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Resurrecting the Carboxyl Polyether Ionophores

Periodic Reporting for period 2 - RECYPION (Resurrecting the Carboxyl Polyether Ionophores)

Reporting period: 2021-09-01 to 2023-02-28

RECYPION aims to investigate a class of complex natural products, collectively referred to as carboxyl polyether ionophores (CPIs), as antibiotics to target resistant bacterial infections. The CPIs have been known for >50 years, but despite numerous studies in the past, its remains unclear exactly how the different members of this class exert their antibiotic potency. It is clear that the CPIs act directly on bacterial membranes. This type of mechanism, however, carries the risk of the compounds also acting on mammalian cells and, as a consequence, having a therapeutic window that is too narrow for development as antibiotics for human use. Paradoxically, a handful of CPIs are employed extensively in the agricultural industry as anti-parasitic agents which demonstrates that the compounds from this class can indeed be safe. The industrially-produced CPIs are a cornerstone of the objectives of RECYPION.

RECYPION aims to leverage the methods of organic chemistry and advanced analytics to widen the therapeutic window of the CPI-antibiotics through preparation of novel derivatives. The project seeks to afford breakthroughs in how compounds from this class can be chemically constructed. Specifically, an approach involving degradation and then reconstruction will be pursued as this allows new variations to be accessed from the abundant, industrially produced, family members. RECYPION also aims to provide fundamental insight into: (1) the behavior of selected polyether ionophores in contact with membranes or other hydrophobic surfaces and (2) their effects on cellular systems. These insights may provide a more clear picture on the different mechanisms of the CPIs which can feed directly back into optimizing the structures for antibiotic activity and selectivity.

The antimicrobial resistance (AMR) crisis, that humanity has unfortunately only just started to experience, will be one of the largest healthcare challenges in the 21st century. No doubt that antibiotics with mechanisms that are not readily subject to resistance development will be needed. The CPIs fit this profile to the point and RECYPION can therefore have an important impact if it can deliver compounds with properties exceeding those of the naturally occurring polyether ionophores. Along this way, RECYPION will attempt to develop methods for breaking down the complex 'polyether backbone' of the CPIs. Although destined for use within the project, these methods may ultimately reach beyond RECYPION and contribute to sustainable protocols for recycling of certain types of materials.
Work within the first half of the project has provided the following two important insights.

(1) We have demonstrated that the antibiotic selectivity of the natural CPIs can be exceeded by our synthetic derivatives (Nature Chemistry 2021). This is incredibly important because it validates a fundamental hypothesis of RECYPION. In the same work, we introduced the use of the method called ‘cell painting’ to study the effects of CPIs on mammalian cells which we demonstrated to be a superior approach rather than measuring toxicity.

(2) Acting in response to the 2020 pandemic, we discovered remarkable anti-SARS-CoV-2 activity of selected CPIs (Antiviral Res 2021). Interestingly, we found that even within a small collection of 12 CPIs, the anti-viral effects differed >100-fold and we identified a CPI called X-206 having a 500-fold selectivity-window for inhibiting replication of SARS-CoV-2 over toxicity to mammalian cells.

In addition, we have made significant progress in the following areas/projects:

(3) We have discovered an enzyme that can perform selective oxidative modifications deep within the polyether backbone of some of the CPIs. This type of transformation cannot be realized through purely chemical means. We are characterizing this new enzyme in detail. This discovery is important as it may allow for development of selective degradation processes for the abundant CPIs which will facilitate recycling of their components.
(4) We are developing a new method of chemical synthesis of CPIs where pre-made (or recycled) building blocks can be coupled together one at a time. By now we have assembled all required building blocks to test the coupling protocol.
(5) We have completed synthesis of the full collection of isotope-labelled derivatives of the CPI called lasalocid. This now puts us in position to conduct advanced spectroscopic investigations which we expect to provide the most detailed picture yet of the structure of lasalocid at a hydrophobic interface.
(6) We have prepared a mirror-image CPI (called ent-routinneocin) and have begun to characterize its biological properties relative to the parent natural product (routiennocin). We are particularly interested in any differences that may suggest a different antibiotic selectivity window of the mirror-image product. So far, we have found that a mirror-image CPI can maintain antibiotic activity.
(7) We have, serendipitously, discovered surprising stability of certain alpha-lactams in biological systems. This is a rather elusive class of compounds with is structurally related to beta-lactams that are constituents of several important human antibiotics.
(8) We have, through systematic studies, uncovered hitherto unknown antibiotic effects of some of natural CPIs. This for instance include ability to target bacterial biofilms.
-We have demonstrated that the antibiotic selectivity of the CPIs can be enhanced through chemical modifications. This marks a key turning point because it suggests that systematic optimization of this class of antibiotics is not only possible but should also be a high priority. I anticipate that we will be able to produce a candidate compound with a sufficiently interesting selectivity profile to warrant future testing in animal infection models.

-The enzyme, we have discovered, has a selectivity profile unlike any other that is known to us. With this, we will be able to break new ground for performing selective oxidative-modifications of the CPIs, but potentially also for modification of substrates that fall outside of the CPI family. I anticipate that we will be able to understand the fundamental mechanism of this enzyme but also learn how this type of oxidative modification alters/changes the antibiotic activity of the compounds. Interestingly, we will be able to know if the compounds become more or less active if we remove this enzyme from the producing bacterial cells.

-The lipid membrane (along with the rest of the cell) is asymmetric from a molecular perspective, because it consists of molecules that are not superimposable upon their mirror-images. From this perspective, it is not at all clear how a mirror-image CPI will behave when exposed to cellular systems. We have demonstrated that a mirror-image CPI can remain antibiotically active which is a fundamental observation with an interesting perspective: for instance if side-effects/toxicity of CPIs to mammalian cells may be caused by direct interactions with cellular proteins, this is very likely to be substantially different for the mirror-image compound. I expect that we will be able to understand if preparation of the mirror-image CPIs is a surprising way to increase the therapeutic index of the compounds. I also expect, that we will be able to understand which proteins these compounds actually do interact with (which remains largely unknown) by using the mirror-image forms as highly sensitive control compounds.
Interplay between chemical synthesis, microbiology and complex natural products on RECYPION