordered proteins, and that's awesome. And then when I moved on to my post doc, what I was looking for really wasn't one particular subject. I'm not married to any particular niche. What drew me to Dr. Akhtar's lab was his connection to various industry partners. And the idea that something that I did in lab could directly benefit people and the public could actually go on to become something and like not that the rest of our work can't do that. The Dr. Akhtar was linked very closely to this. So, it was a very immediate connection. And I also figured I might want to move out of academia and into industry so that would be a good way to kind of dip my toes into that field without leaving the nest of the university.

Michael Lim

I do think that we don't really get a lot of exposure outside of academia whether it be industry [Kelly Boddington: at all] ... I was trying to be generous in university. I do think it's definitely a smart play to try to branch a little bit more and learn a little bit more about industry ties in terms of research. So, what is the moment in your past that you're like, "it is something I really want to get my hands into and learn a little bit more about".

Kelly Boddington

Maybe not necessarily in general with industry and maybe not, that drove me to that beginning. But there was a moment actually like as I was working on this project. So one of the really interesting things about bibenzyls, and their derivatives is their potential as anti-inflammatory drugs. And there's a lot of conversation going on, or at least there was before the pandemic happened [scared all of that] about the opioid crisis and what can be done and what other options we can have on the market aside from NSAIDs and opioids. And bibenzyls are promising in that respect. And I hadn't really had a lot of personal experience with the opioid crisis until my little sister actually saved a man's life. So, I'll take a second to brag. Yeah, it was one of those phone calls that you really remember she called me out of the blue which is super weird in today's day and age of texting. And I could just immediately hear the tears in her voice. She was fine. She was safe. It was just like the shock and adrenaline going on. She'd been out hiking and at the trailhead, there was like a building where you start and someone called for help and she and her friend had done CPR on a man in the bathroom who had overdosed from fentanyl or opioids laced with fentanyl. Thankfully, he survived. The ambulance got there and took over and all that but it's just one of those sort of weird moments where ...

Michael Lim

.... Crazy out of like a TV show or something you'd never experience in real life. Right?

Kelly Boddington

Also, my little sister's a badass. It's not something I expected to brush up against personally. It's just something you hear on the news a lot.

Michael Lim

Focusing more in terms of the actual research have been doing in the lab, you recently published a study titled "Bibenzyl synthesis in Cannabis sativa Linnaeus". Can you briefly describe what bibenzyls are and why they've been mostly overlooked until now?

Kelly Boddington

Bibenzyls, they're secondary metabolites that are found in cannabis. So essentially, that means they're small molecules the plant produces in response to particular kinds of stress. Most likely in this specific case in response to fungal infection. But the interesting part is their potential as natural products or drugs, like we just talked about, but as to why they've been overlooked, I'd suggest probably a couple of different factors. First, they're relatively rare in the plant kingdom. So, while all plants make some of the precursor molecules, only a handful of plants, (orchids, liverworts, cannabis) make bibenzyls. So, none of those plants are particularly well studied. Certainly not compared to like arabidopsis, crop plants, or such. And then, on top of that, cannabis has only recently become legal. So proper research on its therapeutic potential has also only recently begun. That's not quite true. There was some research being done previously before it was made illegal and that all kind of just ground to a halt. And then, when it comes to cannabis research, CBD really just overshadows everything else by a landslide. The PR team for CBD is fantastic. So, we attribute a lot of benefits to CBD, that actually may or may not be appropriate. So generally, when people are taking CBD, what they're taking is an extract or an oil. That extract would contain a whole bunch of other similar molecules that were pulled out of that extraction process from a plant that if we don't know about them, we don't think to test for quantifying the oil. The really interesting thing, is that studies that were done on pure CBD haven't been able to fully recapitulate the effect of the extract which tells us that there are other molecules in the extract having a therapeutic effect. And this is my personal theory as to why people taking CBD oil can sometimes experience a really big difference between batches or between brands. Because there can be a difference in the rest of these molecules. Obviously, they're making sure that there's a standardized amount of CBD, but if they're not measuring the rest of it, it could fluctuate wildly.

Michael Lim 

Okay, you just mentioned there are a whole bunch of other types of not widely known compounds other than CBD and THC, even beyond bibenzyls. Why have you focused bibenzyls over any of the other kinds of secondary metabolites?

