RIPTACs: A Groundbreaking Approach to Drug Discovery

Abstract

Regulated Induced Proximity Targeting Chimeras (RIPTACs), a new class of heterobifunctional molecules, show promise in specifically targeting and eliminating cancer cells, while leaving healthy cells unharmed. RIPTACs, a groundbreaking drug discovery approach, work by forming a stable complex with two proteins – one specifically found in cancer cells (Target Protein, TP), and the other pan-essential for cell survival (Effector Protein, EP), selectively disrupting the function of the EP in cancer cells and causing cell death. Interestingly, the TPs need not be linked to disease progression, broadening the spectrum of potential drug targets. This review summarizes the discovery and recent advances of the RIPTAC strategy. Additionally, it discusses the associated opportunities and challenges as well as future perspectives in this field.

Introduction

A New Era of Targeted Oncology Drug Development

The approval of imatinib mesylate, a breakpoint cluster region-abelson murine leukemia (BCR-ABL) tyrosine kinase inhibitor, for the treatment of patients with chronic myeloid leukemia (CML) by the U.S. FDA in 2001, has ushered in a new era of targeted oncology drug development.^1–5 Compared to traditional radiotherapies and chemotherapies, targeted drugs may selectively kill tumor cells versus normal cells, thereby mitigating the potential adverse off-target side effects. The superiority of targeted therapies is manifested by lower safety risks and enhanced therapeutic index, which greatly benefits patients suffering from various diseases.^6–8 However, the success rate of these therapies remains low due to poor efficacy and significant toxicity.

Challenges in Classical Targeted Cancer Therapy

Classical targeted cancer therapy is aimed to inhibit an oncology-driving protein, such as androgen receptor (AR), mutant epidermal growth factor receptor (mEGFR), and Kirsten rat sarcoma virus G12C mutation (KRAS^G12C), or selectively block a cancer cell survival dependent signal pathway exemplified by inhibiting proteasome, inhibitor of apoptosis protein (IAP), and poly (ADP-ribose) polymerase (PARP). While many targeted therapeutic agents have advanced into phase I clinical trials for cancer, most of them failed in the clinical trials, showing a low success rate (10%), likely owing to two primary reasons, including poor efficacy and significant toxicity.^9–11 Approximately more than 300 proteins have been identified as oncology-driving proteins, but only about 50 of them have been successfully validated as “druggable” targets with drugs approved by the U.S. FDA.^12–14 During the drug discovery process, some genes have been found to play critical roles in cancer cell survival, and their encoded proteins are identified as cancer-selective targets.¹⁵ Consequently, small molecule therapeutic agents have been developed and evaluated in clinical patients, selectively targeting the essential proteins for the survival of tumor cells, such as mitotic kinases (e.g., PLK1 and AURKA/B), cell cycle regulators (e.g., CDK4/6 and CDK9), and epigenetic regulators (e.g., BRD4 and HDACs). Despite some success with several approvals by the U.S. FDA, many of such agents have failed in clinical trials due to a lack of an acceptable therapeutic index, indicating a significant gap in developing targeted therapies.

Emergence of Precision Oncology

Over the past decades, precision medicine has attracted lots of attention, especially precision oncology,^16,17 promoting the development of several novel therapeutic modalities, such as antibody-drug conjugates (ADCs),^18–20 T-cell engages (TCEs),^21–23 and radiopharmaceuticals.^24–26 These therapies specifically induce cancer cell death via selectively targeting cancer cells and delivering more effective anti-proliferative effects. ADCs are a class of targeted therapeutic agents for cancer and other human diseases with high potency and selectivity.^27,28 Currently, more than 10 ADCs have been approved for market use by the U.S. FDA, with over 100 ADCs under clinical trials worldwide.^27,29 ADCs are composed of three components: a monoclonal antibody specifically binds with the antigen located at the membrane of cancer cells, a cytotoxic payload that is usually referred to as a cytotoxic agent, and a chemical linker connecting these two components.²⁷ The ADCs can be selectively delivered to cancer cells and then release the cytotoxic agent, causing the death of cancer cells.²⁸ After the ADC is intravenously injected, it circulates in the bloodstream, and binds to the receptor (antigen) that is selectively expressed on the cancer cell membrane to form an ADC-antigen complex, which is then internalized by receptor-mediated endocytosis. The internalized complex becomes mature and fuses with the lysosome, degrading ADC and releasing the cytotoxic payload, eventually leading to cancer cell death and apoptosis while sparing normal cells.^19,27 T cell recruiters or CAR-T cells selectively kill cancer cells through activating the T cells to secrete perforin and granzyme nearby the cancer cells.^30–34 Radiopharmaceuticals can selectively recognize cancer cells and trigger cell death through emitting radiation within a short distance. Several radiopharmaceuticals have been approved, with a few in clinical trials at different stages for the treatment of various cancers.²⁶ For these therapies, the target protein or antigen acts as a guided receptor, with no need to be the driver of diseases, thereby expanding the scope of potential drug targets. It is well known that lineage-specific antigens, such as CD19, have been widely used as the targets for ADCs and CAR-T therapy, achieving great success in the field of treating hematological malignancies.^35,36 Nevertheless, these approaches bind with the target proteins or antigens on the cell membrane or extracellularly but not with the most cancer-selective proteins intracellularly.^22,26,27,34 Moreover, their wide application is significantly limited by the associated high costs and complexity.

