RIPTACs for Precision Cancer Therapy: A Novel Modality with the Inspiration of HLD-0915 as the First Candidate in Clinical Trials

Chemically induced proximity-based approaches are promising alternatives to small molecule inhibitors, attracting increasing attention in the drug discovery field. Small molecule chemical inducers of proximity (CIPs) can induce molecular proximity of two proteins in living cells. CIPs are developed to recapitulate diverse biological processes in living cells and organisms, including transcription, post-translational modification, and signal transduction. (1) Based on an “event-driven” model instead of “occupancy-driven” model by small molecule inhibitors, CIPs are able to drive a cellular event with a substoichiometric binding of proteins. (2) As an important member of CIPs, proteolysis-targeting chimeras (PROTACs) catalytically induce the proximity between cellular protein-of-interest (POI) and the E3 ligase, causing the polyubiquitination and degradation of the POI. (3) Over the past two decades, the emergence and prosperity of PROTAC technology opened new avenues for drug discovery and development with about 20 PROTAC molecules in human clinical trials. (4−11) Inspired by the great success of PROTACs, (12) several new CIPs beyond degradation have been developed, (13,14) such as regulated induced proximity targeting chimeras (RIPTACs), (15,16) deubiquitinase-targeting chimeras (DUBTACs), (17−20) enhancement-targeting chimeras (ENTACs), (21) RESTORACs, (22) phosphorylation-inducing chimeras (PHICs), (23) phosphatase-recruiting chimeras (PhoRCs), (24,25) phosphorylation-targeting chimeras (PhosTACs), (26) dephosphorylation-targeting chimeras (DEPTACs), (27) acetylation tagging system (AceTAG), (28,29) and transcriptional/epigenetic CIPs (TCIPs). (30−32) RIPTACs are emerging as a new therapeutic modality for various cancers (Figure 1). Like PROTACs, RIPTACs are also a class of heterobifunctional molecules, comprising a ligand for the tumor-specific target protein (TP) that is selectively expressed in cancer cells, a ligand for the pan-Essential Protein (EP) that is required for cell survival, and a linker connecting these two components. RIPTACs selectively kill cancer cells expressing tumor-specific TP while sparing non-TP expressing healthy cells. RIPTACs selectively accumulate in the cancer cells expressing tumor-specific TP and form a stable ternary complex between the TP and EP (TP:RIPTAC:EP). The stable ternary complex induces or enhances the protein–protein interactions (PPIs) between the TP and EP, abrogating the EP function and thus causing selective cancer cell death. RIPTACs role as a class of bivalent molecular glue inhibitors by bringing the TP and EP into proximity in cancer cells, delivering a “hold and kill” blow. Figure 1. Schematic diagram of RIPTAC therapeutic modality. RIPTACs are a novel class of heterobifunctional molecules, comprising a TP ligand, an EP ligand, and a connecting linker. RIPTACs selectively accumulate in cancer cells expressing tumor-specific TP, form a stable ternary complex with the TP and EP (TP:RIPTAC:EP), and abrogate the EP function, leading to selective cancer cell death while sparing healthy cells. RIPTACs can induce neo-PPIs or enhance existing PPIs, acting as bivalent molecular glue inhibitors with a “hold and kill” mechanism. RIPTACs have the potential to mitigate the on-target off-tumor toxicity caused by the EPs. As a paradigm-shifting drug discovery approach, RIPTACs are attracting more and more attention from both academic and industrial settings. Recently, Halda Therapeutics, founded by Crews and co-workers, announced that the first patient with metastatic castration-resistant prostate cancer (mCRPC) is administrated with HLD-0915, an androgen receptor (AR)-targeting RIPTAC, in a Phase 1/2 clinical trial (NCT06800313). (33) The purpose of this first-in-human clinical trial is to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of orally dosed single-agent HLD-0915. (33,34) This study includes an initial Phase 1 and a subsequent Phase 2 clinical trials. The Phase 1 clinical trial is a dose escalation study to determine the maximum tolerated dose (MTD) and/or recommended dose(s) of HLD-0915 as a monotherapy. The Phase 2 expansion cohort is aimed to further evaluate the efficacy and safety of HLD-0915. The Phase 1/2 clinical study plans to enroll up to 80 mCRPC patients. HLD-0915 is a heterobifunctional molecule, comprising an AR ligand as the TP, an EP ligand, and a connecting linker. So far, the EP and chemical structure of HLD-0915 have not been disclosed. HLD-0915 is orally effective in preclinical prostate cancer models, including drug resistant models, resulting in significant tumor shrinkage and prostate-specific antigen (PSA) reduction, while showing a favorable therapeutic index. Prostate cancer is the second most common malignancy and the fifth leading cause of