Periodic Reporting for period 4 - RECYPION (Resurrecting the Carboxyl Polyether Ionophores)
Reporting period: 2024-09-01 to 2025-08-31
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 or modified. 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 and new methods for constructing them. Although destined for use within the project, these methods may ultimately reach beyond RECYPION and contribute to sustainable protocols for synthesis of complex drug candidates
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 or modified. 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 and new methods for constructing them. Although destined for use within the project, these methods may ultimately reach beyond RECYPION and contribute to sustainable protocols for synthesis of complex drug candidates
Work within the project has provided the following key results.
(1) We have demonstrated that the antibiotic selectivity of the natural CPIs can be exceeded by 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. Systematic characterisation of a complete panel of natural CPIs provided novel activities, e.g. inhibition of biofilms and bacterial persister cells (Microbiol. Spect 2023).
(3) We have discovered a non-canonical P450 enzyme (LyoI) that can perform selective oxidative modifications deep within the polyether backbone of some of the CPIs and which plays a key role in their bioactivity (ACIE 2025). The oxidstive transformation by LyoI cannot be realized through purely chemical means. Bioinformatics have pin-pointed key sequence alterations in LyoI that are responsible for its unique oxidative catalysis.
(4) We have developed a new synthetic method to directly construct attached, saturated - C(sp3)-C(sp3) - oxacyclic rings which comprise key motifs in CPIs, e.g. in lasalocid (Pre-print 2025). This method enabled us to prepare lasalocid in 11 steps LLS vs. >30 steps previously.
(5) We have prepared two different mirror-image CPIs (ent-routinneocin and ent-lasalocid) and characterized their biological properties relative to the parent natural product (routiennocin and lasalocid) (ChemBioChem 2024, Pre-print 2025). We have found that mirror-image CPIs maintain antibiotic activity and thus are candidates for future pre-clinical evaluation.
(6) We have, serendipitously, discovered surprising stability of certain alpha-lactams in biological systems and demonstrated biologically relevant covalent reactivity and selective protein targeting (ACIE 2023). This is a rather elusive class of compounds with is structurally related to beta-lactams that are constituents of several important human antibiotics.
(7) We have completed synthesis and initial biological assessment of the cyclohelminthol antibiotics, demonstrating that these possess unique covalent reactivity (Chem Sci 2025).
(1) We have demonstrated that the antibiotic selectivity of the natural CPIs can be exceeded by 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. Systematic characterisation of a complete panel of natural CPIs provided novel activities, e.g. inhibition of biofilms and bacterial persister cells (Microbiol. Spect 2023).
(3) We have discovered a non-canonical P450 enzyme (LyoI) that can perform selective oxidative modifications deep within the polyether backbone of some of the CPIs and which plays a key role in their bioactivity (ACIE 2025). The oxidstive transformation by LyoI cannot be realized through purely chemical means. Bioinformatics have pin-pointed key sequence alterations in LyoI that are responsible for its unique oxidative catalysis.
(4) We have developed a new synthetic method to directly construct attached, saturated - C(sp3)-C(sp3) - oxacyclic rings which comprise key motifs in CPIs, e.g. in lasalocid (Pre-print 2025). This method enabled us to prepare lasalocid in 11 steps LLS vs. >30 steps previously.
(5) We have prepared two different mirror-image CPIs (ent-routinneocin and ent-lasalocid) and characterized their biological properties relative to the parent natural product (routiennocin and lasalocid) (ChemBioChem 2024, Pre-print 2025). We have found that mirror-image CPIs maintain antibiotic activity and thus are candidates for future pre-clinical evaluation.
(6) We have, serendipitously, discovered surprising stability of certain alpha-lactams in biological systems and demonstrated biologically relevant covalent reactivity and selective protein targeting (ACIE 2023). This is a rather elusive class of compounds with is structurally related to beta-lactams that are constituents of several important human antibiotics.
(7) We have completed synthesis and initial biological assessment of the cyclohelminthol antibiotics, demonstrating that these possess unique covalent reactivity (Chem Sci 2025).
-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. The developed synthetic methods, in particular a novel coupling reaction to prepare the complex attached ring scaffolds of the CPIs, will allow us in the coming time to explore novel candidates for future testing in infection models.
-The enzyme (LyoI), 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 future studies will explore the fundamental mechanism and applications of this, and related, non-canonical P450s enzymes.
-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 mirror-image CPIs can remain antibiotically active which provides the interesting (and non-obvious) perspective that CPI-analogs for future studies may be based on the unnatural antipode.
-We are introduced two new covalent reactive groups (CRGs), alpha-lactams and cyclohelminthols, for chemical biology applications. These are important contributions as covalent mechanisms in drug discovery and medicinal chemistry is of increasing interest. Future studies will seek to understand the fundamental proteomic reactivity of these groups and their incorporation Into complex molecular scaffolds.
-The enzyme (LyoI), 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 future studies will explore the fundamental mechanism and applications of this, and related, non-canonical P450s enzymes.
-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 mirror-image CPIs can remain antibiotically active which provides the interesting (and non-obvious) perspective that CPI-analogs for future studies may be based on the unnatural antipode.
-We are introduced two new covalent reactive groups (CRGs), alpha-lactams and cyclohelminthols, for chemical biology applications. These are important contributions as covalent mechanisms in drug discovery and medicinal chemistry is of increasing interest. Future studies will seek to understand the fundamental proteomic reactivity of these groups and their incorporation Into complex molecular scaffolds.