Rational PROTAC design enabled by integrated in silico molecular modeling and in vitro biomimetic affinity assessment Proteolysis targeting chimeras, or PROTACs, have received considerable attention in recent years as a new class of drugs as compared to traditional inhibitors. These small molecules target selective degradation of proteins of interest utilizing cell’s native protein degradation machinery including proteosomes and lysosomes. The benefits of PROTACs stem from an entirely different paradigm of protein targeting, which provides a unique path to target previously “undruggable” proteins and allows for smaller doses and thus lower side-effects. However, the complex mechanism of action has left a large knowledge gap towards the understanding of molecular interactions in different stages, especially on factors that control and stabilize the ternary complex that leads to ubiquitination and removal. In addition, existing technology lacks of strategies to model and confirm the linker region of the PROTAC for their roles in affecting the affinities of the warheads and contributing to the stability of the complex. There has been no report that includes the dynamic membrane in molecular recognition modeling. A new technical platform that can identify key parameters that impact formation of stable ternary complex and has the capability of screening molecular interactions with detailed information on structural insights is highly desired. To fill the unmet need, we propose to develop a collaborative work plan via a combination of in silico modeling and in vitro surface plasmon resonance (SPR)-based affinity assessment. We aim to identify features that lead to formation of stable ternary complexes for efficient PROTAC design. To establish and prove the technical feasibility, we will study anaplastic lymphoma kinase (ALK), a transmembrane receptor tyrosine kinase that is an important drug target for a variety of cancers, and an E3 ligase CRBN which has been used to promote protein degradation. Specifically, we propose three aims: Aim 1. Establish a molecular modeling platform for rational PROTAC design. The platform incorporates protein dynamics and inputs from experiments and can adapt various experimental settings such as membrane environment used in SPR. Aim 2. Build and characterize modeled PROTACs and biomimetic membranes. This includes generating PROTAC-compatible membrane mimics and structural characterization of the interfaces for membrane-bound proteins for SPR analysis. Aim 3. Investigate interaction properties of PROTAC candidates’ in biomimetic membranes. This includes on-line in vitro experiments and screening for the PROTACs at stabilizing the ternary complexes. The identification of stable PROTAC complexes will be used into further exploration into the understanding of the structure-function relationship of the system.

PROJECT NARRATIVE The overall objective of this proposal is to develop a workplan for investigating biophysical dynamics of PROTAC interactions by the combination of in silico modeling and in vitro assay screening. The research aims to identify structural features that lead to stable ternary complex formation of drug candidates. The results will provide molecular basis and a technical platform for effective search of therapeutics with a PROTAC strategy.