Scientists have found a way to create artificial sugars that could lead to more accurate ways to diagnose and perhaps treat diseases. The collaboration of academic and industry scientists in Europe, including teams at the University of Manchester and the University of Leeds, developed a method to create “fluoro-sugars,” or “glycofluoroforms”—specific glycan sugars containing fluorine molecules—that could block pathogens. The team also demonstrated the potential diagnostic utility of their approach for detecting a bacterial toxin using lateral flow technology.
The study provides evidence that the artificial fluoro-sugars can be used to fine-tune pathogen or biomarker recognition or even to discover new drugs. They also offer an alternative to antibodies in low-cost diagnostics, which do not require animal tests to discover and are heat stable. “At Manchester, we are very interested, and currently exploring, how we can deploy natural and unnatural glycans in diagnostics,” said Matthew Gibson, PhD, a professor at the University of Manchester Institute of Biotechnology, speaking to GEN. “Our platform has several benefits such as not needing to use animal technologies to raise new antibodies, and the glycosylated nanoparticles are very stable against heat and other stressors so could find use in lower resource environments and for pandemic preparedness.”
Reporting in their published paper in Nature Communications (“Synthesis and screening of a library of LewisX deoxyfluoro-analogues reveals differential recognition by glycan-binding partners,”) Gibson and colleagues wrote, “We anticipate that our preliminary data demonstrating application of the glycofluoroform approach for lateral flow detection of a bacterial toxin will accelerate the development of glycan-based diagnostics, and therapeutics.”
Sugars play a crucial role in human health and disease, far beyond being just an energy source. Complex sugars called glycans coat all our cells and are essential for healthy function. These sugars are also often hijacked by pathogens such as influenza, SARS-CoV-2, and cholera, to infect us. “Glycan-mediated interactions play a crucial role in biology and medicine, influencing signaling, immune responses, and disease pathogenesis,” the authors wrote, “… and significant advances have been made in understanding these processes through studies of the recognition of glycan sequences by glycan-binding proteins.”
However, the same glycan can bind to many different proteins, and this makes it difficult to use glycans as targets for precise diagnostics or therapeutic approaches. “… the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognized by multiple biologically distinct protein receptors,” the team continued. “This raises the challenge of how one can develop small molecule probes for diagnostics or therapeutics that can distinguish between the interactions of different proteins with the same glycan.”
“LewisX is a glycan motif that is a ligand for recognition in numerous contexts with many different proteins,” the authors further explained. The trisaccharide is expressed, for example, on tumor cells, and on milk sugars (the authors cited studies indicating that it may protect against toxins and pathogens involved in some childhood diseases), and on the surface of some pathogens, including gastric cancer-causing Helicobacter pylori, and the parasite schistosoma mansoni, which causes schistosomiasis. “Given the biological importance of LewisX it would be desirable, for diagnostic and therapeutic purposes, to have chemical probes that could distinguish between each of the diverse proteins with which this glycan can interact,” the investigators further noted.
For their reported study, Gibson and colleagues used a combination of enzymes and chemical synthesis to create a library of 150 LewisX glycofluoroforms, to which they had added fluorine atoms at specific sites. Fluorine is very small, which means that the resulting fluoro-sugars kept their same 3D shape, but the fluorines interfered with how proteins bind to them. The team then characterized and investigated 24 glycans from the library, which they incorporated into nanoparticles or microarrays, to see whether the different fluorination patterns were associated with binding by specific LewisX-binding proteins, including antibodies and bacterial toxins.
Kristian Hollie, PhD, who led the production of the fluoro-sugar library at the University of Leeds, said, “We used enzymes to rapidly assemble fluoro-sugar building blocks to make 150 different versions of a biologically important glycan. We were surprised to find how well natural enzymes work with these chemically modified sugars, which makes it a really effective strategy for discovering molecules that can bind selectively.”
