The global objective of CARBOCENTRE is the design and application of activity-based probes for characterization, detection and inhibition of glycoprocessing enzymes, focusing on three major strands of increasing complexity: (1) retaining glycoside hydrolases (ret-GHs, 1st research strand), inverting glycoside hydrolases (inv-GHs, 2nd research strand, challenging as no ABP ideas exist) and glycosyltransferases (GTs, 3rd research strand, extremely challenging as even inhibitors for this class are lacking).
These compounds are then applied across two domains of application: Biotechnology and Medicine. In biomedicine, we expect the reagents and methods developed to find application in diagnosis (such as for lysosomal storage diseases, and other diseases where lack of enzyme activity is key), as readouts for drug discovery (such as in competitive and high throughput approaches), as enzyme stabilisers (pharmacological chaperones) and as drugs themselves, such as in anti-cancer and anti-viral applications. In biotechnology we expect application in enzyme discovery in complex systems (soil, microbiota, secretomes etc), industrial enzyme characterisation, as readouts for directed evolution; essentially to provide functional data to the wealth of sequence data.
The strands and the application domains are based around a common ethos of understanding and exploiting enzyme mechanism.
Since the start of the program, most progress has been made on strand 1, retaining glycoside hydrolases. By harnessing 3-D structure and computation, ABPs have designed and synthesized for a considerable area of enzymes. In biomedicine, we have diagnostics and chaperones for very many families of enzymes involved in genetic disease, we have readouts for high throughput fluorescence polarization assays allowing for high throughput drug screening. We have developed enzyme inhibitors, based upon probe designs, for application in anti-cancer applications through heparanase inhibition (a spin-out company has been established, aided by ERC proof of concept funds), we have developed inhibitors of human enzymes involved in virus maturation − initially as anti-Sars-Cov2 compounds − and achieved first-in class neuraminidase inhibitors (IP protection in progress). In the biotechnology arena, we have focused our work on discovery (enzymes in bacterial and fungal secretomes, enzymes in animal microbiota), on the enzyme responsible for the degradation of highly recalcitrant plant polysaccharides (cellulose, xylan, xyloglucan, starch) and on the key industrial challenges of enzyme stability and engineering. In research strand 2 (inverting glycoside hydrolases), we need to overcome the challenges of the absence of a catalytic nucleophile. We are working on human glucosidase I (again an anti-HIV/Covid target) and screening newly designed libraries for compounds – including computer modeling − which are looking promising. In strand 3 (glycosyltransferases), we have been focusing on expressing enzymes for study, which is being very challenging and fundamental studies of reaction coordinate, though computation and 3D structures – including AI-generated structures. We also designed libraries of inhibitors, competitive in the first instance and will work on introducing covalency going forward. We have successfully designed competitive inhibitors of glycogen phosphorylase (a “simplified” glycosyltransferase) and α4-galactosyltransferase and hope to build upon this in the future.