Kelly Boddington

Yes, so as I just mentioned there was previous research done before cannabis became illegal. And some of that early research has shown some promising candidates for bibenzyls and their derivatives. They've shown anti-cancer, anti-diabetic, anti-inflammatory activities in in-vitro assays. But the low concentrations that are found within the plant makes taking that to the next phase of research really, really difficult especially back then, which is where biosynthesis and understanding the enzymatic pathway. It's not feasible to extract these compounds, but we know that they're interesting. There's like promise shown already and that's the perfect place for us to come in and discover how to make them enzymatically and then produce them in quantities to be able to take them on to further testing

Michael Lim

Right, so out of curiosity what I don't know if you know this, but what's the relative amount of say by bibenzyls versus CBD within say, a kilogram of fresh cannabis.

Kelly Boddington

I do not know that number off the top of my head, but we're talking like significant orders of magnitude. Probably thousands and thousands of times less. Also, partially because now that people know about CBD and THC they've been breeding these plants to make these molecules. So, that's even somewhat artificially elevated compared to what is normally there. But yes, very, very small.

Michael Lim

So, speaking of your study, obviously a major part of it focuses on looking at the molecular structure of compounds like we touched on before, kind of like playing with Lego blocks and taking things off reattaching things creating new compounds. For our audience the main focus of our talk today is on bibenzyls which are characterized by two benzene rings as sort of other named bibenzyl, and linked with an ethyl bridge. For those of you who have taken organic chem and can think back to that. Recall that benzenes are a six carbon hexagon ring which are characterized by the three double bonds that kind of interchange between pairs of carbons within the ring. Benzenes are then combined with that ethyl bridge which is essentially a two carbon link between a carbon from one benzene ring to the second benzene ring. Do you want to expand on or clarify about that description?

Kelly Boddington

I think you nailed it, that was great. I guess the only thing I could add is, maybe to put into context the alternative names so bibenzyls or stilbenes. So, resveratrol is really commonly known molecule and resveratrol is still being this is the one that people are quoting all the time from red wine (aggressively marketed to me at the mall). So, that's a stilbene, and that's where there's the two benzene rings strung together with an ethyl bridge that has the double bond dihydrostilbenes or bibenzyls are pretty much the exact same structures resveratrol but just instead of that double bond working the two groups it's a single bond and then decorated benzene rings however afterwards.

Michael Lim

So, there's something super healthy about having double benzene rings and compounds.

So, you mentioned earlier that your work is a little bit hard to describe to the generic audience because it requires a lot of background in terms of you know, organic chemistry or even just basic chemical compounds in terms of reactions. And I think we'd be here for hours later just discussing every step in your paper that's needed to create bibenzyls

Kelly Boddington

I've spent hours on that thing, so yeah.

Michael Lim

An expert, even an expert in the field would take hours and hours to explain. So, I don't know if it's possible, but can you highlight some of the major steps or things you had to work out to get from its initial structure from hydroxycinnamic acids, all the way to bibenzyls, in terms of how do we know what compounds we're looking at? How do we know how to transform these compounds?

Kelly Boddington

Well I'll give it a go. It's my favourite job to try to describe chemical structures when we don't have a picture. But yeah, of course. So, to give you a high level overview, we started with hydroxycinnamic acids. So, that's where we're starting. We didn't start from just like carbon atoms. We're starting already with one benzene ring. And that benzene ring already has some of the hydroxyl groups that we need, and then attached sort of sticking off the end of that there's a three-carbon chain. Why did we start here? Because hydroxycinnamic acids are a common precursor in the phenylpropanoid pathway. And because some previous research in orchids had pointed towards this. Starting off with step one, we need to prime the hydroxycinnamic acid. So, this is done in a variety of other plants by something known as a 4-coumarate ligase and this was really more of a like a phylogenetic puzzle that we were looking at. We know it's going to be done by 4CL, but which 4CL is going to do it. Some of my colleagues did some great bioinformatics work, and we found that there are three clades of 4CL (4-coumarate ligase; so, it's a short form that I'm using) enzymes: class one, two, and three. When we look at cannabis, CS 4CL four which was one of the 4CL enzymes that we had was the only cannabis 4CL that grouped into this clade. And after that, so we've primed it we've put the CoA, stuck it onto the end of the hydroxycinnamic acid so that the downstream enzymes can recognize it. And this enzyme actually does two of the jobs. It's going to take that molecule that we got from our 4CL enzyme and it's going to elongate the chain and then cyclize the end of it. We had two candidates in cannabis but only one was functional with the substrates that we were looking at. So, it was a pretty clear winner.

Michael Lim

And rarely ends up being that easy?

Kelly Boddington

So, this makes it sound easier than it is.

