De novo design of miniaturised G-CSFR agonists

• Applicants: Dr. Mohammad ElGamacy, Ph.D.; Dr. Birte Hernandez Alvarez; Professorin Dr. Julia Skokowa, Ph.D.
• Subject Area: Biophysics ; Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 500215849

Protein therapeutics have effectively broadened the range of manageable disorders; owing to their structural properties, protein molecules are capable of carrying out more complex and more specific functional roles when compared to small-molecule drugs. Cytokine-based therapies represent one such central class of therapeutic proteins used to treat several hematopoietic, hemostatic, immunoregulatory disorders of acute or chronic nature. While the advent of recombinant forms of natural cytokines has rendered many disorders and conditions manageable, these cytokines are at present clinically deployed directly in their native form or with minor modifications to their sequence or structure. Such native forms possess relatively complex folds, loosely packed structures, and entrain several post-translational modifications. These properties often result in several pharmaceutical shortcomings; these can range from short serum half-life, poor tissue distribution, poor solubility and stability, low recombinant production yield, and short shelf-life. In contrast to classical engineering approaches that can introduce incremental improvements to an existing protein, de novo protein design offers a means for creating functional proteins with completely novel sequences, and folds, where the molecular properties of the protein are rationally predefined to most efficiently carry out its function. This cutting-edge approach allows direct control over molecular stability, size, and solubility, as design objectives. Moreover, while the ligand-activated configuration of several cytokine receptors are so far unclear, de novo design can generate a range of receptor modulators with controlled receptor association geometries, which can help to unravel the structural determinants of receptor activation. Here we aim to extend our previous work on creating designer cytokines. In this project we further our work on G-CSF, a critical immunotherapeutic for several neutropenia disorders, where our main objectives are: 1) design, biophysically characterise, and pharmacologically test miniaturised (<10 kDa) G-CSF agonists with idealised structural and pharmacological features. 2) Acquire quantitative insights into the structure-function-relationship of the GCSF: G-CSFR complex, which is currently poorly understood. This is achievable by designing a range of receptor dimerisers with controlled geometries to achieve a guided scan of receptor subunits spacing and relative orientation and its impact on the G-CSF receptor internalisation and downstream signal transduction output. This may also potentially reveal possible means of different modes of receptor activation, partial agonists, or even inhibitors.

• DFG Programme: Research Grants