Co-author Sabine Flitsch, PhD, a researcher from the Manchester Institute of Biotechnology at the University of Manchester, added, “One of the key technologies used in this work is biocatalysis, which uses enzymes to produce the very complex and diverse sugars needed for the library. Biocatalysis dramatically speeds up the synthetic effort required and is a much more green and sustainable method for producing the fluorinated probes that are required.”
The results of the researchers’ studies showed that some of the glycofluoroforms prepared could be used to detect the cholera bacteria toxin, indicating that they might be used in simple, low-cost tests, similar to lateral flow tests that are widely used for pregnancy, or SARS-CoV-2 testing. The authors reported, “… incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognize LewisX … Overall, our microarray and nanoparticle binding studies reveal that different proteins with a LewisX binding site have distinctive recognition modes for this important trisaccharide.”
Gibson noted to GEN, “… my team previously filed patents on lateral flow glyco assays, showing how glycans can be introduced into gold particles to enable them as antibody alternatives in lateral flow. I anticipate there is quite a lot of space for innovation non-antibody-based diagnostics … In terms of clinical applications, early in the COVID-19 pandemic, my team led a collaboration to make a SARS-COV-2 rapid diagnostics based on glycans: we validated this in clinical samples showing good performance. The new work that we have published today is important to allow us to get more selectivity and specificity, and we did some model studies for cholera-toxin detection.”
One of the paper’s lead authors, Bruce Turnbull, PhD, from the School of Chemistry and Astbury Centre for Structural Molecular Biology at the University of Leeds, added, “Glycans that are really important for our immune systems, and other biological processes that keep us healthy, are also exploited by viruses and toxins to get into our cells. Our work is allowing us to understand how proteins from humans and pathogens have different ways of interacting with the same glycan. This will help us make diagnostics and drugs that can distinguish between human and pathogen proteins.”
The study provides evidence that the artificial fluoro-sugars can be used to fine-tune pathogen or biomarker recognition or to discover new drugs. “This validated ability of glycan fluorination patterning to deliver significant changes in protein binding whilst maintaining glycan conformation, heralds great promise for the development of diagnostic kits or new anti-adhesion therapies to combat microbial infections,” the team concluded. “We anticipate that our preliminary data demonstrating application of the glycofluoroform approach for lateral flow detection of a bacterial toxin will accelerate the development of glycan-based diagnostics, and therapeutics.”
Gibson further explained to GEN that several members of the team are expanding the methods to additional glycans, and others are integrating these into innovative sensing platforms with new signal generation technologies. “In terms of orthogonal areas, the site-specific integration of fluorine into polysaccharides could bring real benefits to make advanced materials based on sustainable platforms,” he suggested. There is also an opportunity to use fluorinated glycans in biomedical areas as new inhibitors, building on the established use of fluorination in clinically approved drugs. “This is part of why we are excited: the chemo-enzymatic strategies can be applied to a large range of other glycans: for example, in our earlier work we showed some smaller fluoro-glycans could be prepared and had selective interactions with proteins with cancer diagnostic potential.”
Gibson stated, “During the COVID-19 pandemic, our team introduced the first lateral flow tests which used sugars instead of antibodies as the ‘recognition unit.’ But the limit is always how specific and selective these are due to the promiscuity of natural sugars. We can now integrate these fluoro-sugars into our biosensing platforms with the aim of having cheap, rapid, and thermally stable diagnostics suitable for low-resource environments.”
There are a huge number of potential diagnostic targets from bacterial to viral pathogens to cancer markers, he added in his comments to GEN. “I would also stress the opportunity for these glycosylated nanosensors for biosensing, to support research or monitoring of how infectious agents evolve their glycan-binding preferences, such as during influenza zoonosis … The team is currently looking at how this technology can be integrated into other sensing platforms, to expand beyond lateral flow, but are also excited to connect to parties interested in glycan-binding events for sensing/diagnostics.”
