Conversations with John E. Moses: Novel shapeshifting vancomycin shows potential against resistant bacteria.

Recently, a study on a novel class of antibiotics called shapeshifting vancomycin dimers (SVDs), synthesized through click chemistry, appeared in the Proceedings of the National Academy of Sciences. These SVDs are effective against bacteria resistant to conventional antibiotics, such as vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant S. aureus (VRSA), collectively referred to as ESKAPE pathogens. The shapeshifting capability of SVDs is attributed to a triazole-linked bullvalene core, which allows for dynamic rearrangements, creating ligands that inhibit bacterial cell wall biosynthesis. SVDs seem unaffected by a typical method of vancomycin resistance and show little tendency for the bacteria to develop resistance against them. They could also potentially function in a new way, as they appear to destabilize the interaction between bacterial flippase MurJ and lipid II.

The significance of this study lies in its potential to combat the global health issue of antibiotic resistance. With the rise of superbugs, or bacteria resistant to multiple antibiotics, new treatments are urgently needed. This research reveals a promising new class of antibiotics, the SVDs, which are not only effective against these superbugs but also seem to be resilient against the development of resistance. Additionally, these antibiotics might operate via a new mechanism, which could offer fresh insights into the battle against antibiotic resistance.

John E. Moses, from Cold Spring Harbor Laboratory (CSHL) and one of the scientists involved in the study, set aside some time to discuss their creative approach to antibiotic development. (NOTE: This interview has been edited for length and clarity.)

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Can you just briefly sort of give a background on how click chemistry works? 

Click chemistry is an approach; it is not merely a reaction. It began as a philosophy, a way to contemplate the synthesis of molecules. What occurred was the association of chemistry with a specific reaction, known as the click reaction. The concept behind it is to efficiently and consistently construct molecules, ensuring reliable outcomes. Click reactions are devised to function universally in every instance, yielding guaranteed results.

In a sense, it has to be easy and modular, similar to Lego blocks that simply fit into place. That audible “clunk” is very reassuring – you know the blocks have connected. This is the same kind of reassurance that click chemistry provides. It began as an idea, and was proven through experimentation and the development of highly reliable, robust, high-yielding reactions that guarantee a product. That’s essentially it.

When seeking functionality, all searches should focus on easily synthesizable molecules, rather than those that demand numerous steps. If you want to explore structure-activity relationships, it entails a lot of work. If you can perform chemistry and achieve function in very few steps, then you have a significant advantage. This can accelerate the drug discovery process. So, that was truly the driving force behind it.

How long did it take to find the configuration of the shapeshifting vancomycin dimers (SVD) that was effective? 

We utilized vancomycin as a starting point. We knew there would be some activity because vancomycin has been used, almost like a warhead – although I’m not sure if that’s the best term for it. Certainly, the group that binds to the Lipid II complex is already known. So we knew that using that as a starting point, we could potentially improve the activity. Does this mean achieving efficacy at lower concentrations? Yes, and it works against tricky bugs. The starting point was already known. So the hypothesis was whether introducing unusual dynamic fluctuations or chemistry would improve vancomycin. In fact, I wondered if it might just confuse the system if something’s a bit unusual?

The concept of shapeshifting is mind-boggling to me. We are accustomed to dealing with molecules that exhibit considerable rigidity. While bonds do rotate and molecules can flex and change conformation, they do not frequently undergo significant structural changes. To truly alter their core structure, one must break and reform bonds.

However, vancomycin does not behave in that manner, despite bond rotation. Its structure remains consistent when represented on paper. With shapeshifting, the positions of the groups change relative to one another, resulting in the creation of unique structures. This is a significant departure. I pondered whether it would introduce chaos into the system, as nature, to the best of my knowledge, does not exhibit such behavior. The idea was simple, yet intriguing. The beauty was that, due to our ability to employ click chemistry, we were confident in our ability to achieve success.

That’s precisely what click chemistry allows you to do. It provides you with the opportunity to roll up your sleeves, delve into the work, and obtain the genuine answer. The experiment itself yielded the true answer.

Space-filling model of the vancomycin molecule, C66H75Cl2N9OO24, as found in the crystal structure of vancomycin acetate dichloride hydrate. Note that the two amino groups are protonated and the carboxyl group is deprotonated. Color code: Carbon, C: grey; Hydrogen, H: white; Chlorine, Cl: green; Nitrogen, N: blue; Oxygen, O: red. (Structure determined by X-ray crystallography, reported in M. Schäfer, T. R. Schneider, G. M. Sheldrick, ‘’Structure’’, ‘’’1996’’’, ‘’4’’, 1509-1515 (CSD refcode TUCMEJ01).)

Do the two turrets result in an entirely different kind of reaction? Or does it enhance the current mechanism’s activity?

I think there are two answers. I think vancomycin still binds to Lipid II. Probably. I can’t say for sure. There are many answers to that question that remain unanswered. But I’m certain that vancomycin still binds to Lipid II, in fact, because we have modified vancomycin in a way that precludes that.

It is well known from previous studies on vancomycin dimers that their linkers – what we refer to as the non-shapeshifting – are flexible and can rotate. However, the structure of the linker remains unchanged. So we knew that it wouldn’t preclude its function, but we didn’t know whether that linker would improve or generate new functionality.

