A critical assessment of the synthesis and biological activity of p53/Hdm2 stapled peptide inhibitors

The covalent linkage of amino acid side chains to conformationally constrain -helices has been employed as a powerful mean to enhance the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cell entry including cytoplasmic targeting. In particular, stapled peptides inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein were of interest for this approach. Here, we scrutinized to which degree the pharmacokinetic characteristics are a function of the staple and differ from those of a standard cationic cell-penetrating peptide (CPP). Stapled peptides and their linear counterparts were synthesized to verify activity in biochemical and cellular assays. All peptides showed potent sub-nanomolar potency to Hdm2. Assessing uptake for carboxyfluorescein-labeled variants, for short incubation times, there was only little difference in uptake efficiency for the stapled peptides and their linear counterpart and both were taken up less efficiently than the prototypic CPP nonaarginine (R9). Fluorescence was restricted to vesicular structures. Only following long-term incubation, and for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides and also the linear counterparts, albeit to a lesser degree, showed an enhanced cytoplasmic and nuclear accumulation. For HeLa cells, lacking target expression no such accumulation was observed. These findings demonstrate that the cytosolic and nuclear accumulation are not an intrinsic property of the stapled peptide but result from capture by the target Hdm2 once leaking out of the endo-lysosomal compartment

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects

Bioorthogonal Probes to study MDM2-p53 inhibitors in cells and to develop high content screening assays for drug discovery

To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high content screening assays. Using automated image analysis and single cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. In addition, we developed a high throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications

Discovery of new binders for DCAF1, an emerging ligase target in the Targeted Protein Degradation field

In this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected based on internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration

Structural states of Hdm2 and HdmX: X-ray elucidation of adaptations and binding interactions for different chemical compound classes

Hdm2 (human MDM2) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Here, we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds. We also reveal the key additive 18-crown-ether, which we have discovered to enable HdmX crystallization and show its stabilization of various Lys-residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insights into the possible adaptations and structural states of Hdm2 (e.g. flip of F55; flip of Y67; reorientation of H96) and HdmX (e.g. flip of H55; dimer induction), enabling key binding interactions for different compound classes. In order to make comparisons easier, we have used the same numbering for Hdm2 and HdmX. Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors

PAX8 and MECOM are interaction partners driving Ovarian Carcinogenesis

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian carcinogenesis. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. This includes PRDM3 (also called MDS1-EVI1) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulate a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, PTXs and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in Ovarian Cancers and we show that the MECOM locus and PAX8 sustain in-vivo ovarian cancer growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer