Ulf Ellervik

Gaucher disease is a rare lysosomal storage disorder caused by autosomal recessive mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase. Gaucher disease is classified in 3 different subtypes depending on the presence and severity of neurological involvement, with type 2 resulting in fatal early-onset neuropathology and patients exhibiting developmental delays, seizures and early death. Studies investigating disease mechanisms of neuronopathic Gaucher disease are mainly based on animal models and focus predominantly on late neuronal phenotypes. Here, we established healthy control and Gaucher disease patient-derived iPSC lines and engineered them to obtain isogenic control and disease lines. Using these lines, we generated cortical and subpallial brain organoids in which we identified early-onset lipid dysregulation in form of glucosylceramide accumulation, highly elevated glucosylsphingosine, and a later increase in ganglioside levels, recapitulating clinical findings. Furthermore, single-cell transcriptomic profiling uncovered novel phenotypes in both cortical and subpallial forebrain organoids. Subpallial alterations consisted of an early increase in migrating interneurons in subpallial organoids, which upregulated cholesterol metabolism. Cortical alterations showed early upregulation of mitochondrial genes and a downregulation of proliferation, with a subsequent switch from GABAergic to glutamatergic neuron fate with a striking increase in gene expression related to the synaptic assembly. Functional assays demonstrated a marked hyperexcitability of cortical organoids and reduced response to GABA-A receptor blockage in Gaucher disease. Additional 2D neuronal network models confirmed the organoid data and showed that both glutamatergic and GABAergic neurons contribute to the phenotype, with hyperexcitability of Gaucher glutamatergic neurons and incapacity of Gaucher GABAergic neurons to balance the excessive excitation. This alteration represents a clinically significant phenotype as many patients exhibit an excitation/inhibition imbalance leading to treatment-resistant seizures, hastening their decline. In conclusion, our defined human models of Gaucher disease identify novel and clear phenotypes that can be used for drug screening or aid in development of new therapeutic strategies to ameliorate Gaucher disease.

Dermatan sulfate epimerase 1 (DS-epi1) is a key enzyme in the biosynthesis of the glycosaminoglycan chondroitin sulfate/dermatan sulfate, catalyzing the conversion of glucuronic acid to iduronic acid at the polymer level. Chondroitin sulfate/dermatan sulfate chains are found on at least 32 proteoglycans, many of which are implicated in human diseases and syndromes, as well as in both malignant and normal cell development. DS-epi1 therefore represents a promising target for drug development, and recent structural studies have provided insights into its active site and catalytic mechanism. Here, we report the synthesis and biological evaluation of inhibitors based on 1,4-disubstituted glucuronic acids. These compounds were synthesized from glucose through a divergent approach, yielding 19 derivatives that were tested in a functional assay. To explore the importance of the carboxylic acid moiety, we also tested the methyl ester analog and the analogous xylose derivative. The most potent compound exhibited an IC50 of 42 ± 4 μM. Molecular dynamics simulations showed a strong interaction with the active site of DS-epi1.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of glycosaminoglycans (GAG) that transfers the first galactose unit to xylose in the linker region. Searching for new inhibitors of the GAG biosynthesis, we used saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy to evaluate the binding interactions between β4GalT7 and several pentosides in the presence of UDP donors. These investigations verified the glycosylation specificity of β4GalT7 and revealed that the naphthalene and the uridine moieties were significant contributors to the binding of the acceptor and the donor, respectively, while the galactose part was less important. Based on these findings, we set out to investigate conjugates of UDP and naphthoxylosides to function as transition state analogues. These compounds were synthesized using a one-pot procedure and tested as inhibitors in a β4GalT7 assay. Interestingly, one truncated analogue, a bisphosphonate-xyloside construct, showed a significant inhibition (IC₅₀: 188 μM). These findings open for the design of a new class of inhibitors of the GAG biosynthesis.

In galactosylation by β4GalT7 of 2-naphthyl xylosyl sulfoxides, higher affinity for the enzyme was observed for the acceptor substrate having the (R)_S-configuration than for the diastereomer with the (S)_S-configuration.

In search for novel antibacterial compounds, bacterial sialic acid uptake inhibition represents a promising strategy. Sialic acid plays a critical role for growth and colonisation of several pathogenic bacteria, and its uptake inhibition in bacteria was recently demonstrated to be a viable strategy by targeting the SiaT sodium solute symporters from Proteus mirabilis and Staphylococcus aureus. Here we report the design, synthesis and evaluation of potential sialic acid uptake inhibitors bearing 4‐N‐piperidine and piperazine moieties. The 4‐N‐derivatives were obtained via 4‐N‐functionalization with piperidine and piperazine nucleophiles in an efficient direct substitution of the 4‐O‐acetate of Neu5Ac. Evaluation for binding to bacterial transport proteins with nanoDSF and ITC revealed compounds possessing nanomolar affinity for the P. mirabilis SiaT symporter. Computational analyses indicate the engagement of a previously untargeted portion of the binding site.

Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.

A direct one-step nucleophilic substitution of the 4-OAc of acetyl protected Neu5Ac is presented.

Fluorescently labeled xylosides are taken up by cells and initiate priming of labeled GAG chains of various length.

We present a xylosylated naphthoxyloside carrying a terminal azide functionality that can be used for conjugation using click chemistry. We show that this naphthoxyloside serves as a substrate for β4GalT7 and induces the formation of soluble glycosaminoglycan (GAG) chains with physiologically relevant lengths and sulfation patterns. Finally, we demonstrate its usefulness by conjugation to the Alexa Fluor 647 and TAMRA fluorophores and coupling to a surface plasmon resonance chip for interaction studies with the hepatocyte growth factor known to interact with the GAG heparan sulfate.

Proteoglycans have important biological activities. To improve the overall synthetic efficiency, a new chemoenzymatic route has been established for the proteoglycan linkage region bearing a galactose-xylose disaccharide. The xylosylated glycopeptides were synthesized via solid phase synthesis, which was followed by the addition of the galactose unit by the galactosyl transferase β4GalT7. This work leads to a better understanding of the acceptor preference of β4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.