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Abstract: The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC53608 from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC100–23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC53608 became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.

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Abstract: The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.

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Abstract: Observing transient structural intermediates remains a central challenge in enzymology. Little is known about these despite their pivotal role in catalytic function. Time-resolved serial crystallography at synchrotron and X-ray free electron laser (XFEL) sources offers a promising avenue to capture these dynamic events. However, experimental success hinges on rigorous control of crystallisation, ligand delivery, and integration with beamline infrastructure. This thesis explores the use of droplet microfluidic and microcrystal strategies to address these bottlenecks and establish pipelines for future time-resolved studies. A high-throughput crystallisation system was developed to generate uniform microcrystals within discrete aqueous droplets, leveraging a seeding strategy to overcome the low probability of nucleation at diminishing volumes. In parallel, a droplet micromixing device was engineered to initiate ligand-triggered reactions on millisecond timescales by exploiting convection within droplets as a means for rapidly mixing microcrystals with ligands. Flow parameters and mixing efficiency were characterised, followed by iterative design and fabrication of a X-ray transmissible device suitable for deployment at a synchrotron beamline. Arabidopsis thaliana Pyridoxal 5'-phosphate synthase subunit 1.3 (AtPdx1.3) microcrystal slurries were validated for time-resolved studies using static serial femtosecond crystallography (SFX) at the SPring-8 angstrom compact free electron laser (SACLA). High-resolution radiation damage-free structures of apo and ligand-bound AtPdx1.3 were obtained at room temperature, representing the first XFEL structures of this enzyme. Diffraction from 20 μm crystals yielded resolution comparable to or better than previous larger crystals at cryogenic temperatures. Notably, only minimal structural differences were observed relative to cryotrapped structures, indicating strong conformational consistency. Soaking protocols enabled rapid ligand incorporation, capturing R5P, PLP and the crucial I320 intermediate within 15 minutes. These result establish robust workflows for intermediate state trapping and future dynamic studies. Taken together, the platforms developed in this thesis represent a significant step towards realising dynamic structural studies of enzymes at synchrotron and XFEL sources.

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Abstract: Kirsten rat sarcoma viral oncogene homologue (KRAS) is a frequently mutated oncogene in multiple types of cancer and is a high priority target for oncology drug development. There are many different KRAS mutations, including mutations that favor the GTP-loaded hydrolysis-incompetent “active” state of KRAS, KRAS(on), that can lead to tumorigenesis. However, small molecule interventions thus far have predominantly targeted single mutations of “inactive” GDP-loaded KRAS, KRAS(off), such as KRASG12C. Here, we address this gap through the development of heterobifunctional VHL-based PROTACs capable of engaging and degrading KRAS(on), thus addressing a wider range of KRAS mutations. By studying ternary complex affinity, stability, and binding modes using SPR and X-ray cocrystal structures, we identified PROTACs that exhibit high positive cooperativity in forming ternary complexes with VHL and GCP-loaded KRAS as representative of KRAS(on) variants. Degrader activity profiling in relevant cancer cells supported the discovery of ACBI4, a PROTAC which forms a highly stable and cooperative ternary complex between VHL and GTP-bound KRAS and which potently degrades KRASG12R, leading to antiproliferative effect in KRAS mutant-driven cancer cells. ACBI4 provides a new chemical tool for studying the impact of degrading KRAS(on) mutants, which is not possible with current pan-KRAS inhibitors or degraders.

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Abstract: Casein kinase 2α (CK2α) is an oncology drug target that acts as a positive regulator of many tumorigenic signaling pathways. We previously reported that CK2α has a unique cryptic binding site, the αD pocket, that offers the potential for inhibitors with improved kinase selectivity. The prototype bivalent molecule CAM4066 (6) confirmed that improved selectivity could be achieved while binding in both the ATP-binding site and the αD pocket. A drug discovery project to develop a new series of bivalent CK2α inhibitors with increased cell potency and selectivity identified 61f (APL-5125), a highly potent, ATP-competitive CK2α inhibitor with exquisite kinase selectivity and cellular potency. Compound 61f demonstrates in vivo inhibition of p-AKT S129 in tumors (HCT116) following once-daily oral administration and shows a clear PK–PD relationship with unbound drug exposure. 61f has a superior preclinical profile to existing CK2α inhibitors and is currently under evaluation in patients with advanced solid tumors.