Michael Lim

it sounds are easy, but I mentioned

Kelly Boddington

And then we did some experiments and it still worked. It's a little bit messier than that. What was really the puzzle for us though, was at what point in time does that double bond get reduced? And this was where the substrate preference of the bibenzyls and these actually came into play because that was very definitive. It refused to use anything that had not already had the double bond gotten rid of it. And for that when we found that there was actually a change in the shape of the substrate binding pocket. So, if you think about a single versus a double bond: a double bond makes molecule more rigid and then a single bond will allow it to be more flexible. So, there's basically been a narrowing of the substrate binding pocket such that the substrate had to squeeze its way into the enzyme and the more rigid double bond just couldn't do it. And whereas the single bond which is more flexible could you know, wiggle its way in there.

Michael Lim 

These compunds ... they look pretty rigid, but obviously they're a lot more flexible. They look like they're not just like solid structures like those organic chem plastic model kits.

Kelly Boddington

I loved those, those so much. Maybe I'm just a really visual learner I had a blast with those things.

Michael Lim

I strongly recommend to all our listeners to please get the paper and just play this audio at the same time as you're looking at the diagrams ... it will make perfect sense.

Kelly Boddington 

There's a pretty figure I spent a long time on and it has the entire pathway visually laid out for you there that would be figure four if you're following along at home.

Michael Lim

You also mentioned your study was focused on looking in and defining specific compounds needed, I guess enzymes needed to create bibenzyls, such as say CsDBR2. So, what do you think are some of the major benefits provided to your field by having identified some of the possible pathways and possible enzymes involved in creating bibenzyls? Is it difficult to synthesize these enzymes and maintain them for long periods of time?

Kelly Boddington

Yes, so the biggest benefit is definitely the potential to use biosynthesis to make enough of these enzymes to study them in an actual, you know, proper clinical trial, in vitro assays, preclinical clinical trials, because of how sparse they are in nature. Taking that to the level of having enough to give you know all your test subjects in those different trials, it was just sometimes impossible. But knowing what we know now about the pathway, if we can put that towards biosynthesis actually generate larger quantities of them, then we can really start to see what we can do with them. So, are they difficult to synthesize? Competitively to some of the other enzymes that I've worked with - not really. They're soluble enzymes. They're not membrane bound or anything, which is a massive pain if they were, but they're not. So that's good.

Michael Lim

Is your hope that having identified this pathway and the ancillary steps to get to that, that perhaps you might see some companies stocking some of these compounds you don't make from scratch anymore?

Kelly Boddington

Yeah, not only hope but, to let the cat out of the bag I guess, I'm excited because I've actually moved on to join a company, Canurta as a research associate, and that's the exact thing we're working on [Michael Lim: Congratulations!] Yeah, thank you! So, it's really exciting for me to be able to take the research that I was doing previously; we're not using bibenzyls immediately we're using some of the other compounds also from cannabis that have been previously identified although bibenzyls also are on the slate. But it's really cool to be able to take the work that I've done at an academic level, at an academic scale, and scale that up and actually make it you know, feasible. Make a platform to create these things and see that taken further.

Michael Lim

We say that's your favorite part in a research project that you can feel or you can see a final product in the horizon, "Ah, I'm aiming for that goal. That's what I'm looking for".

Kelly Boddington

Definitely. I think it can be hard in academia, sometimes when you're working to you know, keep your motivation up and it's really just my PhD is a testament to the amount of failure that I went through it. My projects and, you know, you keep moving and eventually find the right answer, but it's a lot of failure. That's what science is. "Oh, that wasn't it. That didn't work". And that could be kind of tough to push through. But knowing that it's actually going to become something - like you're going to make something that's really cool.

Michael Lim

I think science is just an exercise in failure [Kelly Boddington: I mean, I love it]. I like to ask this to all people I interview, if you go back in time, and change one thing about your study what would it be and why?

Kelly Boddington

That's a really difficult question. I think partially because I spent so long revising this paper and bashing my head against it. But it's pretty much a loose shape, but I was happy with. There's always more so I guess that's one of the hardest things about writing a paper is that there's not an end to science and you know this as a scientist, like I don't know if anyone really gets to a point where they're "alright, that's all my questions so see you later". There's always like, "okay, that's cool, but knowing that now I have a question about this". And there's always different sort of directions that you could go. One of the directions that we didn't really pursue partially because we weren't set up for it was the transcriptomic side of things. The data that we got was from a public database that we analyzed for our purposes. If we had done it ourselves, if we had been set up for that, I think it would have been interesting to see the different levels of expression under different stress conditions. I think sort of that other side of how it works with the plant and when the plant produces these things would have been interesting as well. But our priority was kind of on the people side, I guess the selfish side of the science.

