This column will cover a variety of topics culled from a number of recent publications including: potential methodologies against resistant microbes involved in biofilms; recent reports on boron-containing potential antibiotics, some old, some new; organo-metallic (ferrocene-containing) against fungi and neglected tropical diseases (NTDs); a short excursion into the genetics of didemnin B production; and finally a very interesting protein kinase inhibitor, from a marine sample collected under the NCI’s marine collection program, further fractionated under the later NCI’s Program for Natural Products Discovery (NPNPD), and then synthesized-yielding aplithianines A and B.
By David J. Newman, DPhil
December 31, 2024 | This article appears in Volume 61, Issue 1
Skin Infection Treatments
The authors1 chose three common antibiotics used against methicillin-resistant Staphylococcus aureus (MRSA) skin infections, viz the lipopeptide daptomycin, the glycopeptide vancomycin (used initially against MRSA until resistance was seen in the 1960s), and the synthetic antibiotic linezolid whose base structure (oxazolidinone) as an antibiotic came from the DuPont antibiotic program in the late 1970s. The base structure was then used by Pharmacia/Upjohn leading to FDA approval in 2000. Their antibiotic choices were made due to the following mechanisms of action of the agents: daptomycin targets the microbial cell membrane, vancomycin targets the growing peptide chain within the cell wall, and linezolid inhibits the formation of the 70S initiator complex. Of definite interest (could even be considered as “thinking outside the box”) was in the authors’ choice of the three antibiofilm agents. These were the cationic peptide IDR-1018 (1),2 which has antimicrobial activity; the food flavoring agent 5-dodecanolide (2), known to inhibit two S. aureus regulator genes; and proteinase K (MW, ~29,000 daltons; no structure), a serine protease known to inhibit some specific MRSA clinical isolates and originally isolated from the fungus Parengyodontium album.
Using a series of crossover studies measuring the viability of the MRSA biofilms, two combinations demonstrated significant synergy. These were Proteinase K plus daptomycin with manipulation of the protein content, thus increasing access to the cell membrane of the biofilm bacteria, and then the cationic peptide IDR-1018 plus vancomycin which caused bacterial damage and prevented repair.
Author Comment: Of these two results, although vancomycin exhibited synergy and might even be superior to the other combination, from a clinical aspect it normally has to be given via injection and/or catheterization. Thus, the daptomycin / Proteinase K might be the route to further explore.
Boron-Containing Agents3
The first bioactive boron-containing compound from nature, boromycin (3), was published in 1967 by Hutter et al.3 It was isolated from Streptomyces antibioticus and had significant activity against certain viruses, Gram-positive bacteria and protozoan parasites. Over the years, a small number of boron-containing molecules have been approved as drugs such as Velcade in 2003 and tavaborole (Kerydin) a topical antifungal in 2014, crisaborole against atopic dermatitis (2016) and the beta-lactamase inhibitor vaborbactam in 2017.
Using a series of molecules that were modelled upon the successful benzoxaborole (4) chemotype, Zain et al.4 chose four subclasses, two benzoxazaborine (5a) and two benzodiazaborine (5b) that differed only by the substitution of an “NH” group in place of the oxygen between the boron and nitrogen atoms in the second ring. With syntheses following similar basic substitution patterns, only two molecules related to the benzoxazaborine structure (5a) demonstrated significant biological activity with one showing strong antifungal and mild antibiotic activities and its companion reversed the activities. It will be interesting to see where this class of molecules ends up.
Antiparasitic Organometallic Substitutions
In 2023, a very intriguing paper was published in the Journal of Medicinal Chemistry by Lin et al.5 This multidisciplinary and multinational group (France, USA and Italy) reported their latest results when one of the triazine (azole) moieties of the well-known antifungal drug fluconazole (6) was replaced by ferrocene (7). This report was a follow-up of earlier work where they had used a ferrocenyl substitution on the well-known antimalarial chloroquine and had taken the compound to Phase II clinical trials.
In the 2023 paper, they reported the use of both ferrocene and ruthenocne substitution to produce agents that were very effective when tested in vivo not only against pathogenic fungal infections but also highly potent against parasitic worms such as Brugia, which causes lymphatic filariasis, as well as the soil-transmitted helminth Trichuris that infects millions of people globally in less developed countries. In addition, further work with resistant fungi demonstrated that one of the later variants (8), which only has minor changes from the first ferrocenyl agent, was extremely effective against the FCZ-resistant C. albicans strain CAAL28 in an immunocompromised Balb/c mouse model.
Thus, modification of older antifungal agents, using what to the organometallic chemist are simple compounds to work with, has significantly expanded the biological arenas in which they might function. I might also add that in another life (in the UK in the early-middle 1960s), I worked extensively with compounds such as the ferrocenes, and they are not a problem for any well-trained organic chemist with access to the necessary equipment.
Figure 1. Structures (1 to 9)
Biosynthesis of Didemnins
In the 2011-2012 timeframe, two groups, one working in the Red Sea and the other in Hong Kong with work also performed at the Scripps Oceanographic Institute, proved definitively that didemnin B, first reported by the Rinehart group in the US many years earlier following isolation of the compound and congeners from a marine tunicate, was in fact produced by a free-living microbe. Recently a group at Berkely, working with the Department of Ocean Science at the Hong-Kong University of Science and Technology, reinvestigated the biosynthesis of didemnin B.6
In this paper the multinational group demonstrated that the initial view that the prodrug activation mechanism that had been proposed whereby a non-toxic precursor would be synthesized via the ribosome, expressed into the cytoplasm, and then transported from the cell after conversion to the “active product” was incorrect, even though that route is well-known for other compound sources.
