Regulated Induced Proximity Targeting Chimeras (RIPTACs): a Novel Heterobifunctional Small Molecule Therapeutic Strategy for Killing Cancer Cells Selectively

Abstract

While specific cell signaling pathway inhibitors have yielded great success in oncology, directly triggering cancer cell death is one of the great drug discovery challenges facing biomedical research in the era of precision oncology. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but they are limited by the number of targets given the inability to target intracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) have paved the way for protein proximity inducing therapeutic modalities. Here, we describe a proof-of-concept study using novel heterobifunctional small molecules called Regulated Induced Proximity Targeting Chimeras or RIPTACs, which elicit a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein:protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein. This approach not only opens new target space by leveraging differentially expressed intracellular proteins but also has the advantage of not requiring the target to be a driver of disease. Thus, RIPTACs can address non-target mechanisms of resistance given that cell killing is driven by inactivation of the essential protein. Using the HaloTag7-FKBP model system as a target protein, we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013 or dinaciclib. We show that these RIPTACs exhibit positive co-operativity, accumulate selectively in cells expressing HaloTag7-FKBP, form stable target:RIPTAC:effector trimers in cells, and induce an anti-proliferative response in target-expressing cells. We propose that RIPTACs are a novel heterobifunctional therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein.

Fig.1. The RIPTAC hypothesis and the HaloTag-FKBP model system.

a) RIPTACs leverage target-dependent intracellular accumulation and positive co-operativity in ternary complex formation to selectively inhibit the proliferation of target protein expressing cells. b) The HaloTag-FKBP fusion protein is selectively expressed in 293_HFL cells and not in the control 293_GFPL cells. c) Quantitation of cellular HaloTag-FKBP concentration using recombinant HaloTag-GST standard curve. d) RIPTAC Effector Ligands (ELs) JQ-1, BI2536, TMX-3013, Dinaciclib, and the FKBP Target Ligand (TL) HLDA-001.

Fig. 2. RIPTAC differential biology in 293_HFL model system.

a) Structures of HaloTag-FKBP fusion protein targeting RIPTACs with JQ1, TMX3013, BI-2536, or dinaciclib as effector ligands b) Differential anti-proliferative activity of select covalent RIPTACs in target protein expressing 293_HFL cells in a 7-day Cell TiterGlo assay c) Differential anti-proliferative activity of select non-covalent RIPTACs in a 7-day Cell TiterGlo assay. d) GI₅₀ values and fold-shift over 293_GFPL cells, obtained under continuous 7-day treatment with the optimal linker length from each RIPTAC series. All data representative of 3 independent experiments (N=3)

Fig. 3:. Mechanisms underlying RIPTAC differential biology

a) The FKBP ligand accumulates selectively in 293_HFL cells b) Non-covalent RIPTACs also accumulate selectively in 293_HFL cells c&d) RIPTAC activity in 7 day viability assays can be competed off by pre-treatment with 10μM HLDA-001 in the case of non-covalent RIPTACs and 300nM TAMRA-CA in the case of covalent RIPTACs e) RIPTAC HLDA-119 is a 20-fold more potent CDK9 inhibitor in 293_HFL cells than in control 293_GFPL cells.

Fig. 4. Ternary Complex Formation with RIPTACs.

a) 3h treatment of 293_HFL cells with indicated compounds followed by HaloTag immunoprecipitation using HaloTrap beads demonstrates cellular ternary complex formation with both the non-covalent RIPTAC HLDA-222 and the covalent RIPTAC HLDA-121, but not with the control molecule HLDA-125 that does not bind BRD4. b) Ternary complex formation with HLDA-121 and HLDA-222 can be competed away with 30min pre-treatment with TAMRA-CA and HLDA-001, respectively. 30min JQ1 pre-treatment competes away complex formation with both RIPTACs partially. c) 293_HFL cells were treated with the indicated compounds for 3h (T₀), following which the compound treated medium was washout out and replaced with normal growth medium for up to 72h. The HaloTag-FKBP fusion protein was immunoprecipitated at the timepoints shown and the BRD4 protein levels in the complex were detected by immunoblotting.d) AlphaLISA assay measuring BRD4-BD1 inhibition demonstrates positive co-operativity in biochemical RIPTAC ternary complex formation in presence of the non-covalent RIPTAC HLDA-221 and FKBP. e) 7 day CellTiter Glo viability assay with indicated covalent RIPTACs in cell lines expressing the HaloTag-FKBP fusion protein selectively in the nucleus (293_NLS2HF), plasma membrane (293_MYRHF), or both (293_HFL). f) 7 day CellTiter Glo viability assay in the same cell lines using non-covalent RIPTACs.