Synthetic biology offers significant potential for advancing our understanding of fundamental biological processes and developing next-generation cell-based therapies. A key component of synthetic biology is the ability to precisely control cellular activities, often achieved through small-molecule-controlled protein switches. These switches typically involve the assembly or disassembly of protein subunits in response to a small molecule. While the rapamycin-controlled FKBP:FRB system is widely used, many chemically-controlled protein switches suffer limitations, especially in translational applications, due to issues like drug toxicity, side effects, unfavorable pharmacokinetics, and immunogenicity. The need to expand the repertoire of protein switches and the chemical space used to control engineered cellular activities is therefore critical. Previous approaches, primarily focusing on chemically induced dimerization, have yielded some success, but challenges remain in precisely designing key interaction residues to control small molecule interactions. This study introduces a strategy to design chemically-controlled switches by repurposing protein components and small molecules involved in inhibiting protein-protein interactions (PPIs). Many PPI inhibitors are in clinical development or already approved, making them attractive candidates for synthetic biology applications. The authors' previous work demonstrated the design of chemically disruptable heterodimers (CDHs) by transferring peptide motifs to globular proteins, successfully using a Bcl-XL inhibitor as a chemical disruptor. This current study builds upon that foundation.

The authors review existing methods for expanding the panel of available protein switches. They highlight the work of Hill et al. using *in vitro* evolution to engineer antibodies for CID systems, and the work of Foight et al. using computationally designed mutants of a *de novo* protein scaffold. They also note the rational design of a CID by Glasgow et al., which involved transplanting ligand binding sites to an existing protein dimer. These approaches focus on chemically induced dimerization, whereas chemical disruption systems, though important, are less explored. The authors' previous work on CDHs, using Bcl-XL:BIM-BH3 complex as a basis, is also detailed, highlighting the successful creation of a switch controlled by the Bcl-XL inhibitor A-1155463. This previous system demonstrated dose-dependent, dynamic, and reversible control of CAR-T cell activity *in vivo*. The current work expands on this.

The researchers employed a computational protein design strategy to create novel chemically-controlled protein switches using clinically relevant drugs. They designed three new CDHs: CDH-2 (Bcl2:LD3) disrupted by Venetoclax, CDH-3 (mdm2:LD6) disrupted by NVP-CGM097, and CDH-1 (Bcl-XL:LD3) using a previously reported system. Surface plasmon resonance (SPR) was used to determine binding affinities and drug competition assays were performed to determine IC50 values. The CDHs were tested in two cellular systems: transcriptional gene regulation (using a split transcription factor design, CDH-TFs) and control of endogenous signaling pathways (using the GEMS platform, CDH-GEMS). To investigate the effect of biochemical parameters, a suite of CDHs with tailored affinities was designed and tested. A novel strategy was developed to create ON switches (activation by inhibitor release, AIR switches) by repurposing the CDHs. AIR switches utilize a split architecture with a drug-sensitive fused CDH and a rationally designed drug-insensitive receptor. The drug releases the designed binder from the fused CDH, allowing dimerization with the insensitive receptor. Multistate design was employed to create the drug-insensitive receptors. Mammalian cells were engineered with multi-input/output control modes to demonstrate the broad applicability of the designed protein switches. X-ray crystallography was used to determine the structure of CDH-3, confirming the accuracy of the computational design. The methodology included detailed descriptions of computational design parameters, protein expression and purification, compound preparation, CD spectroscopy, SEC-MALS, SPR, cell viability assays, cell transfection procedures, SEAP detection assays, reversibility assays, and flow cytometry. Specific details are provided regarding the computational design of CDH-3 and AIR switches, the design of weaker affinity variants, and the prediction of drug-insensitive receptor mutations using Rosetta.

The study successfully designed and characterized three novel CDHs controlled by clinically relevant drugs: Venetoclax (CDH-2), NVP-CGM097 (CDH-3), and A-1155463 (CDH-1). These CDHs functioned as effective OFF-switches in both intracellular (transcriptional gene regulation) and extracellular (GEMS platform) contexts, demonstrating dose-dependent and reversible responses. The researchers showed that the sensitivity of CDHs could be tuned by altering their binding affinities. They developed a novel AIR switch architecture, which successfully created ON-switches controlled by Venetoclax and A-1155463, demonstrating high sensitivity and dynamic response ranges. The X-ray crystal structure of CDH-3 confirmed the accuracy of the computational design. The AIR switches were also tested in transcriptional regulation and CAR expression, exhibiting controlled drug response. Finally, the researchers demonstrated the successful implementation of multi-input/multi-output control modes in mammalian cells using combinations of CDHs and AIRs, showcasing the potential for complex logic devices. Specific quantitative data, including Kd values, IC50s and EC50s, are provided throughout the paper to support these findings.

The findings address the research question by providing a robust computational design strategy to expand the limited repertoire of chemically-controlled protein switches. The successful design and characterization of CDHs and AIRs, using both FDA-approved and investigational drugs, significantly expands the chemical space and logic capabilities of synthetic biology tools. The ability to tune CDH sensitivity through affinity adjustments and the development of the AIR architecture demonstrate versatile control over cellular responses. The successful integration into complex multi-input/multi-output systems highlights the potential for sophisticated cellular control and its implications for advanced applications. The authors acknowledge a non-linear relationship between heterodimer affinities and drug IC50s in cells, emphasizing the need for testing a range of switches for optimization. The study's contribution lies in providing a generalizable design blueprint for protein switches, which bridges the gap between computational protein design and next-generation cell-based therapies.

This research presents a significant advancement in the field of synthetic biology by developing a rational blueprint for designing chemically-controlled protein switches. The creation of both OFF-switches (CDHs) and ON-switches (AIRs) controlled by clinically relevant drugs provides versatile tools for controlling cellular activities. The successful implementation of multi-input/multi-output control systems in mammalian cells demonstrates the potential for building sophisticated logic devices with broad applications. Future research directions include expanding the range of drugs and protein targets for switch design, further optimizing switch sensitivity and response kinetics, and exploring new applications in areas such as cell-based therapies and biosensing.

The study primarily focuses on HEK293T cells, a commonly used cell line but not necessarily representative of all cell types. The observed non-linear relationship between heterodimer affinities and cellular drug responses suggests that optimizing drug responsiveness might require extensive testing. While the AIR switches showed high sensitivity, the absolute levels of output varied between different AIR designs, potentially impacting the effectiveness in certain applications. The generalizability of the multistate design approach to other drug targets might need further investigation.