Affinity and cooperativity modulate ternary complex formation to drive targeted protein degradation

Abstract

Targeted protein degradation via “hijacking” of the ubiquitin-proteasome system using proteolysis targeting chimeras (PROTACs) has evolved into a novel therapeutic modality. The design of PROTACs is challenging; multiple steps involved in PROTAC-induced degradation make it difficult to establish coherent structure-activity relationships. Herein, we characterize PROTAC-mediated ternary complex formation and degradation by employing von Hippel–Lindau protein (VHL) recruiting PROTACs for two different target proteins, SMARCA2 and BRD4. Ternary-complex attributes and degradation activity parameters are evaluated by varying components of the PROTAC’s architecture. Ternary complex binding affinity and cooperativity correlates well with degradation potency and initial rates of degradation. Additionally, we develop a ternary-complex structure modeling workflow to calculate the total buried surface area at the interface, which is in agreement with the measured ternary complex binding affinity. Our findings establish a predictive framework to guide the design of potent degraders.

Discovery of Novel Potent and Fast BTK PROTACs for the Treatment of Osteoclasts-Related Inflammatory Diseases

“…Contemporary empirical evidence increasingly underscores that a stable and cooperative ternary complex, elicited by a PROTAC, can significantly expedite the degradation of the POI. 42,47,48 However, scenarios exist where the stabilization of less favored PPIs within the ternary complexes may attenuate the degradation efficacy, emphasizing the nuanced interplay between complex stability and degradation potency. 40,43 To substantiate our in silico findings, experimental characterization of the ternary complex mediated by compound 23 is warranted in the future.…”

“…Formation of a favorable ternary complex induced by a PROTAC is considered crucial for valid degradation. Variations in the spatial orientation of PROTACs may result in the formation of distinct ternary complexes. , Interfacial buried surface area (BSA) of PROTAC-mediated ternary complex represents the stability cooperativity of the ternary complex and could be a key parameter to predict and rank the ability of the PROTACs to induce the formation of ternary complexes. − To further elucidate the structural differences between the ternary complexes induced by 23 and other ibrutinib-based BTK PROTACs (such as 35 ), in silico modeling was performed using protein–protein docking and molecular dynamics simulation as reported in previous protocols . Initially, the BTK-PROTAC-CRBN ternary complexes induced by 23 or 35 were generated through protein–protein docking using the PRosettaC platform .…”

PROTACable Is an Integrative Computational Pipeline of 3-D Modeling and Deep Learning To Automate the De Novo Design of PROTACs

“…However, unlike SMIs, designing PROTACs presents significant challenges, including the complex structure–activity relationship optimization, the synthetic difficulty of heterobifunctional molecules, and the need for a detailed understanding of ternary complex formation to enhance the drug-like properties and pharmacokinetics of these large molecular weight compounds. , For instance, a link between E3 ligase choice and PROTAC activity has been observed. , E3 ligase bioavailability is influenced by tissue expression, while varying thermodynamic cooperativity is observed with POI-E3 binding and PROTAC molecule saturation . Similarly, the design of the linker is also crucial to ensure the stability and specificity of the ternary complex. , …”

PEG: The Magic Bullet for Biophysical Analysis of Highly Aggregating Small Molecules in Aqueous Solutions

“…So far, ITC studies of ternary complex formations have been limited to relatively soluble hSMs and by using so-called “reverse” titration, where the protein at a higher concentration is first titrated to an order of magnitude lower concentration of hSMs, followed by a titration of a second protein. , This protocol allows determination of the cooperativity factor, which has a significant impact on the cell-based degradation efficiency of a particular hSM and has been instrumental in the optimization of lead candidates toward a therapeutic hSM . This protocol is not applicable for 1 ; however, the aggregation in buffer does not allow detection of a meaningful isotherm sufficient for extraction of a binding constant for the binary interactions (Figure S3).…”

“…6,7 This protocol allows determination of the cooperativity factor, which has a significant impact on the cell-based degradation efficiency of a particular hSM and has been instrumental in the optimization of lead candidates toward a therapeutic hSM. 8 This protocol is not applicable for 1; however, the aggregation in buffer does not allow detection of a meaningful isotherm sufficient for extraction of a binding constant for the binary interactions (Figure S3). In contrast, in the presence of PEG the binary ITC titrations of 1 with SMARCA2 or VBC showed full binding isotherms yielding accurate determination of binding constants (Figure 2).…”