The Advent of Heterobifunctional PROTACs and Monomeric MGs

In recent years, the advent and prosperity of heterobifunctional molecules, especially proteolysis targeting chimeras (PROTACs), have opened new avenues for drug development, especially helpful for traditionally “undruggable” targets.^37–49 PROTACs have several advantages over traditional small-molecule inhibitors. PROTACs can exert their effects at a catalytic amount based on an “event-driven” model to replace the “occupancy-driven” model of small molecule inhibitors, which may avoid or reduce the off-target side effects. Moreover, PROTACs can exert their effects without binding tightly to the active site of proteins of interest (POIs), greatly expanding the target protein space. Therefore, some proteins playing critical roles in various diseases but lacking potent active sites can also be targeted by PROTACs, such as transcriptional factors NF-κB and E2F, providing new therapeutic strategies.^50,51 However, several potential limitations exist for PROTACs,^42,52 including that 1) The relatively large molecular weights are beyond Lipinski’s rule of five (Ro5), leading to poor permeability, oral bioavailability, and pharmacokinetic properties in general; 2) The mutations that occurred in E3 ligase may result in drug resistance.⁵³ Studies have demonstrated that continuous efforts may eventually overcome these limitations. Recently, RNK05047, a BRD4 PROTAC developed by Ranok Therapeutics, has been advanced into phase I/II clinical trials (ClinicalTrials.gov Identifier: NCT05487170). RNK05047 induces the degradation of BRD4 without directly interacting with E3 ligase through chaperone-mediated E3 recruitment and following polyubiquitination of BRD4, which may overcome drug resistance caused by mutations in E3 ligase.⁵⁴ To date, approximately 20 PROTACs (structures shown in Figure 1A) have been advanced into clinical trials, especially for treating various cancers (Table 1).⁴⁶ The most advanced is ARV-471 (an ER degrader) in phase III clinical trials for the treatment of advanced-stage ER⁺/HER2^- breast cancer (ClinicalTrials.gov identifier NCT05654623). Recent positive clinical trial results reported by pharmaceutical companies Arvinas, Kymera Therapeutics, and Nurix Therapeutics, assuage concerns about the poor druggability of PROTACs with relatively high molecular weights. Other than PROTACs, some emerging target protein degradation (TPD) approaches such as Molecular Glues (MGs), AUtophagy-Targeting Chimeras (AUTACs), and AUTOphagy-TArgeting Chimera (AUTOTACs) have also been developed.^38,55 In recent years, some MGs have been discovered by rational discovery and/or rational design beyond serendipity, including degraders inducing the degradation of CDK12, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, HDAC1/3, and SMARCA2/4.^55–60 Excitingly, some MGs (structures shown in Figure 1B) have been successfully approved or advanced into clinical trials (Table 1).^61,62 TPD strategies provide new therapeutic potentials for unmet clinical needs, attracting more and more attention in both academic and industrial setting.

Figure 1.

Chemical structures of representative small molecule degraders 1–18 in clinical trials or received the US FDA-approval. A) Chemical structures of PROTACs 1–8; and B) Chemical structures of molecule glues (MGs) 9–18.

Table 1.

Representative Small Molecule Protein Degraders in Clinical Trials or Approved in Clinic^{46,61–63 a}