cancer death among men worldwide, with about 1.5 million new cases and 397,000 deaths in 2022. (35) Almost all prostate cancer patients develop drug resistance to AR signaling inhibitors, which is driven by many heterogeneous bypass resistance mechanisms, including genetic mutations in AR and increased expression of AR levels. The AR gene, which codes for AR, or the upstream enhancer region of DNA are aberrantly amplified in more than 80% mCRPC patients. (36) Clinical needs remain unmet for prostate cancer patients, especially those with advanced, drug resistant, and the most lethal type. Thus, novel therapies are urgently needed to tackle such challenging diseases. AR-targeting RIPTACs (e.g., HLD-0915) with a unique mechanism of action are emerging as a promising alternative precision cancer therapy for mCRPC. To develop novel therapeutics for treating prostate cancer resistant to AR antagonists, such as enzalutamide, researchers at Halda Therapeutic designed and synthesized a series of AR-targeting heterobifunctional molecules. Excitingly, two compounds, H001 and H003, were demonstrated to be promising candidates for treating prostate cancer. (33) H001 and H003 are bona fide RIPTACs, acting through a chemically induced proximity mechanism. H001 and H003 form a stable ternary complex with the TP (AR) and EP, which is revealed by the X-ray cocrystal structure of the AR:RIPTAC:EP ternary complex, but the PDB code has not been disclosed. The determination of the X-ray crystal structure of the AR:RIPTAC:EP ternary complex enables structure-based drug design (SBDD) of AR-targeting RIPTACs. Both H001 and H003 form an intracellular ternary complex with AR and the EP in VcaP cells with EC₅₀ values of 24 nM and 9 nM, respectively. However, neither the AR ligand nor the EP ligand alone induces the formation of the intracellular ternary complex, indicating that both the AR ligand and the EP ligand within RIPTACs are involved in forming the ternary complex. Additionally, AR-targeting RIPTACs exhibit positive co-operativity in AR binding and TP binding, contributing to forming a more stable ternary complex. H001 and H003 display potent antiproliferation activity (H001: IC₅₀ < 10 nM; H003: IC₅₀ < 1 nM) and EP inhibition in Trex293 cells expressing doxycycline-inducible AR. Compared to the parental 22RV1 cells expressing low levels of FL-AR, H001 and H003 significantly induce cell apoptosis in 22RV1 cells overexpressing FL-AR (H001: EC₅₀ = 113 nM; H003: EC₅₀ = 48.5 nM), as determined by the Caspase 3/7 GIo assay. 22RV1/AR cells overexpressing FL-AR are derived from the parental AR-V7⁺ cells, an Enzalutamide-resistant cell line. H001 induces the formation of AR:RIPTAC:EP ternary complex in Trex293 cells expressing clinically relevant AR mutants (AR^Mut) induced by doxycycline. Doxycycline-induced AR^Mut leads to the substitution of lysine residue at position 702 by histidine (L702H), histidine residue at position 875 by tyrosine (H875Y), and threonine residue at position 878 by alanine (T878A). H001 and H003 (30 mg/kg, p.o., QD) display superior oral in vivo efficacy to Enzalutamide, an AR signaling inhibitor, in an AR-amplified (AR^amp), V7⁺ VCaP prostate tumor model in castrated mice. H001 and H003 cause tumor regression in a VCaP tumor model in castrated mice, an Enzalutamide-insensitive prostate cancer model. Moreover, H001 and H003 significantly reduce the plasma PSA. Furthermore, H001 and H003 can form a ternary complex with AR and EP in the tumor tissue and resulted in EP inhibition, transforming into robust in vivo efficacy. Predosing with Enzalutamide that occupies the AR ligand binding domain (AR-LBD) competes off AR binding with H001, leading to significant attenuation in the ternary complex (AR:H001:EP) formation and EP inhibition in a VCaP tumor model in castrated mice. This finding indicates that the in vivo pharmacodynamic (PD) modulation of H001 is dependent on AR binding. H001 induces “BRCAness” in both in vitro and in vivo VCaP models. H001 dramatically suppresses the expression of breast cancer gene 1 (BRCA1), breast cancer gene 2 (BRCA2), and related homologous recombination repair (HRR) genes, such as RAD51-associated protein 1 (RAD51AP1), RecQ-mediated genome instability protein 2 (RMI2), and RAD54-like protein (RAD54L), in VCaP cells in a dose-dependent manner. Moreover, H001 (30 mg/kg) is orally effective for inhibiting the expression of these HRR genes in a castrate VCaP tumor model. PARP inhibitors can cause synthetic lethality in prostate cancer cells bearing BRCA1/2 deficiency. Therefore, H001 in combination with PARP inhibitors may exert synergetic effect for treating prostate cancers, showing the potential to overcome drug-resistance to PARP inhibitors. Combination therapy studies of AR-RIPTACs and PARP inhibitors will be anticipated. RIPTACs have several unique features in comparison with existing therapeutic modalities as outlined in Table 