How does the triazole-linked bullvalene contribute to the shapeshifter modality?

You can think of bullvalene as a Rubik’s Cube; that is the core of the molecule. There are two vancomycin groups, forming a chain, connected via this bullvalene hub. Just like you can rotate a Rubik’s Cube, imagine if you had two pencils glued to the faces of each cube.

The two pencils are positioned 180 degrees apart. Now, what if you rotate it? By doing so, you change the relative positions of the two pencils. That’s exactly what bullvalene does. The vancomycin groups themselves don’t actually change; they can rotate and perform the typical movements that molecules do. However, the Rubik’s cube, which is the bullvalene, alters the relative position of the two vancomycin groups.

If you’re a bacteria and you’re about to bind to vancomycin, you will sense this moving entity, and the vancomycin could initially grab onto one area, then it might discover its optimal position elsewhere. If it finds another spot on the bacterial cell wall that fits comfortably, then you have something that has found its comfortable position. The core enables such proximity.

When does the shape shifting occur? Is the molecule always in flux?

When you have interactions, structures, or conformations, there’s an energy penalty, and some are more favored. If things pass one another, there’s a penalty for that. But if there’s a more favorable interaction, those tend to have lower energy. Is that more preferable? 

Well, we hypothesize the same about the shapeshifting – we think, but we don’t know because the vancomycin groups are quite large. There could be some structures from the shapeshifter that are unfavorable, higher energy, and therefore less likely to occur given certain temperatures and parameters. There would be certain structures and my inclination is that the vancomycin molecules prefer to be as far away from each other as possible. This allows them to move around, but they still want to maintain a significant distance. 

So, I think there’s limited shapeshifting. Therefore, there would still be some predisposed structures, or perhaps, a preferred structure, which is probably more accurate.

Under a very high magnification of 20,000x, this scanning electron micrograph (SEM) shows a strain of Staphylococcus aureus bacteria taken from a vancomycin intermediate resistant culture (VISA). (CREDIT: CDC/Janice Haney Carr)

What does that mean for resistance?

Resistance? I mean, I think nature, in its own right, is smart, and it will always find a way around things. What we can do is try to slow that process down. Slowing it down is the best approach, but I don’t believe we can completely evade resistance. Otherwise, the entire concept of natural selection goes out the window, doesn’t it?

What is the mechanism of how this works? Also, does the shape shifting hinder the way bacteria latch on when they they’re developing resistance?

Historically, it’s referred to as a d-ala-d-lac transition. An amino acid residue is modified in VRE, weakening its interaction with vancomycin. However, it has been shown in the past, several years ago, that by covalently tethering two or even three vancomycin molecules, one can overcome that resistance.

In our study, we discovered through our own syntheses a shapeshifting vancomycin. We also created some control ligands where they were linked but unable to shapeshift. These compounds were active as well and slowed down resistance, but they didn’t perform as well as the shapeshifter. The shapeshifter was two to three times better than the controls and even better than vancomycin.

Something is definitely happening. It appears that bullvalene is playing a role, but we are still uncertain. It’s too early to tell. I’m certain that someone, if not us, will come along and try to unravel all of this. Maybe we don’t even need bullvalene; it could be lipophilicity. We truly don’t know yet.

At the moment, it was an idea that seemed to work, and we have conducted as much as possible within the project’s allotted time to demonstrate that there is certainly something occurring with the flippase enzyme and the lipid complex. It behaves differently from vancomycin, whether due to shapeshifting or the addition of a chain. We have yet to determine that. We need to perform further experiments and confirm it down the line. There are still many unknowns.

What evidence supports the claim that SVDs show little propensity for acquired resistance? And how does this durability make them potentially more effective solution against the resistance?

Well, if it is indeed a fact that the dimers themselves exhibit less propensity and greater durability against resistance, I believe that should be acknowledged. It’s not solely due to being a shapeshifter. However, if being a shapeshifter gives it an edge and an additional boost, then that’s great. The experiment we conducted supports this claim. We performed a serial passaging experiment, exposing the bacteria to a gradient of vancomycin.

After serial passaging, we plated the bacteria again and observed the effects on vancomycin-susceptible strains and VSE (Vancomycin-Susceptible Enterococci) strains. The potency of vancomycin decreased by 30 times after serial passaging in the VSE strains. This outcome is quite significant. That experiment alone demonstrates the presence of additional benefits. The dimers definitely slowed down the development of resistance, but the shapeshifter appears to provide an extra boost.

Does it have the same specificity as vancomycin or does that change?

Well, vancomycin is less active due to the transition from D-alanine to D-lac in the lipid, which reduces binding. However, vancomycin must have an additional mechanism because our compound remains active even though this transition occurs.

So it’s presumably not just binding to the lipid complex through hydrogen bond interactions. There must be something else at play.

We expect it to behave similarly to vancomycin. However, when you modify a molecule by adding a sizable group, like in our case with the shapeshifter, there will be different structural behaviors because it is not vancomycin anymore; it is a completely different molecule. Nonetheless, there are still embedded features in the shapeshifters that may or may not affect its activity. But as I mentioned, something is happening because it is definitely interacting with vancomycin-resistant species that undergo this transition.

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