Degrader	Pharmaceuticals	Target	E3 ligase	Phase stage	Indication	NCT Identifier
ARV-110 (1)	Arvinas	AR	CRBN	I/II	mCRPC	NCT03888612
				I	mCRPC with rising PSA values on abiraterone	NCT05177042
ARV-766 (2)	Arvinas	AR	CRBN	I/II	mCRPC	NCT05067140
CC-94676 b	Celgene/Bristol-Myers Squibb	AR	CRBN	I	mCRPC	NCT04428788
AC176 b	Accutar Biotech	AR	Undisclosed	I	mCRPC	NCT05673109
				I	mCRPC	NCT05241613
HP518 b	Hinova Pharmaceuticals	AR	Undisclosed	I	mCRPC	NCT05252364
ARV-471 (3)	Arvinas/Pfizer	ER	CRBN	I	Advanced or metastatic ER+/HER2- breast cancer.	NCT05501769
				I	ER+/HER2- locally advanced or metastatic breast cancer	NCT05463952
				II	ER+/HER2- localized breast cancer	NCT05549505
				I	Advanced ER+/HER2- breast cancer	NCT05732428
				I/II	ER+/HER2- locally advanced or metastatic breast cancer	NCT04072952
				II	Advanced or metastatic breast cancer	NCT05573555
				I/II	Advanced or metastatic breast cancer	NCT05548127
				III	Advanced metastatic breast cancer	NCT05654623
AC682 b	Accutar Biotech	ER	CRBN	I	ER+/HER2- locally advanced or metastatic breast cancer	NCT05489679
				I	ER+/HER2- locally advanced or metastatic breast cancer	NCT05080842
NX-2127 (4)	Nurix Therapeutics	BTK	CRBN	I	Relapsed/refractory B-cell malignancies	NCT04830137
NX-5948 b	Nurix Therapeutics	BTK	CRBN	I	Relapsed/refractory B-cell malignancies	NCT05131022
BGB-16673 b	BeiGene	BTK	CRBN	I	B-cell malignancies	NCT05294731
				I	B-cell malignancies	NCT05006716
HSK29116 b	Haisco Pharmaceutical	BTK	CRBN	I	Relapsed/refractory B-cell malignancies	NCT04861779
RNK05047 b	Ranok Therapeutics	BRD4	Chaperone complex	I/II	Advanced solid tumors/DLBCL	NCT05487170
CFT8634 (5)	C4 Therapeutics	BRD9	CRBN	I/II	Synovial sarcoma; SMARCB1-null tumors	NCT05355753
FHD-609 (6)	Foghorn Therapeutics	BRD9	CRBN	I	Advanced synovial sarcoma or advanced SMARCB1-loss tumors	NCT04965753
DT2216 (7)	Dialectic Therapeutics	BCL-XL	VHL	I	Relapsed/refractory malignancies	NCT04886622
KT-413 b	Kymera Therapeutics	IRAK4	CRBN	I	Relapsed or refractory B-cell NHL	NCT05233033
KT-333 b	Kymera Therapeutics	STAT3	Undisclosed	I	Refractory lymphoma; LGL leukemia; Solid tumors	NCT05225584
ASP3082 b	Astellas Pharma	KRAS G12D	Undisclosed	I	Locally advanced, unresectable or metastatic KRASG12D mutant solid tumors	NCT05382559
CFT1946 (8)	C4 Therapeutics	BRAF V600X	CRBN	I/II	BRAF V600 mutant solid tumors	NCT05668585
Thalidomide (9)	Celgene/Bristol-Myers Squibb	IKZF1/3	CRBN	Approved	MM
Pomalidomide (10)	Celgene/Bristol-Myers Squibb	IKZF1/3	CRBN	Approved	MM; Kaposi’s sarcoma
Lenalidomide (11)	Celgene/Bristol-Myers Squibb	IKZF1/3, CK1α	CRBN	Approved	MM; FL; MZL; MDS; MCL
Avadomide (12)	Celgene/Bristol-Myers Squibb	IKZF1/3, ZPF91	CRBN	II	Advanced melanoma	NCT03834623
				I/II	Unresectable HCC	NCT02859324
				I/II	CLL; SLL	NCT02406742
Iberdomide (13)	Celgene/Bristol-Myers Squibb	IKZF1/3, ZPF91/98	CRBN	III	Relapsed or refractory MM	NCT04975997
				III	Newly diagnosed MM	NCT05827016
				I/II	MM	NCT02773030
Mezigdomide (14)	Celgene/Bristol-Myers Squibb	IKZF1/3	CRBN	III	Relapsed or refractory MM	NCT05552976
				III	Relapsed or refractory MM	NCT05519085
				I/II	Relapsed or refractory MM	NCT03374085
				I/II	Relapsed or refractory MM; Newly diagnosed MM	NCT03989414
				I/II	Relapsed or refractory MM	NCT05372354
Eragidomide (15) 63	Celgene/Bristol-Myers Squibb	GSPT1	CRBN	I	Relapsed or refractory AML or higher-risk MDS	NCT02848001
				I	AML	NCT04336982
Golcadomide (16)	Celgene/Bristol-Myers Squibb	IKZF1/3	CRBN	II	NHL	NCT05283720
				I/II	Relapsed or refractory NHLs	NCT03930953
				I/II	Relapsed/refractory B-cell malignancies	NCT03310619
CFT7455 (17)	C4 Therapeutics	IKZF1/3	CRBN	I/II	Relapsed/refractory NHL or MM	NCT04756726
DKY709 (18)	Novartis	IKZF2	CRBN	I	Advanced NSCLC or melanoma	NCT03891953

^aData collected from http://www.clinicaltrials.gov; Data last updated June 20, 2023.

^bStructures are not disclosed.

