Gideon J. Davies

Neutral glucosylceramidase GBA2 is a retaining β-d-glucosidase involved in sphingolipid metabolism and implicated in neurological lysosomal storage disorders, yet the lack of highly selective chemical inhibitors has hindered efforts to disentangle its function from that of glucosylceramide synthase (GCS) and lysosomal glucosylceramidase (GBA1). Here, we report the development of the first brain-penetrant, covalent inhibitor that selectively inactivates GBA2 in vivo. Building on β-d-arabinofuranose-configured (β-d-Araf) cyclitol aziridine activity-based probes previously shown to covalently and selectively label GBA2, we designed and synthesized a focused library of electrophile-bearing cyclitols, including epoxides, cyclic sulfates, and a diverse set of N-alkyl aziridines. Biochemical evaluation of their inhibitory activity against GBA1, GBA2, GBA3 and GCS revealed that several N-alkyl aziridines are highly selective, nanomolar GBA2 inhibitors. Lead compounds also demonstrated metabolic stability in murine plasma, robust cellular activity and high selectivity in living cells, with no detectable GCS inhibition. Notably, octyl-alkyne aziridine 5 selectively inhibited GBA2 in mouse brain, representing the first covalent GBA2 inhibitor with demonstrated in vivo activity and brain permeability. Together, these results establish β-d-Araf cyclitol aziridine 5 as a versatile inhibitor for selective, covalent GBA2 inhibition, well suited to dissect GBA2 biology in vivo, particularly in neurological lysosomal storage disorders.

This paper describes the development of mannobiose, mannotriose, and glucomannose-based probes and their use in reporting on mannanase activities in secretomes from saprophytic bacteria and fungi grown on mannan-containing biomass polysaccharides.

Xylanases are central to lignocellulosic biomass degradation, yet current methods lack the specificity to resolve how enzymes distinguish complex xylan structures decorated with arabinofuranose (Ara f ) and 4- O -methyl-glucuronic acid (MeGlcA). Here, we report a suite of chemically-defined activity-based probes (ABPs) that enable the selective detection of arabinoxylan- and glucuronoxylan-specific xylanases (AXXs and GXXs). These cyclophellitol-derived ABPs covalently label retaining xylanases at their active sites, allowing precise mapping of substrate specificity across diverse glycoside hydrolase families. Crystallographic and mass spectrometric analyses reveal the molecular basis of probe selectivity, while in-gel and pull-down assays demonstrate their effectiveness in profiling xylanase activities in complex bacterial and fungal proteomes, including cellulosomes. By integrating activity-based protein profiling (ABPP) with sequence similarity networks (SSNs), we further show that xylanase specificity can be predicted from sequence alone, enabling rapid functional annotation of uncharacterized xylanases. This chemoproteomic strategy provides a powerful platform for discovering and engineering substrate-specific enzymes for biomass valorisation, microbial ecology, and biotechnological applications.

Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by noncovalent binding and emerging resistance. We hypothesized that Oseltamivir aziridines would unite transition-state mimicry for tight binding, with aziridine-enabled covalent capture of the catalytic tyrosine, thereby supporting both therapy and activity-based quantification. Here, we present oseltamivir-based aziridines, inspired by cyclophellitol chemistry, that act as covalent inhibitors and activity-based probes via an N -acylaziridine warhead. Free-energy calculations, and NMR observations, indicate a ⁴ H ₅ half-chair preference consistent with the NA transition state, and selected analogues inhibit multiple NA subtypes with low nanomolar binding constants. Diverse evidence establishes covalency: time-dependent inactivation, inhibitor washout, intact-mass shifts, MS/MS identification of a tyrosine adduct, and QM/MM reaction profiles, while cryoEM of N1 aligns with the proposed binding mode, revealing an elimination product. The inhibitors demonstrate formidable activity against diverse viral neuraminidases, including H5N1, and further enable imaging and quantification of active NA. With their dual therapeutic and diagnostic potential, these first-in-class inhibitors indeed benefit from transition state mimicry and covalency, and thus offer a powerful platform for antiviral development and neuraminidase imaging, addressing urgent global health needs in influenza treatment and prevention.

Sulfoquinovose (SQ) is a major biogenic sulfonated sugar whose degradation fuels microbial sulfur and carbon cycling. In the sulfoglycolytic Entner–Doudoroff (sulfo‐ED) pathway, 2‐keto‐3,6‐dideoxy‐6‐sulfogluconate (KDSG) is cleaved by KDSG aldolase to yield pyruvate and sulfolactaldehyde, yet the structure and mechanism of this enzyme have remained unclear. We report the biochemical and structural characterization of a metal‐dependent KDSG aldolase from Pseudomonas putida using chemo‐enzymatically synthesized KDSG. The enzyme forms a homohexamer, with a (β/α) ₈ TIM‐barrel monomer assembling as a ‘dimer‐of‐trimers’. The enzyme exhibits optimal activity in the presence of Co ²⁺ or Mn ²⁺ , consistent with other class II aldolases. Kinetic analysis revealed millimolar‐range K _M values for KDSG and modest cross‐reactivity with the related glycolytic intermediate, 2‐keto‐3,6‐deoxy‐6‐phosphogluconate (KDPG). Crystal structures of the apo and Co ²⁺ •pyruvate‐bound forms (2.85 Å and 2.80 Å) show a metal‐coordinated active site at the subunit interface, with conserved residues mediating metal binding and catalysis, providing insights into the mechanism of sulfonate‐specific aldol cleavage. Sequence‐similarity network and genome‐context analyses show that KDSG aldolases are widespread among Proteobacteria and typically cluster with sulfo‐ED pathway genes. These results define the structural and mechanistic basis of KDSG aldolases and inform on their roles in bacterial sulfur metabolism.