1. First, RIPTACs exert their function independent of the TP function, which is similar to bispecific antibodies (bsAbs), antibody–drug conjugates (ADCs) and CAR-T-mAb, but different with small molecule inhibitors and protein degraders. Second, RIPTACs are effective for drug-resistant cancer cells caused by nontarget-based resistance mechanisms, which is similar to bsAbs, ADCs, and CAR-T-mAb, but different with small molecule inhibitors and protein degraders. Third, RIPTACs show potential to treat cancers by targeting tumor-driving “undruggable” oncoproteins, which is similar to protein degraders, bsAbs, ADCs, and CAR-T-mAb, but superior to small molecule inhibitors. Fourth, RIPTACs can induce the formation of TP:RIPTAC:EP ternary complex, inducing or enhancing the PPIs between the TP and EP, which is similar to protein degraders, such as molecular glue degraders (MGDs), but different with small molecule inhibitors, bsAbs, ADCs, and CAR-T-mAb. Fifth, like small molecule inhibitors and protein degraders, RIPTACs can be developed as oral dosage with a low cost of goods sold (COGS), which is not accessed by bsAbs, ADCs, and CAR-T-mAb. In addition, like small molecule inhibitors and protein degraders, RIPTACs target intracellular TPs, which is different with bsAbs, ADCs, and CAR-T-mAb relying on the antigen on the cell membrane. RIPTACs present several advantages over existing therapeutic modalities. First, RIPTACs selectively kill cancer cells while sparing normal cells, delivering a high therapeutic index. Second, RIPTACs take effect independent of the TP function, largely expanding the scope of TP. To this end, some “undruggable” targets specifically expressing in tumor cells can be used as TPs for developing RIPTACs. Third, based on the positively cooperative binding mechanism, RIPTACs do not need to have a high binding affinity with the TPs, showing the potential to overcome drug resistance caused by TP mutations. Moreover, RIPTACs also have the potential to overcome drug resistance caused by nontarget-based resistance mechanisms. Nevertheless, challenges also exist for the development of RIPTACs. Like PROTACs, RIPTACs also have a relatively large molecular weight, which is beyond the Lipinski’s “Rule of Five” (Ro5), limiting the overall druglike properties such as cell permeability, aqueous solubility, and metabolic stability. Positive clinical results of PROTACs partially attenuate such a concern through systematic and continuous structural optimizations. Moreover, the successful advancement of HLD-0915 into clinical trials inspires the endeavor of RIPTAC modality for the drug development. Given the unique molecular glue mechanism, the requirements for the ligand selection of the TP and EP, as well as the type and length of the linker are stricter for RIPTACs than PROTACs. Thus, the success rate of developing RIPTACs is lower than that of PROTACs. Few of the designed and synthesized bivalent heterobifunctional molecules as potential RIPTACs are bona fide RIPTACs, while most of them may simply act as potential dual inhibitors without inducing proximity. Furthermore, the number of TPs explored for developing RIPTACs is largely limited. Currently, only AR, ER, FKBP, and P53 Y220C proteins have been determined as suitable TPs for RIPTACs. (15,16,37,38) More diverse TPs need to be explored for developing novel RIPTACs. (38) The EPs used for RIPTACs mainly include cyclin-dependent kinases (CDKs), bromodomain and extra terminal domain (BET) proteins, and polo-like kinase 1 (PLK1). (15,16) In addition, RIPTACs are currently exploited primarily for treating cancers, while this strategy may also be expanded and applicable for other threatening diseases in the future. In conclusion, RIPTAC technology is a novel chemically induced proximity-based therapeutic strategy, opening new avenues for innovative drug discovery and development. RIPTACs with a unique selective and wide applicable cancer cell-killing mechanism have the potential to mitigate side effects including on-target off-tissue toxicity and overcome drug resistance. As an emerging technology, developing RIPTACs is still in an early stage, but inspired by the milestone of successful advancement of a first RIPTAC HLD-0915 into Phase 1/2 clinical trial for treating mCRPC (NCT06800313). (34) While we look forward to the positive clinical outcomes of these studies, RIPTACs as a novel modality may offer an unparalleled opportunity for developing precision medicines, particularly for cancer therapy. This work was partially supported by R01CA226001 and R01CA231150 grants from the National Institutes of Health, Breast Cancer Research Program (BCRP) Breakthrough Awards W81XWH-17-1-0071 and W81XWH-17-1-0072 from the Department of Defense (DoD), the John D. Stobo, M.D. Distinguished Chair Endowment, and the Edith & Robert Zinn Chair Endowment in Drug Discovery. 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