The Rise of RIPTACs and Other Heterobifunctional Molecules

Motivated by the success of PROTACs and MGs, some other bifunctional molecules have been developed and reported, including regulated induced proximity targeting chimeras (RIPTACs), deubiquitinase-targeting chimeras (DUBTACs), enhancement-targeting chimeras (ENTACs), phosphorylation-inducing chimeras (PHICs), phosphatase-recruiting chimeras (PhoRCs), dephosphorylation-targeting chimeras (DEPTACs), phosphorylation-targeting chimeras (PhosTACs), acetylation tagging system (AceTAG), and transcriptional/epigenetic chemical inducers of proximity (TCIPs).^64–66 As a groundbreaking drug discovery strategy, RIPTACs with a unique mechanism present attractive advantages over other drug discovery approaches such as small molecule inhibitors and other bifunctional molecules. Developing RIPTACs may improve the therapeutic window attributed to their high selectivity and enhanced efficacy by the induction of targeted protein-protein interactions (PPIs). Herein, this review summarizes the advances of RIPTAC strategy and discusses the relevant challenges, opportunities, and future perspectives toward viable therapeutics.

The Proposition of the Concept of RIPTACs

It is known that MGs may induce or enhance the PPIs by forming a stable ternary complex. Other than MGs, some natural molecules have been serendipitously found to display robust efficacy through the mechanism of inducing PPIs, such as rapamycin (an mTOR kinase inhibitor), cyclosporin A (a calcineurin phosphatase inhibitor).^55,67,68 Excitingly, KRAS inhibitor RMC6236, rationally developed by Revolution Medicines based on this mechanism, has been advanced into phase I clinical trials (ClinicalTrials.gov Identifier: NCT05379985).⁶⁹ Deeply motivated by the great success of PROTACs, MGs, PPI inducers, and other bifunctional molecules, as a pioneer in the field of TPD, Crews and his team initiated a more recent campaign to develop RIPTACs through a groundbreaking approach designing heterobifunctional small molecules with a goal of selectively inducing the death of cancer cells expressing a specific intracellular TP.⁷⁰ RIPTACs are made up of three components: a ligand binding with the TP specifically expressed in cancer cells, a ligand binding with the pan-essential Effector Protein (EP) required for cell survival, and a linker connecting these two ligands. RIPTACs exert their functions via specific accumulation inside cells expressing TP, and the formation of a cooperative ternary complex (TP:RIPTAC:EP), abrogating the functions of the EP and eventually leading to selective cell death (Figure 2). The effect event process of RIPTACs is briefly classified into five steps: 1) accumulation in cancer cells by selectively binding with the TP; 2) formation of a stable ternary complex by simultaneously binding with the TP and EP; 3) induction of PPIs between the TP and EP; 4) enhancement of the inhibitory activity against the EP; and 5) causing cell death by abrogating the function of EP. The selective accumulation of RIPTACs in cancer cells relies on the effective TP binding and/or the simultaneous involvement of pan-essential EP. The unique mechanism of action adopted by RIPTACs may significantly improve the therapeutic window by eliminating or reducing the toxic side effects on TP-absent cells. Detailed proof-of-concept study and mechanism exploration of RIPTACs are described in the following sections.

Figure 2.

The general mechanism of action for RIPTACs involves the formation of a stable ternary complex between a Target Protein (TP) and an Essential Protein (EP), thereby selectively killing the cancer cells, while leaving healthy cells unharmed.

Proof-of-concept Study on RIPTACs

To verify the rationality of the relevant hypothesis, a proof-of-concept study was initiated by constructing a model system with well-characterized target proteins and ligands.⁷⁰ A 293_HFL cell line, derived from HEK293 cells, was engineered with lentiviral overexpression of Flag-tagged HaloTag7-FKBP^F36V (HaloTag-FKBP) containing a C-terminal P2A-EGFP sequence, forming a HaloTag-FKBP fusion protein, which was employed as a TP in the successive experiments. This TP can recruit covalent and non-covalent ligands via its HaloTag and FKBP domains, respectively. HaloTag-FKBP fusion protein is selectively expressed in 293_HFL cells but not in the control 293_GFPL cells, indicating the successfully engineered 293_HFL cells. Moreover, the expression of HaloTag-FKBP was not restricted to any specific compartment of the cell. Furthermore, the intracellular concentration of HaloTag-FKBP was also determined based on the recombinant HaloTag-GST as a standard curve. After an extensive and systemic characterization, engineered 293_HFL cells were determined as suitable tool cells. Other than TPs, the EPs, essential for cell survival, are also critical for RIPTACs to exert their effects. The selection of suitable EPs and corresponding ligands is also crucial for the design of RIPTACs. Based on the published CRISPR dropout screening, combined with the potency, selectivity, and extent of validation of their ligands, BET bromodomain protein, PLK1 kinase, and CDK kinase were eventually chosen as the suitable essential EPs. Meanwhile, small molecule inhibitors 19 ((+)-JQ1, a BET inhibitor), 20 (BI2536, a PLK1 inhibitor), 21 (TMX3103, a multi-CDK inhibitor), and 22 (dinaciclib, a multi-CDK inhibitor) were determined as the effector protein ligands (ELs). In addition, compound 23 (HLDA-001) was defined as a non-covalent FKBP-binding ligand, and chloroalkane (CA) was defined as a covalent HaloTag-binding ligand. The chemical structures of compounds 19–23 are shown in Figure 3A.

Figure 3.