By constructing a genetic modification system in the didemnin-producing microbe Tistrella mobilis, they reinvestigated the production of didemnin B. Using that genetic tool, they confirmed the presence of NRPS and that the complex gene named DiDA was involved in the maturation of didemnin B. They then discovered a previously unknown gene in the didemnin B production cascade. This was an Abi family transmembrane protease (DidK) that was part of the maturation step in didemnin B biosynthesis.
Analyzing other production systems for microbial non-ribosomal peptides, they identified over 20 homologues of this protein, indicating that such an involvement of Abi-linked proteins in the biosynthesis of natural products may not be rare. Thus, this finding might well alter “current biosynthetic dogma.”
PKA Inhibitors from the NPNPD Program at NCI Frederick
The last paper7 to be discussed demonstrates the continued value of the initial marine collections by NCI plus the corresponding other collections of flora and fauna. These are now being further fractionated by current techniques, using monies from the Cancer Moonshot applied to the initial fractions (aqueous and organic) from the marine and plant collections to produce an excellent source of novel bioactive agents, the NCI Program for Natural Product Drug Discovery (NPNPD).
In this report, having identified potential bioactive agents, follow-up syntheses produced the active agent(s) which were then tested extensively biologically. The initial collection of the tunicate Aplidium sp. was made in South African waters by the then-NCI marine collectors, the Coral Reef Foundation in Palau, sent frozen to NCI Frederick and extracted using the then customary techniques at NCI Frederick, with no activity being found in the initial testing of the unfractionated extract(s).
However, using the current fractionation system which has been published in the last few years by the NCI Frederick group, over 300K fractions from the current NPNPD were assayed using a high throughput kinase assay system, with the results leading to the kinase inhibitor aplithianine A (9) which inhibited wild type PKA with an IC50 of 84 nM, with further activities shown against various serine/threonine kinases yielding IC50 values ranging from 11-90 nM. Due to having only small amounts of the active material and no mechanism for recollecting, a simple four-step synthetic program led to an overall 10% yield providing material for further investigations.
Author Comment: What is of significant interest is that of all of the hundreds of millions of dollars (a very conservative figure) expended in production and biological assays/analyses of combinatorial chemistry agents over the last approximately 30 years, no more than four compounds from those attempts led to approved drugs up through 2020. Yet here, potential agents for further development have been found from the NCI marine collection efforts, mainly from the Coral Reef Research Foundation collections from 1991~2010 and their subsequent work-up. The ultimate aim of the current fractionation program is to produce ~1 million fractions that are made available for investigators worldwide in due course.
I must also admit to a “slight bias” as I was the NCI Project Officer for the CRRF program from their first to last collections from 1991 to ~2010, when funding ceased.
Literature Cited
- Deusenberry, C.; Carneiro, O.; Oberkfell, C.; Shukla, L. Synergistic coupling of antibiotics and antibiofilm agents against methicillin-resistant S. aureus biofilms. ACS Infect. Dis. 2023, doi.org/10.1021/acsinfecdis.3c00239.
- Wieczorek, M.; Jenssen, H.; Kindrachuk, J.; Scott, W. R. P.; Elliott, M.; Hilpert, K.; Cheng, J. T. J.; Hancock, R. E. W.; Straus, S. K. Structural studies of a peptide with immune modulating and direct antimicrobial activity. Chem. & Biol. 2010, 17, 970-980. doi: 10.1016/j.chembiol.2010.07.007
- Hutter, R.; Keller-Schierlein, W.; Knusel, R.; Prelog, V.; Rodgers, Jr., D. C.; Suter, P.; Vogel, G.; Voser, W.; Zahner, H. Stoffwechselprodukte von mikroorganismen. Boromycin. Helv. Chim. Acta. 1967, 50, 1533-1539. doi: 10.1002/hlca.19670500612
- Zain, M.; Kazmi, H.; Schneider, O. M.; Hall, D. G. Expanding the role of boron in new drug chemotypes: Properties, chemistry, pharmaceutical potential of hemiboronic naphthoids. J. Med. Chem. 2023, 66, 13768-13787. doi.org/10.1021/acs.jmedchem.3c01194.
- Lin, Y.; Jung, H.; Bulman, C. A.; Ng, J.; Vinck, R.; O’Beirne, C.; Zhong, S.; Moser, M. S.; Tricoche, N.; Peguero, R.; Li, R. W.; Urban Jr, J. F.; Le Pape, P.; Pagniez, F.; Moretto, M.; Weil, T.; Lustigman, S.; Cariou, K.; Mitreva, M.; Sakanari, J. A.; Gasser, G. Discovery of new broad-spectrum anti-infectives for eukaryotic pathogens using organometallic chemistry. J. Med. Chem. 2023, 66, 15867-15882. doi: 10.1021/acs.jmedchem.3c01333.
- Jia, K.; Wang, J.; Zhai, R.; Du, Y.; Kira, J.; Wu, C.; Pei-Yuan Qian, P.-Y.; Zhang, W. Abi family protein, DidK, is involved in the maturation of anticancer depsipeptide, didemnin B. ACS Chem. Biol. 2023, 18. doi: 10.1021/acschembio.3c00393.
- Du, L.; Wilson, B. A. P.; Li, N.; Shah, R.; Dalilian, M.; Wang, D.; Smith, E. A.; Wamiru, A.; Goncharova, E. I.; Zhang, P.; O’Keefe, B. R. Discovery and synthesis of a naturally derived protein kinase inhibitor that selectively inhibits distinct classes of serine/threonine kinases. J. Nat. Prod. 2023, 86, 2283-2293. doi: 10.1021/acs.jnatprod.3c00394.