WRN
The Werner Syndrome RecQ helicase (WRN) is a multifunctional enzyme from the DNA helicase family. It is involved in many cellular processes that maintain genome stability, such as DNA repair.
WRN has been identified as a promising target for cancers with microsatellite instability (MSI). These cancers are characterized by mutations in DNA mismatch repair genes, which alter the DNA structure within microsatellite regions across the genome. They are often resistant to existing treatments and require alternative therapeutic approaches.
A deficiency in WRN activity is lethal for MSI cancer cells but not for cells without alterations in the mismatch repair genes - either normal or from other cancer types. This behavior is called "synthetic lethality" and can be exploited to develop selective therapies for MSI cancers.
No approved medications targeting WRN or other human DNA helicases are currently on the market. A recent Novartis study reported the development of HRO761, the first non-covalent inhibitor of WRN helicase, which entered a clinical trial. Structural studies unraveled the inhibition mechanism of this molecule.
The Novartis team developed a non-covalent, allosteric molecule, leading to the clinical candidate HRO761. In the co-crystal structure of HRO761 in a complex with WRN, the molecule was found to bind the interface between the D1 and D2 domains of the protein (Image 1A). When comparing this structure with the one in complex with the ATP-analog ATPγS (Image 1B), the scientists observed that ATPγS binds at the D1-D2 interface too, but that HRO761 stabilizes a conformation with a 180° rotation of the D1 and D2 domains relative to the ATPγS-bound one.
Image 1. A) The WRN helicase complex with HRO761 (PDB: 8PFO, protein in grey and ligand in orange). B) The WRN complex with ATPγS (PDB: 8PFP, protein in pink and ligand in blue). Protein domains are highlighted using the 3decision® Annotation Browser function and colored as follows: D1 in green, D2 in blue, and the zinc-binding domain in yellow. The ligand surfaces, produced with the 3decision® software, are in orange for HRO761 and blue for ATPγS.
This difference was attributed to a conformational change in the flexible hinge between the D1 and D2 domains (composed by the residues 728-732), stabilized by HRO761 binding in a unique conformation compared to the ATP-bound state (Image 2A). In this conformation, HRO761 displaces the Walker motif (residues 571–578), including its catalytic residue Lys577 (Image 2B). This displacement prevents ATP binding and inhibits ATPase activity, resulting in allosteric inhibition of WRN.
Image 2. Overlay of the WRN helicase in complex with HRO761 (PDB: 8PFO, protein in grey and ligand in orange) and ATPγS (PDB: 8PFP, protein in pink and ligand in blue). A) The residues composing the flexible hinge are indicated and the labels are in grey for the HRO761-bound structure and in pink for the ATPγS-bound one: Thr728, Gly729, Phe730, Asp731, Arg732. The picture is produced using the 3decision® highlight mode: the residues with an RMSD higher than 1.4 are displayed as lines and cartoons, while for the residues with a lower RMSD, only the backbone line is represented. This visualization mode allows easy spotting of the conformational change of the flexible hinge induced by the allosteric inhibitor HRO761. B) The Walker motif and Lys577 are highlighted and colored in orange for the HRO761-structure and in blue for the ATPγS-bound one. The Walker motif annotation was highlighted using the 3decision® Annotation Browser.
HRO761 was extensively studied in vitro and in vivo and showed potent and selective inhibition of tumor cell growth in MSI cells, thereby validating WRN as a therapeutic target in MSI cancers. These findings provide a foundation for further exploration of novel treatments for MSI cancers.