Anne-Christin Sarnow

Background: Increasing resistance against protein kinase inhibitors used in cancer therapies enforces the search for novel target structures to be addressed with favourable small-molecule inhibitors. One of these novel target structures is the tyrosine kinase Brk that is known to play a prominent role in breast cancer progression. Moreover, Brk overexpression in various kinds of cancer is associated with poor outcomes, making Brk an interesting target structure for potential treatment. So far, no class of promising Brk inhibitors has been identified. Methods: We synthesized novel functionalized pyrrolopyridines in one- and two-step reactions under substitution of the molecular scaffold and the 4-aniline residue, respectively. They were evaluated as inhibitors of Brk and HER2 in a radiolabelled enzyme assay. Results: The most favourable substituents for Brk inhibitory activity at the aniline residues were 3-hydroxy functions combined with either bromo or nitro substituents at the molecular scaffold. Those compounds, as well as bromo- and nitro-substituted compounds, also showed the best HER2 inhibitor activities. Conclusions: Novel pyrrolopyridines were discovered to be a promising class of nanomolar Brk inhibitor with additional HER2 activities to further strengthen Brk inhibitory activity in prospective anticancer therapies. Thus, the first class of Brk inhibitors could be identified.

Zusammenfassung: Das Protein Checkpoint‐Kinase‐1 (CHK1) steuert die Replikationsgeschwindigkeit und die Reparatur der DNA. Tumorzellen sind in ihrem Wachstum von CHK1 abhängig, dessen hohe Expression mit einer schlechteren Prognose von an Tumoren erkrankten Menschen assoziiert ist. Wir entdeckten ein Molekül, dass die Proteolyse von CHK1 auslösen kann (sogenanntes PROTAC für CHK1). Dieses PROTAC MA203 besteht aus dem Typ‐I‐Kinaseinhibitor Rabusertib, der bevorzugt aktiviertes CHK1 hemmt, sowie dem Cereblon‐Liganden Pomalidomid. MA203 beschleunigt den von dem Strukturprotein Cereblon (CRBN) abhängigen Abbau von CHK1 über den zellulären Proteasomen‐Komplex. Dies gilt für von soliden Tumoren isolierte Zellen und für akute Leukämiezellen. Durch Chemotherapie induzierter DNA‐Replikationsstress und die damit einhergehende Aktivierung von CHK1 fördern diesen Prozess, der DNA‐Schäden auslöst und Tumorzellen dem programmierten Zelltod (Apoptose) zuführt. Biochemische und zelluläre Bindungsstudien bestätigen die Potenz und Selektivität von MA203. Gesunde, reife und junge hämatopoetische Zellen, Stromazellen und Retina‐Epithelzellen werden jedoch nicht von MA203 geschädigt. MA203 ist seinem korrespondierenden Inhibitor hinsichtlich DNA‐Schädigung, Regulation von Apoptose‐regulierenden BCL2‐Proteinen und der Induktion von Apoptose in Tumorzellen überlegen. Diese Prozesse können unabhängig vom tumorsuppressiven Transkriptionsfaktor p53 erfolgen. Die Eliminierung des CHK1‐Proteins als Strukturelement ‐nicht jedoch seine Hemmung‐ löst eine proteasomale Degradation von DNA‐Replikationsproteinen und DNA‐Reparaturproteinen aus. Ein genetisch vermitteltes Ausschalten von CHK1 bestätigt, dass diese neu identifizierten Funktionen von CHK1 über seine bekannte katalytische Aktivität hinausgehen. Somit lassen sich neue Funktionen von CHK1 als Strukturelement der transformierten Zelle durch innovative pharmakologische Ansätze ausnutzen.

Checkpoint kinase‐1 (CHK1) controls DNA replication and repair. Tumor cells depend on CHK1, whose high levels are associated with worse patient prognosis. We define a bona fide proteolysis‐targeting‐chimera (PROTAC) for CHK1. PROTAC MA203 contains the type I kinase inhibitor rabusertib, which preferentially inhibits activated CHK1, and the cereblon (CRBN) ligand pomalidomide. MA203 accelerates CRBN‐dependent proteasomal degradation of CHK1 in solid tumor‐derived cells and acute leukemia cells. Chemotherapy‐induced DNA replication stress and a consequent activation of CHK1 accelerate this event‐driven process which promotes DNA damage and tumor cell apoptosis. Biochemical and cellular target engagement studies confirm the potency and selectivity of MA203. MA203 does not damage healthy differentiated and primitive hematopoietic cells, stromal cells, and retinal epithelial cells. MA203 is superior to its corresponding kinase inhibitor concerning DNA damage, dysregulation of BCL2 proteins, and apoptosis induction. These processes occur independently of the tumor‐suppressive transcription factor p53. Elimination of CHK1 protein as structural element, but not its inhibition per se, triggers a proteasomal degradation of key DNA replication and repair proteins. Genetic CHK1 elimination confirms that such newly recognized functions of CHK1 rely on functions beyond its well‐known catalytic activity. Thus, kinase‐independent functions of CHK1 can be exploited with innovative pharmacological agents.

Proteolysis-targeting chimeras (PROTACs) offer a novel therapeutic strategy for degrading disease-causing proteins, but designing effective degraders remains challenging. PROTACs function by inducing a ternary complex between the target protein and an E3 ligase, requiring structural insights for rational design. Key factors include linker optimization, attachment points, and warhead refinement. Computational approaches, particularly protein-protein docking, are essential for modeling ternary complexes and predicting critical interactions. However, existing docking methods struggle with cereblon (CRBN)-based ternary complexes. To address this, we introduce a computational approach combining HADDOCK protein-protein docking with induced fit PROTAC docking. Validated against 26 crystal structures from the Protein Data Bank (PDB), this method demonstrated high accuracy, especially for CRBN-based complexes. Additionally, molecular dynamics (MD) simulations of CRBN-BRD4-BD1 complexes (PDB IDs 6BN7, 6BOY) provided insights into complex stability through buried surface area and radius of gyration calculations. This validated approach was then applied to five Ataxia telangiectasia and RAD3-related (ATR) kinase PROTACs, enabling modeling in the absence of experimental structures. Our method provides a robust framework for optimizing and designing novel PROTACs targeting diverse proteins.