Open Access

Show Abstract ▼

Abstract: WRN helicase is an established synthetic lethal target for inhibition in the treatment of microsatellite instability-high (MSI-H) and mismatch repair deficient (MMRd) cancers. The identification of helicase inhibitors is challenging as high-throughput biochemical screening campaigns typically return few validated hits that are often inactive in cell-based assays. Herein, we highlight the power of non-covalent fragment-based lead discovery in locating new druggable allosteric sites on WRN, enabling us to bypass the challenging behavior of WRN during high-throughput screening hampering hit identification. During the fragment optimization process, structures of WRN with key prioritized fragments reveal multiple conformations of WRN with significant domain rotations up to 180°, including a WRN conformation not previously described. Rooted in a combination of biochemical, biophysical, and structural approaches, we present the detailed analyses of optimized chemical matter evolved from screening hits and the unique ability of WRN to accommodate diverse conformations as detailed by structural characterization.

Open Access

Show Abstract ▼

Abstract: Traboulsi Syndrome is an autosomal recessive hereditary disease associated with developmental defects, in particular of the ocular system. Single nucleotide polymorphisms affecting the ASPH gene, which encodes for the 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH), are associated with Traboulsi Syndrome. AspH catalyzes hydroxylations of conserved aspartate/asparagine residues in epidermal growth factor-like domain (EGFD) proteins. We report studies on the clinically-observed Traboulsi Syndrome-associated R688Q, R735Q, and R735W AspH variants. The results reveal that pathogenic active site substitutions substantially reduce, though do not ablate, EGFD hydroxylase activity compared to wildtype AspH. They imply that efficient AspH catalyzed EGFD hydroxylation is important during human development. Crystallographic studies reveal conservation of the overall AspH fold, but that the preferred conformations of 2OG in complex with the R735Q and R735W AspH variants differ from that with wildtype AspH. Screening of potential 2OG cosubstrate substitutes reveals certain 2-oxoacids, including naturally present metabolites, manifest enhanced catalytic efficiency of Traboulsi Syndrome-associated AspH variants compared to 2OG. The results thus provide proof-of-principle for a therapeutic strategy involving rescue of impaired activities of pathogenic active site AspH variants by use of 2-oxoacids, or 2-oxoacid precursors, other than 2OG.

Open Access

Show Abstract ▼

Abstract: Tissue fibrosis is a hallmark of systemic sclerosis (SSc) and results from the persistent activation of fibroblasts and excessive accumulation of extracellular matrix component such as collagen. Recent evidence implicates the matricellular protein Tenascin-C (TNC) in promoting self-sustaining fibroblast activation and fibrosis via its interaction with Toll-like receptor 4 (TLR4). In this study, we utilized Adhiron-guided ligand discovery to identify small molecule inhibitors targeting the fibrinogen-like globe domain of TNC, a key mediator of TLR4 activation. Two lead compounds (464 and 830) demonstrated structural similarity, favourable ADME profiles, and robust anti-fibrotic activity in vitro. Treatment of dermal fibroblasts derived from SSc patients with either compound significantly reduced Transforming growth factor-β-induced expression of fibrotic genes, ACTA2, COL1A1, COL1A2, and CCN2, and inhibited myofibroblast differentiation. These studies may facilitate the development of effective targeted therapy for fibrosis in SSc and support this novel strategy for small molecule development.

Open Access

Show Abstract ▼

Abstract: This study describes the affinity maturation, molecular engineering, and preclinical assessment of depemokimab, an enhanced anti-interleukin-5 antagonist antibody. The molecular design objective for depemokimab was to generate a therapeutic antibody enabling a less frequent dosing regimen of once every 6 months compared with every 4 weeks for mepolizumab. Mepolizumab is a marketed monoclonal antibody used as an add-on prescription maintenance treatment for patients with severe asthma with an eosinophilic phenotype and other eosinophilic-associated disorders. A complementarity-determining region restricted affinity maturation strategy was used where affinity improved interleukin-5 binding antibody variants were subject to affinity driven selective pressure and identified using the Adimab yeast-based platform. Improved complementarity-determining region variants were combined with serum half-life extending amino acid mutations introduced into the fragment crystallizable region of the antibody. When compared with mepolizumab, depemokimab demonstrated improved in vitro interleukin-5 neutralization in a TF-1 (human erythroleukemia) functional cell assay. In vivo, depemokimab displayed significantly extended pharmacokinetic performance and pharmacodynamic duration determined via eosinophil suppression in cynomolgus monkey (Macaca fascicularis). These data provide compelling evidence that a less frequent dosing regimen for depemokimab in humans is possible and supported the advancement of depemokimab into a Phase I study in patients with asthma.

Open Access

Show Abstract ▼

Abstract: Objective: To determine the high-resolution structure of human Copper-Zinc superoxide dismutase (hSOD1), an antioxidant enzyme whose mutations cause amyotrophic lateral sclerosis (ALS), under near-physiological conditions. Because SOD1 is intrinsically dynamic, capturing its structure at ambient temperature is key to understanding how temperature modulates its conformational flexibility, ensemble and functional states relevant to both catalysis and disease. Materials and Methods: Recombinant hSOD1 was expressed in E. coli, purified by affinity and size-exclusion chromatography, and crystallized at ambient temperature. Serial synchrotron crystallography (SSX) data were collected at 293 K at the EMBL P14-2 Time-Resolved Experiments with Crystallography (T-REXX) beamline at PETRA III, and compared with a 100 K cryogenic at the Diamond Light Source beamline (I03). Both datasets were processed and refined using CCP4 suite and PHENIX packages. B-factor distributions, per-residue RMSD values, and conformational differences were analyzed to quantify temperature-dependent effects. Results: The ambient-temperature SOD1SSX structure was determined at 2.3 Å resolution (PDB ID:9XJ0 this work) and closely matched its 2.37 Å cryogenic counterpart (SOD1CRYO, PDB ID:9XJI this work), both obtained from identical crystallization conditions in the hexagonal P6₃ space group. Cryocooling caused a 3.8% contraction in unit-cell volume, consistent with lattice densification and a 5.2% reduction in molecular surface volume. Despite the overall similarities, the ambient-temperature model revealed localized conformational differences in solvent-exposed loop residues, particularly Ser25-Asn26, Leu67-Glu77, Ile99, and the Asp109-His110-Cys111 triad, and a distinct side-chain orientation of Asn53 was observed at the dimerization interface. While the β-barrel core remained rigid, these regions correspond to redox- and metal-responsive sites implicated in aggregation/fiber formation and putative drug binding. Conclusions: Temperature perturbs local dynamics in SOD1 structure without altering its native dimeric form. The ambient-temperature model reveals flexible, chemically accessible regions that act as druggable hotspots and coincide with ALS-linked mutation sites driving misfolding and aggregation. Considering temperature effects is crucial for structure-based drug design, ensuring candidate molecules engage physiologically relevant conformations. This structure lays the groundwork for future time-resolved crystallography of SOD1 folding and ligand interactions.