Chemical structures of compounds 19–50. A) Chemical structures of Effector Protein ligands (ELs) 19–22, and Target Protein (TP) HaloTag-FKBP ligand 23 (non-covalent); B) Chemical structures of covalent and non-covalent RIPTACs 24–47; and C) Chemical structures of negative controls 48–50.

After determining the suitable EPs and ELs, as well as the FKBP ligand, Crews et al. designed and synthesized a series of small molecule RIPTACs (compounds 24–47, Figure 3B) along with three negative control compounds (compounds 48–50, Figure 3C).⁷⁰ The effects of the synthesized RIPTACs on cell growth were tested using a CellTiter-Glo viability assay. This experiment involved two treatment procedures. In one, the 293_HFL and 293_GFPL cells were continuously exposed to the RIPTACs for 7 days. In the other, the cells were treated with the RIPTACs for 4 hours, after which the RIPTACs were washed off, and the cell viability was measured after 7 days. The findings from this experiment revealed that the RIPTACs significantly suppressed the growth of the 293_HFL cells under both treatment conditions. Particularly, 20-based RIPTACs 27 (HLDA-131) and 45 (HLDA-231), and 22-based RIPTAC 35 (HLDA-119) showed significantly enhanced anti-proliferation activity (492-, 145-, and 195-fold, respectively) in the 293_HFL cells compared to the 293_GFPL cells after a 4-hour exposure followed by 7 days of cultivation. This experiment also provided insights into the optimal lengths for the linkers in the RIPTACs.

Based on the structure of ELs, these RIPTACs were divided into four series: 19-based RIPTACs (series I); 20-based RIPTACs (series II), 21-based RIPTACs (series III), and 22-based RIPTACs (series IV). According to the binding mode of the TP ligands, except series IV, the other three series of RIPTACs can be divided into two subseries, namely covalent RIPTACs and non-covalent RIPTACs. Among series I RIPTACs, compounds 25 (HLDA-124) and 40 (HDLA-222) exerted the most potent anti-proliferation activity in 293_HFL and 293_GFPL cells, showing > 50 and 11 folds-shift, respectively, in 293_HFL cells compared to 293_GFPL cells. Among series II RIPTACs, compounds 32 (HLDA-111) and 43 (HLDA-212) were the most potent RIPTACs, showing 44 and > 900 folds-shift in 293_HFL cells compared to 293_GFPL cells, respectively. Among series III RIPTACs, compounds 27 (HLDA-131) and 45 (HLDA-231) displayed the strongest anti-proliferation activity in 293_HFL and 293_GFPL cells, showing 189 and 58 folds-shift in 293_HFL cells compared to 293_GFPL cells, respectively. Among series IV RIPTACs, compound 35 (HLDA-119) exerted the most potent anti-proliferation activity in 293_HFL and 293_GFPL cells, showing 27 folds-shift in 293_HFL cells compared to 293_GFPL cells. The detailed anti-proliferation activities (GI₅₀ values) of compounds 25, 27, 32, 35, 40, 43, and 45 with optimal linker length in 293_HFL and 293_GFPL cells and fold-shift for 293_HFL cells over 293_GFPL cells were described in Table 2. Notably, results showed that both the ELs and the TP FKBP ligands themselves displayed slight differences in anti-proliferation potency between 293_HFL and 293_GFPL cells, suggesting that RIPTACs exerted their effect via a novel unique mechanism. The differential anti-proliferative activity exerted by RIPTACs in the presence of TP was not affected by the cell line background. RIPTAC 25, composed of 19, CA and a linker, can cause a clear, albeit modest, and leftward shift in HaloTag-FKBP (TP)-expressing HT29_HF cell line (an engineered HT29 cell line) compared to HT29 cells. Also, the differential anti-proliferative activity induced by RIPTACs in 293_HFL and 293_GFPL cells did not originate from interacting with the FKBP immunophilin domain of the HaloTag-FKBP fusion protein. RIPTAC 32, composed of 21, CA, and a linker, but not the EL 21 alone, showed an apparent leftward shift in anti-proliferative activity in Akt-HaloTag (TP)-overexpressing 293_AktH cells (an engineered HEK293 cell line) compared to 293_GFPL cells. Likewise, compound 27, composed of 20, CA, and a linker, but not the EL 20 alone, also showed an obvious leftward shift in the HaloTag-BTK-overexpressing 293_HBTKL cells (an engineered HEK293 cell line) compared to 293_GFPL cells. All these findings suggest that RIPTACs can selectively suppress the proliferation of cells expressing TP over the cells in the absence of TP, providing the basis for eliminating or reducing toxic side effects.

Table 2.

