Regulated induced proximity targeting chimeras-RIPTACs-A heterobifunctional small molecule strategy for cancer selective therapies

Abstract

We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.

Keywords: Halda Therapeutics; RIPTAC; anticancer drugs; biotechnology; chemical biology; drug discovery; heterobifunctional molecules; oncology; protein proximity; ternary complex.

Figure 1.. 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. All data representative of 3 independent experiments (N=3)

Figure 2.. RIPTAC activity depends on target protein abundance.

(A) Activity of RIPTACs on cell viability as expression level of HaloTag-FKBP is titrated using doxycycline. (B) Activity of effector protein inhibitors on cell viability as expression level of HaloTag-FKBP is titrated using doxycycline.

Figure 3.. Mechanisms underlying RIPTAC differential biology.

(A) The FKBP ligand HLDA-001 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 10 μM HLDA-001 in the case of non-covalent RIPTACs or 300 nM TAMRA-CA in the case of covalent RIPTACs. (E) RIPTAC DNE-11PEG-CA is 19-fold more potent a CDK9 inhibitor in 293_HFL cells than in control 293_GFPL cells. (F) RIPTAC BI-2PEG-FKBP is a 18-fold more potent PLK1 inhibitor in 293_HFL cells than in control 293_GFPL cells. (G) RIPTAC JQ1-4PEG-CA is a 60-fold more potent BRD4 inhibitor in 293_HFL cells than in control 293_GFPL cells.

Figure 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 JQ1-4PEG-FKBP and the covalent RIPTAC JQ1-6PEG-CA, but not with the control molecule epi-JQ1-6PEG-CA that does not bind BRD4. (B) Ternary complex formation with JQ1-6PEG-CA and JQ1-4PEG-FKBP can be competed away with 30 min pre-treatment with TAMRA-CA and HLDA-001, respectively. JQ1 pre-treatment for 30 min partially competes away complex formation with both RIPTACs. (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.

Figure 5.. Target protein expression and localization in absence and presence of RIPTAC.

293_HFL cells treated with 1 μM JQ1-6PEG-CA (top) or vehicle (bottom) were immunostained against BRD4 (panels a and b) and the FLAG epitope on HaloTag-FKBP (panels c and d). Merged BRD4 and FLAG channels are shown in panels e and f. Nuclei were counterstained with Hoescht 33342 (panels g and h).

Figure 6.. Cooperative Binding in Ternary Complex Formation by RIPTACs.

(A) AlphaLISA assay measuring BRD4-BD1 inhibition demonstrates positive co-operativity in biochemical RIPTAC ternary complex formation in presence of the non-covalent RIPTAC JQ1-2PEG-FKBP and FKBP. (B) NanoBRET in cellulo assay measuring cooperativity of effector protein engagement by JQ1-based RIPTACs; (C) by BI2536-based RIPTACs; d) by TMX-3013- and dinaciclib-based RIPTACs; e) by effector protein ligands.

Figure 7.. Target Protein Subcellular Localization Affects RIPTAC Activity.

(A) 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). (B) 7 day CellTiter Glo viability assay in the same cell lines using non-covalent RIPTACs.