Abstract by Nicoline Ninn Jensen

Protein phosphorylation is a fundamental regulator of cellular signalling, and its dysregulation contributes to numerous human diseases. While most therapeutic strategies have relied on occupancy-driven pharmacology, the event-driven phosphorylation-inducing chimeric small molecules (PHICS) have emerged as a novel and promising modality for regulating phosphorylation states. However, thus far only a limited number of targets have been modulated using PHICS. This PhD dissertation sought to expand the PHICS approach to two previously unexplored systems: the calcium/calmodulin dependent protein kinase II alpha (CaMKIIα) and the transmembrane sodium/proton exchanger 1 (NHE1), with the future potential to regulate phosphorylation in disease states.

In the first project (Chapter 3), CaMKIIα was investigated as a PHICS‑amenable kinase using newly designed heterobifuncitonal CaMKIIα PHICS and CaMKIIα‑targeting homobifunctional molecules. While the PHICS series showed binary binding to CaMKIIα, reliable evidence of substrate neo‑phosphorylation could not be established, highlighting the need for further assay optimization. In contrast, SAXS analysis demonstrated that the homobifunctional O‑5‑HDC–based molecules induced self‑association of the CaMKIIα hub domain, revealing a promising and previously unexplored strategy for modulating CaMKIIα activity.

The second project (Chapters 4-6) focused on establishing NHE1 as a PHICS target by recruiting adenosine monophosphate-activated protein kinase (AMPK) or protein kinase C (PKC). Initial validation of the literature‑reported NHE1 ‘proteolysis-targeting chimera’ suggested intracellular engagement between the 9t‑derived d2A‑2 and NHE1, which rationally guided the present PHICS design. A first generation of AMPK‑ and PKC‑recruiting PHICS was subsequently synthesized, and although the pharmacological characterization proved challenging – with Western blot results remaining inconsistent – preliminary mass spectrometry identified several phosphopeptides following treatment with PKC‑recruiting PHICS. These findings suggest that the PKC-recruiting PHICS still hold potential to induce NHE1 phosphorylation, though it also underscored the need for further assay optimization to fully assess their phosphorylation‑inducing capacity.

Overall, this PhD dissertation expands the chemical and conceptual landscape of PHICS by generating new heterobifunctional, heterotrifunctional, and homobifunctional molecules, and it presents the pharmacological work undertaken to establish an early‑stage experimental pipeline for their evaluation. Although definitive pharmacological characterization is still incomplete, the work offers valuable insights and lays a strong foundation for future studies aiming to explore NHE1 and CaMKIIα within the PHICS framework.