Unraveling A4GALT Mechanism and Its Modulation With Adamantyl-Galactosylceramide Analogues: Advancing Fabry Disease Therapeutic Strategies.

NDST1 as a substrate-reduction target in Mucopolysaccharidosis type IIIC: virtual screening, microsecond molecular dynamics, and peptide design

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood–brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of −13.36 ± 5.87 kcal mol−1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

Revisiting Enzyme Replacement Therapy for Aspartylglucosaminuria: Truncated Phosphotransferase Enhances Mannose-6-Phosphorylation and Cellular Uptake of Aspartylglucosaminidase.

Novel tetrazole annulation on benzopyridodiazepine derivatives as inhibitor of tropomyosin kinase B (TrkB): rational design, synthesis, biological evaluation, SAR, and in silico studies

Tropomyosin kinase B (TrkB) is an attractive target for cancer treatment and is emerging as a target for the treatment of neurological disorders, specifically epilepsy, due to its role in regulating neuronal degeneration and inflammation. However, current medications frequently suffer from toxicity, resistance, and poor metabolism. In the present study, four new series of tetrazole annulated benzopyridodiazepine derivatives (26 examples) were synthesized in two steps: intramolecular cyclization with various sets of amines to produce benzopyridodiazepinone derivatives, and then, in the second step, the tetrazole ring was introduced by the reaction of imidoyl chloride with azide. All four synthesized series were assessed for in vitro inhibitory activity against tropomyosin kinase (TrkB) using both enzyme and cellular assays. To further evaluate the biological efficacy of the synthesized derivatives, we conducted ROS measurements, fluorescence-based microscopy, and flow cytometry to determine the mode of action and the induction of apoptosis. Molecular docking, ADMET, and DFT studies supported the binding at the TrkB site and suggested favorable pharmacokinetic profiles. Overall, the results indicate that the synthesized compounds are highly promising candidates, especially 14h, which shows significant inhibition of TrkB in neuroblastoma cells. Thus, the tetrazole annulated benzopyridodiazepine is an interesting molecule for exploration in the management of cancer and neurological disorders.

Metabolomic, lipidomic, and N-glycomic analyses of a human cell model of Krabbe disease reveal treatable deficits in glycosylation and serine-ceramide metabolism

Krabbe disease is a rare autosomal recessive lysosomal disease caused by deficiency of galactocerebrosidase (GALC), leading to accumulation of galactosylceramide and formation of the toxic metabolite galactosylsphingosine (psychosine). While psychosine accumulation is well-established as a primary pathogenic mechanism, the broader metabolic consequences of GALC deficiency remain incompletely understood. In this study, we used stable isotope tracing to comprehensively characterize metabolic perturbations in a human oligodendrocellular Krabbe disease model. This approach revealed elevated de novo ceramide synthesis in GALC knock-out cells, characterized by increased incorporation of glucose-derived serine into ceramide biosynthetic pathways. This enhanced ceramide production was amenable to pharmacological intervention by tezacaftor, an inhibitor of sphingolipid Δ4-desaturate (DEGS); tezacaftor administration also normalized psychosine levels, raising the possibility of its use as substrate reduction therapy. Additionally, we identified significant disruption of UDP-hexose metabolism, manifesting as an overabundance of truncated and hypogalactosylated glycans. These findings suggest impaired protein glycosylation as a previously unrecognized pathogenic mechanism in Krabbe disease. Our findings reveal novel metabolic dysregulation in Krabbe disease extending beyond established psychosine toxicity. The identification of enhanced de novo ceramide synthesis presents a new therapeutic target, while the discovery of galactose-deficient glycosylation defects supports galactose supplementation as a potential therapeutic intervention. These metabolic insights provide new mechanistic understanding and therapeutic opportunities for this devastating neurodegenerative disorder.

The immunosuppressant tacrolimus (FK506) inhibits C. glabrata Cdr1 efflux pump function by stabilizing the inward-facing conformation

The pathogenic yeast Candida glabrata is intrinsically resistant to azole antifungals through the overexpression of the multidrug transporter Cdr1. CgCdr1 detoxifies the yeast by expelling azoles out of the cell, thereby decreasing their intracellular concentration. Tacrolimus (FK506), one of the most widely used immunosuppressant medications used world-wide, has been identified as a broad-spectrum inhibitor of Cdr1 homologs in several Candida species. However, its mechanism of action remains unknown. We solved the cryoEM structure of CgCdr1 in complex with FK506, with or without ATP. The structure revealed that FK506 binds within the drug-binding site of CgCdr1, occupying the space occupied by Itraconazole. The hydrophobic face of FK506 stacks against the TMD1 and forms hydrogen bonds with TMD2, stabilizing a different conformation from the one adopted in FK-binding-proteins. FK506 binding triggered structural rearrangements bringing the nucleotide-binding-domains closer to the trans-membrane-domains, while stabilizing the inward-facing conformation. While ATP can still bind to the catalytic nucleotide-binding site, FK506 prevents the conformational transition required for ATP hydrolysis, thereby effectively blocking azole transport. Inter-particle variability analysis (3DVA) revealed significant conformational flexibility of FK506 within the binding pocket, with minimal transporter mobility. It allowed to visualize the conformational space occupied by the inhibitor within its binding-pocket, serving as a useful tool for inhibitor rational design. Overall, these findings demonstrate that FK506’s inhibition extends beyond competitive binding, involving allosteric modulation of the ATPase cycle. Significance statement The pathogenic yeast Candida glabrata exhibits intrinsic resistance to azole antifungals via the multidrug transporter Cdr1, which expels azoles from the cell. Tacrolimus (FK506), a widely used immunosuppressant, inhibits Cdr1 homologs across Candida species, yet its mechanism remained unknown. Here, we resolved the cryoEM structure of CgCdr1 in complex with FK506, revealing that FK506 binds to the drug-binding site, like itraconazole. Its hydrophobic face interacts with TMD1, while hydrogen bonds form with TMD2. FK506 stabilizes the inward-facing conformation, preventing ATP hydrolysis despite ATP binding, thereby blocking azole transport. Variability analysis highlighted FK506’s conformational flexibility within the pocket, offering insights for rational inhibitor design. These findings demonstrate that FK506’s inhibition involves both competitive binding and allosteric modulation of the ATPase cycle.

Novel salicylamido sulfonamides as CD73 immune checkpoint inhibitors: Synthesis and in vitro biological evaluation.

The immune checkpoint enzyme CD73 plays a crucial role in the adenosine (ADO) metabolic pathway by catalyzing the conversion of AMP to adenosine. Dysregulated CD73 activity elevates extracellular ADO in the tumor microenvironment, driving immunosuppression and tumor immune evasion, highlighting CD73 as a key immunotherapeutic target. In this study, we report the design and synthesis of a novel series of salicylamido sulfonamide-based small-molecule CD73 inhibitors. The in vitro CD73 inhibitory assay reveals that SA-41 and SA-26 exhibited potent activity with IC50 values of 2.83 ± 0.45 μM and 2.96 ± 0.46 μM, respectively. Molecular docking and dynamic simulations revealed that SA-26 maintained stable interactions with CD73 throughout a 100-ns simulation with no significant deviation. Furthermore, in silico ADME analysis indicated that both lead compounds possess favourable drug-like properties, with no Lipinski's rule-of-five violations, balanced lipophilicity, and acceptable predicted oral absorption, supporting their pharmacokinetic potential. These findings highlight SA-26 and SA-41 as promising candidates for further development as non- nucleotide-based small-molecule CD73 inhibitors.