Woo, Christina MDippon, Vanessa Narin2026-07-0720262026-05-142026Dippon, Vanessa Narin. 2026. Identification and Characterization of a Cryptic Allosteric Site on the E3 Ligase Adapter Protein Cereblon. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32677205https://dash.harvard.edu/handle/1/42744025Cereblon (CRBN) is the target of thalidomide derivatives that achieve therapeutic efficacy against some hematologic neoplasias by recruiting neosubstrates for degradation. These findings have motivated scientists in the field of targeted protein degradation to hijack CRBN by designing small molecules that bind to CRBN’s orthosteric thalidomide binding site to induce degradation of neosubstrates implicated in diseases. Despite the success of these orthosteric ligands, challenges remain in broadening the repertoire of proteins degradable by CRBN and enhancing the efficacy and selectivity of these drugs. Furthermore, efforts in the field have vastly focused on designing ligands to target the thalidomide binding site, leaving other regions of CRBN largely understudied. Allosteric ligands are significant contributors to key therapeutic protein targets, providing mechanisms to enhance efficacy of orthosteric ligands, overcome resistance mutations, and circumvent off-target toxicities. Here I present the identification and characterization of a cryptic allosteric site on the E3 ligase adapter protein CRBN that is ligandable by small molecule SB-405483 and its derivatives. In Chapter 1, I introduce allostery and the implementation of allosteric ligands in representative cancer targets. I then detail the discovery and development of orthosteric CRBN molecular glues that degrade G-loop containing proteins. Finally, I discuss the role of CRBN allostery for targeted protein degradation modalities as well as the identification of orthosteric ligands that degrade CRBN neosubstrates that lack the canonical G-loop motif. In Chapter 2, I describe the discovery of the cryptic allosteric site and allosteric ligand SB-405483 as well as subsequent in vitro validation and crystal structure studies. I then discuss cellular experiments that reveal SB-405483 enhances orthosteric ligand engagement with CRBN and subsequently potentiates the degradation of neosubstrate CK1α. In Chapter 3, I investigate the therapeutic implications of allosteric modulation of CRBN by SB-405483. I first demonstrate SB-405483 can be used to accelerate the discovery of CRBN neosubstrates by global proteomics. I then elucidate that SB-405483 alters the degradation landscape of orthosteric ligands, potentiating the degradation of some neosubstrates while inhibiting the degradation of others. I finally discuss cryo-EM efforts which establishes the mechanism by which SB-405483 elicits its allosteric effects. In Chapter 4, I discuss ongoing biological testing of SB-405483 analogues with the goal of developing superior allosteric CRBN ligands. I first describe biological assays used to assess allosteric CRBN ligands, testing their affinity for CRBN, impact on orthosteric ligand binding, and influence on protein degradation. I then perform analysis on correlations between different biological assays and their implications. Finally, I shortlist superior allosteric CRBN ligands for future investigation. Throughout the chapter I draw from concepts and findings from allosteric ligands in the GPCR field, establishing these concepts in the framework of CRBN allostery.application/pdfenAllosteryCancer TherapeuticsCereblonChemical BiologyTargeted Protein DegradationChemistryThesis or Dissertation2026-07-070009-0009-8434-732X