2026-09-18T17:38:41Zhttps://publikationen.uni-tuebingen.de/oai/request
oai:publikationen.uni-tuebingen.de:10900/1512712026-01-30T08:12:20Zcom_10900_42126col_10900_42133
Publikationssystem UB Tuebingen advisor Lupas, Andrei (Prof. Dr.) author Ullrich, Timo 2024-02-23T10:59:30Z 2026-01-30 http://hdl.handle.net/10900/151271 Considering the indispensable role played by proteins in maintaining vital life processes, it is not surprising that proteins are linked to a broad spectrum of diseases. Conversely, proteins can be leveraged for effective therapeutic interventions. For example, secreted growth factors known as cytokines have emerged as promising candidates for protein-based therapeutics, primarily due to their potent immunomodulatory properties. An integral element of the innate immune system consists of neutrophils, the maturation of which is coordinated by a hematopoietic subprocess known as granulopoiesis. This process requires a complex interplay among various cytokines and their corresponding receptor molecules. A critical player in these interactions is the granulocyte-colony stimulating factor receptor (G-CSFR), which is activated by its native ligand, G-CSF. Unlike most other cytokines, G-CSF has found clinical use in its native form due to its favorable safety profile and its ability to increase the number of neutrophils in the blood. However, G-CSF application is restricted by its stability, production cost and native activity on G-CSFR. Additionally, there is very limited knowledge regarding non-native G-CSFR modulators, which could be key not only for understanding G-CSFR related diseases, but also for the development of innovative therapeutic applications. In order to unlock the untapped potential of the clinically significant G-CSFR beyond its native activity, my objective was to employ protein design techniques to craft novel ligands capable of modulating G-CSFR activity. In addition to customizing receptor activity, these designs offer enhanced stability and more efficient production compared to their native counterpart G-CSF. To this end, I utilized a recently developed hyper-thermostable de novo designed GCSFR binding module and optimized it with in silico and in vitro high-throughput methods to obtain a broad spectrum of variants with enhanced binding affinity. I demonstrate that these enhanced binding modules can be utilized to generate GCSFR agonists that achieve G-CSF activity in cell-based assays and can also be used to create ligands capable of modulating G-CSFR activity by tuning receptor geometry. These ligands featured fine-tuned intracellular signaling, transcriptomic activity, and primary stem cell differentiation and exhibit in vivo activity in zebrafish and mouse models. In addition to designing agonists, I show that these enhanced binding modules can be used to generate competitive G-CSFR antagonists with nanomolar inhibitory activity. To the best of my knowledge this work is the first demonstration that G-CSFR activity can be tuned by the design of ligands inducing non-native receptor geometries. Additionally, this study presents an array of binding modules with diverse affinities to G-CSFR, along with the newly designed ligands, providing the foundational components for systematic investigation of G-CSFR activity modulation. These findings hold significant promise for advancing the development of innovative protein therapeutics. en ubt-podok Computational Design and Optimization of G-CSFR Modulators PhDThesis URL https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/151271/2/dissertation_TU_final.pdf File MD5 6fea41161e056803bfcd605c7bae54d6 14219644 application/pdf dissertation_TU_final.pdf URL https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/151271/3/dissertation_TU_final.pdf.txt File MD5 ba866f2292b24beb97081ac098b7ed00 362708 text/plain dissertation_TU_final.pdf.txt