Glycoside hydrolases, "glycosidases", catalyze carbohydrate catabolism, remodeling, and signaling by accelerating glycosidic-bond cleavage by more than 17 orders of magnitude. Distributed across every kingdom of life and grouped into over 180 sequence-defined families, these enzymes exhibit exceptional diversity in fold, mechanism, and physiological function, and many also catalyze transglycosylation or phosphorolysis. The classical Koshland paradigms─stereochemical inversion, enzymatic nucleophile-assisted retention, and substrate-assisted retention─are analyzed with an emphasis on the conformational itineraries and oxocarbenium ion-like transition states revealed by kinetic isotope effects, linear free-energy relationships, and high-resolution three-dimensional structures. Attention then turns to noncanonical enzymes that employ NAD⁺-dependent redox hydrolysis or other cleavage mechanisms. Mechanistic insights have inspired the development of engineered glycosidase-derived catalysts for programmed bond construction as well as mechanism-based inhibitors, transition-state analogues, and activity-based probes that are driving advances in chemical biology, biotechnology, and drug discovery.

D-Cysteinolic acid is a prominent sulfur-containing compound in marine and freshwater ecosystems, yet its metabolic fate has remained largely uncharacterized. Here, we describe a newly identified d-cysteinolic acid degradation pathway in a marine model bacterium Ruegeria pomeroyi DSS-3. This pathway involves a PLP-dependent cysteinolic acid racemase (ClaA) that interconverts d- and l-cysteinolic acids, followed by a NAD⁺-dependent l-cysteinolic acid dehydrogenase (ClaB) that oxidizes l-cysteinolic acid to l-cysteate. A cysteate racemase (CuyB) then converts l-cysteate to d-cysteate, the preferred substrate for the sulfolyase CuyA. Bioinformatic analysis of the Tara Oceans gene atlas reveals that ClaA and CuyA homologues are widespread in marine bacterial populations, particularly within the alphaproteobacterial Roseobacter and SAR116 groups and the gammaproteobacterial SUP05 clade. Relative abundance of gene homologues correlates with surface water chlorophyll levels, linking d-cysteinolic acid degradation to photosynthetic primary production. This discovery advances our understanding of marine sulfur cycling and highlights d-cysteinolic acid as an important metabolic currency in microbial networks.

Metalloprotein discovery is often made post hoc , in which activity studies following protein isolation reveal a metal-ion dependence. Herein we take a different approach to finding metalloproteins, by building on the discovery of copper-containing lytic polysaccharide monooxygenases (LPMOs), which include an N-terminal histidine as part of their sequence. This residue acts as a natural chelator for transition metal ions, irrespective of the structure of the protein. We report the method of signal strapping , where sequences of N-terminal signal peptides artificially appended with a histidine residue at their C-terminus are used to bootstrap a proteomic search. These searches return sequences of proteins with an N-terminal histidine capable of coordinating a metal ion. We exemplify the approach by the discovery and characterisation of four classes of bacterial metalloproteins, including two that we denote as anglerases reflecting their potential to capture transition metal ions from the bacterial environment.

Fragment-based drug discovery is widely used in both academia and industry during the early stages of drug discovery. There is a growing interest in the design of 3-D fragments for inclusion in fragment libraries in order to increase chemical space coverage. We present herein the design and synthesis of 58 shape-diverse 3-D fragments that are prepared using just three modular synthetic methodologies. The 3-D fragments comprise a cyclic scaffold (cyclopentane, pyrrolidine, piperidine, tetrahydrofuran or tetrahydropyran) with one aromatic or heteroaromatic ring and possess properties within ‘rule-of-three’ fragment space. 3-D shape is assessed using principal moments of inertia analysis and conformational diversity is achieved by considering all conformations up to 1.5 kcal mol⁻¹ above the energy of the global minimum energy conformer. Due to the modular nature of the fragment syntheses, these 3-D fragments are synthetically-enabled for fragment elaboration follow-on work, a key design feature. This modular, shape-diverse 3-D fragment collection has delivered privileged starting points across a spectrum of targets. Fragments from the set have been crystallographically validated in the SARS-CoV-2 main protease (M^pro) and the nonstructural protein 3 (Nsp3) (Mac1) as well as human glycosyltransferase MGATV, a major enzyme in the mammalian N-glycosylation pathway and a promoter of aggressive metastatic cancers, underscoring the breadth of biological space that can be explored.

Fragment-based drug discovery is widely used in both academia and industry during the early stages of drug discovery. There is a growing interest in the design of 3-D fragments for inclusion in fragment libraries in order to increase chemical space coverage. We present herein the design and synthesis of 58 shape-diverse 3-D fragments that are prepared using just three modular synthetic methodologies. The 3-D fragments comprise a cyclic scaffold (cyclopentane, pyrrolidine, piperidine, tetrahydrofuran or tetrahydropyran) with one aromatic or heteroaromatic ring and possess properties within ‘rule-of-three’ fragment space. 3-D shape is assessed using principal moments of inertia analysis and conformational diversity is achieved by considering all conformations up to 1.5 kcal mol-1 above the energy of the global minimum energy conformer. Due to the modular nature of the fragment syntheses, these 3-D fragments are synthetically-enabled for fragment elaboration follow-on work, a key design feature. This modular, shape-diverse 3-D fragment collection has delivered privileged starting points across a spectrum of targets. Fragments from the set have been crystallographically validated in the SARS-CoV-2 main protease (Mpro) and the nonstructural protein 3 (Nsp3) (Mac1) as well as human glycosyltransferase MGATV, a major enzyme in the mammalian N-glycosylation pathway and a promoter of aggressive metastatic cancers, underscoring the breadth of biological space that can be explored.