Applications of engineered ligand biosensors to elucidate cancer biology and develop novel therapeutics

<p dir="ltr">Developing therapies to treat diseases requires a rigorous process involving conceptualization, design, and testing in biochemical assays, cell-based systems, and animal models, culminating in clinical trials to ensure safety and efficacy. However, understanding drug-target interactions within the complexity of living cells remains a significant challenge.</p><p dir="ltr">This thesis introduces CeTEAM (Cellular Target Engagement by Accumulation of Mutants), a pioneering platform designed to address this gap. CeTEAM leverages destabilizing mutations to generate biosensors that illuminate drug binding and target engagement directly within the cellular environment. Unlike traditional methods focused solely on binding affinity, CeTEAM provides real-time insights into the functional consequences of drug interactions, such as effects on DNA repair or cell cycle progression. By combining high-throughput screening with live-cell imaging, CeTEAM offers a transformative approach to studying drug behaviour with unprecedented precision.</p><p dir="ltr">Beyond its foundational development, this thesis demonstrates CeTEAM's versatility by applying it to characterize novel PROTACs (Proteolysis Targeting Chimeras), thereby offering new strategies for drug design and therapeutic innovation. As the first implementation of CeTEAM, this work highlights its potential to streamline drug discovery, improve our understanding of cellular drug responses, and accelerate the development of safer and more effective treatments.</p><p dir="ltr">In Paper I, CeTEAM is presented as an innovative tool for real-time analysis of drug-target interactions in living cells. It is applied to distinguish on-target from off-target effects to profile PARP inhibitors (PARPi) based on their trapping efficacy and influence on DNA repair, and to explore the role of small molecules targeting MTH1 and NUDT15 in cancer biology. This work also demonstrates CeTEAM's scalability for high-throughput screening and its potential for in vivo applications, highlighting its value in optimizing therapeutic strategies and improving drug specificity.</p><p dir="ltr">In Paper II, CeTEAM is employed as a dual-biosensor system for PARP1 and PARP2, enabling investigation of PARPi selectivity in living cells, overcoming the limitations of conventional in vitro assays. While most PARPi displayed limited selectivity between PARP1 and PARP2, certain compounds, such as AZD5305, showed marked specificity for PARP1, suggesting potential for reduced toxicity. Interestingly, niraparib exhibited a 10-fold higher selectivity for PARP1 over PARP2 in living cells, a difference that was not evident in earlier biochemical studies. We also infer ways of creating selective PARPi based on trends related to compound structures. The study also revealed a role for histone PARylation factor 1 (HPF1) in modulating PARPi binding and enhancing PARP-DNA trapping, a critical mechanism in cancer treatment. These findings emphasize the importance of evaluating drug interactions in physiologically relevant environments.</p><p dir="ltr">In Paper III, targeted protein degradation (TPD) was investigated using a combination of CeTEAM and a complementary degradation assay, both playing central roles in the development and assessment of PROTACs targeting NUDT5, an enzyme involved in nucleotide metabolism and cancer. Using destabilized E3 ligase mutants for VHL and CRBN, CeTEAM enabled real-time tracking of PROTAC binding and degradation efficacy in live cells. Among the tested compounds, PROTAC-2 (P2), a VHL-based PROTAC, showed the highest degradation efficacy, whereas CRBN-based PROTAC variants were less effective. The study also identified critical lysine residues (K210, K218) essential for ubiquitination and degradation, emphasizing the significance of lysine positioning and E3 ligase choice in PROTAC design.</p><p dir="ltr">Together, the work presented in this thesis illustrates how CeTEAM can revolutionize drug discovery by addressing key challenges in understanding drug- target interactions. By delivering real-time, cell-based insights and supporting the development of advanced therapeutic approaches like PROTACs, CeTEAM emerges as a powerful and flexible platform for refining treatment strategies. These findings open new avenues for creating more targeted, effective, and safer therapies, reinforcing the importance of innovative tools in tackling complex diseases such as cancer.</p><h3>List of scientific papers</h3><p dir="ltr">I. <i>Nicholas C.K. Valerie</i>, Kumar Sanjiv, Oliver Mortusewicz, Si Min Zhang, Seher Alam, <b>Maria J. Pires</b>, Hannah Stigsdotter, Azita Rasti, Marie-France Langelier, Daniel Rehling, Adam Throup, Matthieu Desroses, Jacob Onireti, Prasad Wakchaure, Ingrid Almlöf, Johan Boström, Luka Bevc, Giorgia Benzi, Pål Stenmark, John M. Pascal, Thomas Helleday, Brent D.G. Page, Mikael Altun. Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM. Nat Commun 15, 10347 (2024). https://doi.org/10.1038/s41467-024-54415-7<br><a href="https://doi.org/10.1038/s41467-024-54415-7">https://doi.org/10.1038/s41467-024-54415-7<br></a><br></p><p dir="ltr">II. <b>Maria J. Pires</b>, Seher Alam, Alen Lovric, Emanuele Fabbrizi, Dante Rotili, <i>Mikael Altun, Nicholas C.K. Valerie</i>. Duplexed CeTEAM drug biosensors reveal determinants of PARP inhibitor selectivity in cells. Journal of Biological Chemistry, Volume 301, Issue 4 108361 (2025). https://doi.org/10.1016/j.jbc.2025.108361<br><a href="https://doi.org/10.1016/j.jbc.2025.108361">https://doi.org/10.1016/j.jbc.2025.108361<br></a><br></p><p dir="ltr">III. Seher Alam*, <b>Maria J. Pires</b>*, Gabriel Tidestav, Ann-Sofie Jemth, Fredrik Klingegard, Rémi Caraballo, Massimiliano Gaetini, Eri van Berkum, Olov Wallner, Jonas Malmström, <i>Per Arvidsson</i>, <i>Nicholas C.K. Valerie, Mikael Altun</i>. Cell-based technologies for the development and assessment of targeted protein degraders. [Manuscript] * - these authors contributed equally to this study</p><p dir="ltr">Italics - corresponding authors</p>