Keywords: RIPTAC, target selection, ternary complex, positive cooperativity, high expression of target protein, effector protein

The uniqueness of RIPTAC technology lies not only in its "hold and kill" mechanism of action but also in the flexibility and breadth of its target selection. Traditional small-molecule inhibitors require targets with well-defined active pockets and druggable chemical space; PROTACs require target proteins to be recognized and degraded by the ubiquitin-proteasome system; whereas RIPTAC's only requirement for a target protein is—specific high expression in tumor cells. This extremely low threshold for targetability enables RIPTAC to potentially target a vast number of proteins previously deemed "undruggable." This article explores the core scientific challenges in RIPTAC molecular design from two dimensions: target selection logic and the thermodynamics of ternary complex formation.

RIPTAC's target selection can be abstracted as a binary decision problem: choosing which TP (Target Protein) to serve as a tumor-specific "anchor" and which EP (Effector Protein) to act as the "killing executor."

1.1 Selection Criteria for Target Protein (TP)

The selection of TP follows these principles:

(1) Tumor specificity or significantly high expression. TP must be expressed at significantly higher levels in tumor cells than in normal cells. This differential expression can be qualitative (tumor-specific mutant proteins, e.g., p53-Y220C) or quantitative (proteins overexpressed in tumors, e.g., AR, HER2, BCL6).

(2) Can serve as an intracellular "anchor." TP does not need to have any specific function—it can be an oncogenic driver, transcription factor, signaling molecule, or any other type of protein. TP merely acts as a molecular tag for RIPTAC to "dock" and "enrich" in tumor cells.

(3) Intracellular localization. RIPTAC is a small molecule that acts intracellularly, so TP must be an intracellular protein. This allows RIPTAC to target intracellular proteins inaccessible to antibody-drug conjugates (ADCs).

Based on these criteria, RIPTAC's potential target pool is vast. Halda claims its design strategy can utilize "hundreds of tumor-selective intracellular protein markers" as TP sources.

1.2 Selection Criteria for Effector Protein (EP)

The choice of EP directly determines RIPTAC's killing efficacy and safety:

(1) Essential for cell survival. EP's function must be indispensable for cell survival. When EP is "hijacked" by RIPTAC, cells die due to the loss of essential function. BRD4 is the EP chosen for HLD-0915—BRD4 is a core transcriptional regulator essential for prostate cancer cell survival.

(2) Ubiquitous expression. EP should be universally expressed in all cells. This ensures RIPTAC can find available EP targets in any tumor cell expressing TP.

(3) Bindable by small-molecule ligands. EP must have known or developable small-molecule ligands. This limits the scope of EP selection, but there are still many known "pan-essential proteins" to choose from, such as BET family proteins (BRD2/3/4), CDK family kinases, PLK1, etc.

1.3 Logic of Target Pairing

The pairing of TP and EP determines RIPTAC's tumor selectivity and killing efficacy. An ideal combination should meet: TP is highly expressed in tumors but low in normal tissues; EP is expressed in all cells and functionally indispensable; TP and EP are spatially accessible within cells (e.g., both localized in the nucleus or cytoplasm) to ensure efficient ternary complex formation.

RIPTAC's effectiveness depends on the stability and selectivity of the TP:RIPTAC:EP ternary complex. This involves complex molecular recognition and thermodynamic challenges.

2.1 Binary Binding and Cooperativity in Ternary Complexes

RIPTAC's binding to TP and EP is not two independent binary events but exhibits positive cooperativity. When RIPTAC first binds to TP, its conformation may change, enhancing its affinity for EP, and vice versa. This cooperativity makes ternary complex formation thermodynamically more favorable—even if RIPTAC's affinity for individual proteins is modest, the ternary complex can still form stably.

2.2 Determinants of Ternary Complex Stability

Ternary complex stability is influenced by multiple factors:

(1) Ligand-protein affinity. The intrinsic affinity of RIPTAC's two ligands for their respective target proteins is foundational. Although positive cooperativity can compensate for weak individual affinities, excessively low affinity will still prevent effective ternary complex formation.

(2) Protein-protein interface. New protein-protein interactions (neomorphic PPIs) may form between TP and EP in the ternary complex. These newly formed interfaces can significantly enhance complex stability.

(3) Linker length and flexibility. Linker design determines the spatial distance and relative orientation between the two ligands. A linker that is too short may hinder simultaneous binding of both proteins, while one that is too long may reduce effective concentration and cooperativity.

(4) Intracellular concentration. TP expression levels in tumor cells directly affect ternary complex formation efficiency. Studies show that when TP concentration is in the 1μM to 3μM range, RIPTAC activity is significantly enhanced—this explains why RIPTAC selectively targets tumor cells with high TP expression.

2.3 Validation Methods for Ternary Complex Formation

In drug discovery, validating ternary complex formation is central to RIPTAC development. Common detection methods include: HTRF (homogeneous time-resolved fluorescence)-based ternary complex formation assays, ELISA-based intracellular complex detection, and fluorescence polarization (FP)-based binary complex evaluation. These methods provide direct data support for structure-activity relationship studies and optimization of RIPTAC molecules.

p53 is a tumor suppressor gene mutated in over 50% of human cancers. p53-Y220C is a structurally unique missense mutation that reduces DNA-binding ability and leads to loss of function. Traditionally, p53 has been considered "undruggable"—its DNA-binding domain is complex and intracellular, making it difficult to target effectively with small molecules.

At the 2025 AACR annual meeting, the Lupey-Green team reported an orally bioavailable RIPTAC molecule targeting the p53-Y220C mutation. This RIPTAC molecule binds mutant p53-Y220C (TP) at one end and a cell survival-essential protein (EP) at the other, inducing ternary complex formation. Unlike pharmacological chaperone strategies that attempt to "repair" mutant p53 back to wild-type function, RIPTAC "hijacks" mutant p53 as an anchor to block EP function. Studies show that this p53-Y220C RIPTAC exhibits stronger antiproliferative activity than reported pharmacological chaperones and is a potent apoptosis inducer. This case demonstrates how RIPTAC bypasses the limitations of traditional "function restoration" strategies by leveraging mutant proteins themselves as "weaknesses" for selective killing.

RIPTAC's tumor selectivity is fundamentally a quantitative rather than qualitative issue. High TP expression in tumor cells is the "concentration threshold" for RIPTAC activity—only when TP concentration is sufficiently high can ternary complex formation effectively drive EP functional blockade. Low (or zero) TP expression in normal cells makes ternary complex formation thermodynamically unfavorable, allowing EP to remain functionally intact.

This mechanism gives RIPTAC tunable tumor selectivity—by optimizing ligand affinity and linker design, the "threshold" can be adjusted to achieve the best therapeutic window across tumor types with varying TP expression levels.

RIPTAC's target selection logic breaks traditional drug development constraints: TP only needs to serve as an "anchor" for tumor selectivity, requiring no enzymatic activity or druggable pocket; EP acts as the "killing executor" to provide cytotoxicity. Ternary complex formation relies on positive cooperativity and TP overexpression—this is both the source of RIPTAC's tumor selectivity and the core challenge of its molecular design. The p53-Y220C case demonstrates that RIPTAC can target "undruggable" proteins inaccessible to traditional methods, opening a new chemical space for precision oncology.