GI₅₀ values of representative RIPTACs in 293_GFPL and 293_HFL cells and fold-shift over 293_HFL cells

RIPTAC	25 (HLDA-124)	27 (HLDA-131)	32 (HLDA-111)	35 (HLDA-119)	40 (HLDA-222)	43 (HLDA-212)	45 (HLDA-231)
Target Protein (TP)	HaloTag	HaloTag	HaloTag	HaloTag	FKBP	FKBP	FKBP
Effector Protein (EP)	BET BRD	PLK1	multi-CDK	multi-CDK	BET BRD	multi-CDK	PLK1
Effector Ligand (EL)	19 ((+)-JQ1)	20 (BI-2536)	21 (TMX-3013)	22 (Dinaciclib)	19 ((+)-JQ1)	21 (TMX-3013)	20 (BI-2536)
293_GFPL GI50 (µM)	>10	0.377	2.224	0.347	5.442	>10	0.868
293_HFL GI50 (µM)	0.191	0.002	0.050	0.013	0.506	0.011	0.015
Fold-Shift	>50	189	44	27	11	>900	58

Mechanism Study on RIPTACs

Mechanisms Underlying RIPTAC Differential Biology

Crews and colleagues conducted systematic mechanism studies with a clear aim to test the hypothesis that RIPTACs accumulate in cells via the formation of a ternary complex, thereby explaining the observed increase in anti-proliferative activity in cells expressing the TP.⁷⁰ This hypothesis was grounded in previous observations, and the following experiments were designed to explore and potentially validate this proposed mechanism. Firstly, they investigated whether 23 could accumulate in cells via TP-dependent recruitment. It was found that >1000× enrichment of the TP ligand 23 in 293_HFL cells expressing HaloTag-FKBP fusion protein, compared to the control 293_GFPL cells lacking the fusion protein. Secondly, they elucidated whether RIPTACs could be accumulated through TP-dependent recruitment in cells expressing the TP. Several non-covalent RIPTACs were selected as tool compounds to treat two cell lines. Significant accumulation of 40, 43, and 45 were observed in 293_HFL cells expressing the TP, while not in 293_GFPL cells lacking the TP, although their accumulation level was lower than that observed for 23. Next, the accumulation of 43 in 293_GFPL cells was significantly inhibited upon pre-treatment of the cells with excessive 23 (an FKBP ligand), indicating that the accumulation of RIPTACAs was achieved in a TP-dependent manner.

Similarly, the leftward shift in anti-proliferative activity in 293_HFL cells induced by RIPTACs could be eliminated upon pre-treatment with the TP ligands TAMRA-CA (covalent) or 23 (non-covalent), illustrating that the differential biological function of RIPTACs was also realized dependent on the TP recruitment. These results revealed that RIPTACs exerted the differential biological function in a mode of action of heterobifunctional molecules rather than through the combined cytotoxicity of the TP and EP ligands. Additionally, to further validate the role of TP and EP in the anti-proliferation activity of RIPTACs, negative control compounds 48 (HLDA-120) and 49 (HLDA-110) without chloride atom that cannot bind with the TP HaloTag, and 50 (HLDA-125) with a 19 epimer that cannot bind with the EP BRD2/3/4, were synthesized, and tested against 293_HFL and 293_GFPL cells. In line with their expectations, all three negative control compounds showed no differential anti-proliferative activity between 293_HFL and 293_GFPL cell lines, supporting that RIPTACs exerted their activity based on the TP and EP binding.

To determine whether the leftward shift in cell viability of a RIPTAC can be achieved by mere inhibition of the EP function, compounds 35 and 22 with varying concentrations were used to treat 293_HFL and 293_GFPL cells and the levels of phosphorylation at Ser2 of RPB1-CTD were analyzed by western-blotting experiments. RPB1-CTD is a recognized phosphorylation site catalyzed by CDK9, and this process can be blocked by CDK9 inhibitor 22. 35 displays 20-fold more potent phosphorylation inhibitory activity in 293_HFL cells than in 293_GFPL cells (IC₅₀s = 16 nM and 334 nM, respectively), which is highly consistent with its anti-proliferative activity tested in these two cell lines (GI₅₀s = 13 nM and 347 nM, respectively). However, no significant phosphorylation inhibitory activity difference was observed in these two cell lines (IC₅₀s = 30–100 nM), indicating that RIPTACs act through a novel mechanism differing from simply an EP inhibition.

RIPTAC-Mediated Ternary Complex Formation (TP:RIPTAC:EP)

Further mechanism study explored whether the differential biology displayed by RIPTACs originates from the formation of ternary complex (TP-RIPTAC-EP).⁷⁰ The 293_HFL cells were treated with 19-based RIPTACs 40 (non-covalent) and 26 (HLDA-121) (covalent) for 3 h, and then the HaloTag protein was immunoprecipitated with Halo-Trap Agarose beads. BRD4 co-immunoprecipitation was observed with both RIPTACs, but not with the negative control 50. Moreover, the co-immunoprecipitation of BRD4 with 26 and 40 could be prevented upon pre-treating the 293_HFL cells for 30 min with TAMRA-CA (a HaloTag ligand) and 23 (an FKBP ligand), respectively. Furthermore, the formation of ternary complex triggered by both RIPTACs could be partially prevented by pre-treatment 293_HFL cells with 19. In addition, the intracellular TP:RIPTAC:EP were formed after treatment of 293_HFL cells with PLK1-targeted RIPTAC 45 (non-covalent), CDK9-targeted RIPTAC 37 (HLDA-115, covalent), and CDK1/2/5-targeted RIPTAC 32 (covalent), at a concentration of 100 nM, but not with the negative control ELs 20 and 22, and TP FKBP ligand 23, or HaloTag ligand TAMRA-CA. Interestingly, the formed ternary complexes containing RIPTACs, even the non-covalent RIPTAC 40, were still readily detectable 72 h after washout, revealing that the ternary complexes induced by RIPTACs were exceptionally stable. All these findings suggest that RIPTACs act their functions via a mechanism of the formation of a stable ternary complex, inducing or enhancing the PPIs, improving the inhibitory potency against the EP, and causing cell death by abrogating the function of EP (Figure 4).