Michael Lim

Unfortunately, that's where all the funding goes is what's better for people. They don't really care so much about what are the plants doing? So, do you kind of see a developing market for I guess more bibenzyls and related compounds in the future in say the next 10-20 years?

Kelly Boddington

For sure, yeah. I think we're getting to the point where we have more technology in order to be able to make these compounds that previously we just didn't have access to again, because the quantities but there's a whole treasure trove of things that exist in nature that we can start to look into not just in cannabis, but in other plants as well. A lot of potential anti-inflammatory, anti-cancer, all kinds of things, and I think it's going to be a huge market.

Michael Lim

So, that's enough questions for me. We're going to take some or whether you're going to take some taken from our social media and from our miscellaneous listeners. Our first question is: "With the legalization of marijuana, has there been increase in jobs, working with marijuana that require a lot of research experience or research degrees? What do you think the field in the job market will be like in the next five to 10 years?"

Kelly Boddington

Yes. [Michael Lim: Short answer yes.] Short answer yes. There's definitely a lot more room for a bunch of different scientific positions within this field. There's, I guess, if we're looking at what I think the market will be in the next 5 to 10 years, there was a really big boom around CBD and THC products when cannabis was first legalized. Because again, that's what everyone thinks of, that everyone knows about. But that's also a sort of very saturated market of everyone's making them. I do think that the future of this market is going to look more like what we're doing at Canurta with sort of pulling apart some of these other interesting unstudied novel potential pharmaceutical compounds from plants, and really just investigating more of what it has to offer beyond the surface level.

Michael Lim

And our final question in the audience is, "What are some of the most common misconceptions you hear about different cannabis compounds? Has there been a noticeable shift in the past few years?"

Kelly Boddington

Most common misconceptions and a noticeable shift? I guess as far as misconceptions, I don't know if I would call it fully a misconception, but like I touched on before, everything good gets attributed to CBD. We're like, okay, THC gets you high. CBD is the good one. And then we're done. We're gonna go home. But like I said before, there's a lot, a lot more molecules that likely are having effect. And although CBD certainly does have an effect as to whether it has every single one of these effects that are attributed to it, or whether it's responsible for the entirety of the effects that are seen, that's what I would question. Well, as much as I love, you know, investigating the research potential of these molecules. As a scientist, I do want to make sure that when we're marketing these that we're backing it up with good information, that we're communicating that good science that was done to consumers. And that consumers feel equipped and empowered to, I guess, ask those questions about what research was done without feeling kind of attacked or bullied for it. I think sometimes, when science is presented in the media, it's very monolithic. Click someone in a lab coat who's just going to tell you what science thinks. And it's a lot more nuanced than that. And I'd encourage people to ask questions and feel like they're allowed to do that.

Michael Lim

So, do you have any final comments you want to make about your work, or about anything else we've chatted about today, for our listeners to learn or take away one thing from this whole 30 minute long chat. What do you hope it is?

Kelly Boddington

I hope they will remember, a very specific enzymatic pathway (just kidding) ... there will be a test. I think just the idea of keeping an eye out on what's gonna come out of not just the cannabis industry, or the biotech industry of what we can do now with the technology that we have. I think there's gonna be a lot of interesting molecules and pharmaceuticals that will start to be available. And I think, keep an eye out for that. And it's just sort of cool to understand how they're made.

Michael Lim

And that's cool.

Kelly Boddington

That's cool. [Michael Lim: Science is cool, everybody.]

Michael Lim

So, that actually brings us to the end of today's podcast. A big thanks again to our guest, Dr. Kelly Boddington for joining us today, Gryphon CAST is brought to you by your host me, Michael Lim, with editing assistance from Ian Smith. If you're hungry to learn more about different science topics, please check out the SCRIBE research highlights that's scribe S-C-R-I-B-E, research highlights on the University of Guelph website at uoguelph.ca or you can follow us on social media @UofGCBS. You can find us on Instagram, Twitter, and Facebook. Music in the podcast, comes from Upbeat with details in the show notes. And you may also want to check out a new podcast coming out of the Ontario Veterinary College called Vet Sessions. On their podcast you can learn more about what goes on in clinics and what's involved in primary care. That's V-E-T space S-E-S-S-I-O-N-S and until next time, please stay curious.