<p>Kaplan-Meier Survival Curve.</p>

Supplementary figure 1 and 2 describe protein purity and expression. Supplementary Figure 3 shows cell-based cytotoxic assays. Supplementary Figure 4 shows protein activity against several substrates. Supplementary Figure 5 describes levels of individual metabolites.

<p>Strong nuclear staining of mutant TP53-R337H protein in normal tissue of carriers who developed ACC.</p>

<p>Senescence associated β-galactosidase expression and ROS levels in mutant p53-R334H primary MEFs.</p>

<p>Mutant p53-R334H has a prolonged half-life and compromised in forming stable tetramers in primary mouse embryo fibroblasts.</p>

Mouse Cohorts and Associated Pathology

<p>Cell death in response to radiation is impaired in thymocytes and splenocytes expressing Mutant p53-R334H.</p>

Gene set enrichment analysis of control and IR-treated WT and p53-R334H thymocytes.

Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.

Elevated levels of mutant p53 protein in the nucleus of low passage p53-R334H MEFs.

<p>Mutant p53-R334H has a prolonged half-life and compromised in forming stable tetramers in primary mouse embryo fibroblasts.</p>

<p>Erastin treatment of early passage primary MEFs induced cell death in cells with mutant p53.</p>

Supplemental Materials and Methods and Supplementary Figure Legends

<p>Cell death in response to radiation is impaired in thymocytes and splenocytes expressing Mutant p53-R334H.</p>

<p>Mutant p53-R334H is abnormally expressed and functionally impaired in vivo in response to DNA damage.</p>

<p>Mdm2 Knockout Embryonic Lethality</p>

<p>Kaplan-Meier Survival Curve.</p>

<p>Senescence associated β-galactosidase expression and ROS levels in mutant p53-R334H primary MEFs.</p>

p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCFSKP2 (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCFSKP2. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.