Figure 4.

Schematic illustration of the mechanism of RIPTACs by a step-by-step process. (a) Selective accumulation of the RIPTACs in cancer cells by binding with the Target Protein (TP). (b) The formation of the ternary complex by simultaneously binding with the TP and EP (TP:RIPTAC:EP). (c) Induction and/or enhancement of the protein-protein interactions (PPIs) based on a stable ternary complex. (d) Selective induction of cancer cell apoptosis and death by abrogating the function of the Effector Protein (EP).

It was further characterized whether the positive cooperativity during the ternary complex formation partially contributed to the improved anti-proliferative activity of RIPTACs in 293_HFL cells but not 293_GFPL cells. The binding of 19-based RIPTAC 39 (HLDA-221) (non-covalent) to BRD4-BD1 was evaluated in the presence or absence of purified recombinant FKBP via an internally established AlphaLISA-based bioassay. A dramatic improvement (~35-fold) in the binding affinity of 39 to BRD4-BD1 was observed upon pre-incubation with FKBP, while such a potency shift was not observed for either the EL 19 or the TP ligand 23. This finding suggests that, like other bivalent heterobifunctional molecules PROTACs, and monovalent bifunctional molecules MGs, the pharmacological activities of RIPTACs are mainly attributed to the ligand-induced PPIs, which may also partially account for the selective anti-proliferative activity of RIPTACs in 293_HFL cells over 293_GFPL cells.

Given that the selective anti-proliferative potency of RIPTACs primarily originated from the ternary complex formation with the TP and EP, it is necessary to determine whether the subcellular localization of these two important proteins significantly affects the ternary complexes formation and related potency of RIPTACs. To this end, 293_NLS2HF and 293_MYRHF were engineered, expressing a nuclear-localized and a plasma membrane-localized HaloTag-FKBP fusion protein, respectively. Several RIPTACs were then selected to test their anti-proliferative activity in 293_NLS2HF, 293_MYRHF, and 293_HFL cells. Intriguingly, 19, 20, and 21-based covalent RIPTACs 25, 27, 32, 40, 43, and 45 showed decreased potency in 293_MYRHF cells, while among all tested non-covalent RIPTACs, only RIPTAC 40 binding with FKBP (TP) and BRD4 (EP) showed decreased potency in 293_MYRHF cells, indicating that non-covalent RIPTACs are less dependent on the subcellular localization of the TP and EP compared to covalent RIPTACs. As expected, the control compounds, including ELs 19–21 and TP FKBP ligand 23, showed remarkably similar activity in all four tested cell lines.

Orally Bioavailable Androgen Receptor (AR) RIPTACs

To advance the concept of RIPTACs into clinical practice, Halda Therapeutics (https://haldatx.com/), founded by Crews et al., focuses on the discovery and development of small molecule RIPTACs with a primary aim to address various diseases, particularly cancers. Drug resistance often occurs in almost all prostate cancer (PCa) patients after treatment with AR inhibitors for a period of time, primarily owing to the AR gene mutations, causing the overexpression of AR protein. There is an urgent need to develop novel therapeutic agents to overcome drug resistance caused by AR mutations. To achieve the goal of selectively killing the AR-overexpressed cancer cells, researchers from Halda Therapeutics developed a series of novel heterobifunctional molecules (AR RIPTACs), leveraging the feature of overexpressed AR protein.⁷¹ Such AR RIPTACs were found to selectively kill the AR-positive PCa cells while sparing normal cells, although their in vivo safety properties are currently unknown. These AR RIPTACs could cooperatively bind with AR (TP) and EP, and then form a stable ternary complex (AR:RIPTAC:EP) across PCa cell lines with a remarkable EC₅₀ value of ~ 1 nM, thereby blocking the EP function and displaying significant anti-proliferative activity in AR-upregulated PCa cell lines.⁷¹ The AR RIPTACs exerted their selective cell-killing activity largely depending on the presence of upregulated AR protein. These AR RIPTACs can induce the apoptosis of cells with overexpressed AR but not control normal cells at low nM concentrations. Notably, in castrated mice bearing VCaP xenografts, these AR RIPTACs accumulate in the tumor tissues and facilitate the formation of ternary complex (AR:RIPTAC:EP) at a low oral dose, resulting in the specifically EP inhibition in tumor tissues and eventually suppressing the tumor growth. More importantly, these AR RIPTACs showed excellent pharmacokinetic properties and readily achieved efficacious exposures after oral dosing in mice, rats, and dogs.⁷¹ Compared to AR PROTACs acting their function by the elimination of AR, which plays important role in cell growth and survival, AR RIPTACs may display stronger therapeutic efficacy based on their unique mechanism that directly abrogating the function of EP that is essential for cell survival, which needs more preclinical and clinical data to demonstrate. Taken together, these AR RIPTACs with favorable druglike properties and robust antitumor efficacy may serve as promising clinical candidates for further development. However, to date, the chemical structures of these promising AR RIPTACs have not been disclosed. Excitingly, Halda Therapeutics announced that the most promising AR RIPTACs are being evaluated in toxicology studies, and the IND filing with the ideal RIPTACs has been slated for 2024. If these AR RIPTACs progress into clinical trials, it could potentially lead to the development of novel therapeutics. Nevertheless, further extensive studies are required to confirm their efficacy and safety for patients affected by PCa.

