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.

Background: Novel drugs are needed to tackle forms of prostate cancer that demonstrate resistance to hormonal agents. Halda has invented an innovative cancer treatment approach that does not rely on oncogenic drivers. Regulated Induced Proximity Targeting Chimera (RIPTAC™) therapeutics are heterobifunctional small molecules that work via a hold and kill mechanism that has the potential to overcome drug resistance mechanisms. RIPTAC therapeutics function by holding together two proteins, a cancer-specific protein, and a protein with essential function (EP) in a ternary complex, resulting in abrogation of the essential function and subsequent cancer cell death. We exemplify this platform to treat metastatic castration resistant prostate cancer (mCRPC), where the RIPTAC therapeutic utilizes the Androgen Receptor (AR) as a tumor specific protein to selectively inhibit an essential protein involved in transcriptional regulation and provide in vivo efficacy coupled with a therapeutic index. Methods: RIPTACs therapeutics were assayed for their ability to form a ternary complex with AR and the EP using a novel TR-FRET based assay in VCaP prostate cancer cells that harbor AR amplification. Selective apoptosis in ARhigh cells was observed using a Caspase 3/7 Glo assay (Promega). EP pharmacodynamic modulation was ascertained using qRT-PCR and western blotting in both in vitro and in vivo samples. RIPTACs were optimized for oral bioavailability, and tumor ternary complex formation in prostate cancer cell line-derived xenograft models.Results: RIPTAC therapeutics display nanomolar in vitro potency in AR:RIPTAC:EP ternary complex formation, which results in abrogation of the EP function and antiproliferative activity in prostate cancer cell lines, but not in AR-knockout control cells. The prostate cancer RIPTAC therapeutics are active in vitro against clinically relevant AR mutants. Lead molecules utilizing AR as a tumor specific protein are orally bioavailable in multiple preclinical species and induce ternary complex formation in VCaP tumor xenografts grown in mice. We present in vivo data where lead RIPTACs demonstrate significant tumor growth inhibition in several prostate cancer models and induce tumor regressions in VCaP xenografts grown in castrated male mice. Conclusions: Taken together, our in vitro mechanistic data and in vivo PD/efficacy observations in multiple prostate cancer models support further investigation of prostate cancer RIPTAC therapeutics as a novel heterobifunctional therapeutic modality in mCRPC. Citation Format: Xinheng Yu, Kyle J. Eastman, Kanak Raina, Kelli M. Jones, Chris D. Forbes, Abigail Hundt, Marco Garcia, Rebecca Stronk, Katia Howard, Andrew McGovern, Rebekka Chenard, Allison Denny, Mia Forgione, Kyle Bassoli, Ethan Garvin, James J. Mousseau, Hao Li, Madeline P. King, Amit Bhardwaj, Katherine J. Kayser-Bricker, Craig M. Crews. Prostate cancer RIPTAC™ therapeutics demonstrate activity in preclinical models of Enzalutamide-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1629.