Challenges, Opportunities, and Future Directions

Compared to other medicinal modalities, such as small molecule modulators, PROTACs, and MGs, RIPTACs have several advantages, including: 1) RIPTACs selectively kill the cells expressing the TP, while not affecting the TP-absent cells, which may help mitigate the side effects; 2) TPs are not necessary to be the drivers of diseases, the presence or absence of the active binding sites in TPs are not critical, and the agonists and/or antagonists are both acceptable as the TP ligands, thereby greatly expanding the scope of target proteins and providing new therapeutic options for various diseases; 3) RIPTACs do not need to have a high binding affinity with the TPs, which may contribute to overcoming the drug resistance caused by gene variations; and 4) RIPTACs with high selectivity for cancer cells over normal cells may widen the therapeutic window and lower the administration dose, significantly improving the safety issues. Beyond the advantages, RIPTAC have their own limitations, including 1) the number of currently available TPs and EPs, especially TPs, is limited, which significantly restricts the broad application of RIPTACs; 2) RIPTACs exert their function by simultaneously binding with the TP and EP, which sets up strict requirements for the tissue distribution and location of the TPs and EPs; and 3) the potent efficacy of RIPTACs depends on the PPIs induced by the formation of a stable ternary complex, which greatly increases the difficulty for developing RIPTACs based on rational design. Notably, the development of RIPTACs is in a very early stage, and some major challenges must be addressed. 1) the TPs and EPs are critical for RIPTACs to selectively kill the abnormal cells, achieving the purpose of precision treatment. However, the suitable TPs and EPs, especially TPs, are difficult to determine owing to the number of currently available is very limited; 2) the detailed underlying mechanism of RIPTACs has not been comprehensively elucidated to understand their exact action modes; and 3) similar to other heterobifunctional molecules such as PROTACs, the RIPTACs also typically possess relatively large molecular weight, generally limiting their druglike profiles, including aqueous solubility, cell permeability, oral bioavailability and other PK properties for further drug development. More extensive efforts are imperative to address such challenges by optimizing the linker structures with similar approaches in developing PROTACs. In addition, exploring more suitable TPs is also critical for the extensive applications of RIPTAC strategy toward viable therapeutics.

As mentioned above, the development of a series of AR RIPTACs for treating AR-positive PCa represents a milestone success. The preclinical data that some compounds exhibit robust antitumor activities in different PCa cell lines overexpressing AR and VCaP xenografts (an animal model resistant to AR antagonists) appear encouraging. Furthermore, these RIPTACs also display excellent oral bioavailability and efficacious exposures in mice, rats, and dogs. It is exciting that Halada Therapeutics announced the plan of toxicity studies of these promising AR RIPTACs towards the possible IND filing in 2024. It is worthwhile to mention that KRAS^G12C that is selectively expressed in cancer cells may play a role as the TP for designing RIPTACs, contributing to overcoming drug-resistance due to their efficacy may be not affected by the partial loss of binding affinity caused by gene mutations.⁷⁰

Concluding Remarks and Prospects

Over the past decades, precision therapy has attracted increasing attention. Inspired by precision therapies, such as ADCs and PROTACs, developing RIPTACs as a groundbreaking drug discovery strategy is emerging. Initially, a 293_HFL cell line steadily overexpressing HaloTag-FKBP with a C-terminal P2A-EGFP sequence (TP) and a control 293_GFPL cell line expressing only EGFP were constructed for the proof-of-concept study. Subsequently, a series of novel RIPTACs were designed, synthesized, and evaluated. All tested RIPTACs showed enhanced anti-proliferative activity in 293_HFL cells expressing HaloTag-FKBP fusion protein, compared to control 293_GFPL cells. However, neither the ELs nor the FKBP ligand showed significant potency differences in 293_HFL cells and 293_GFPL cells. The finding suggests that RIPTACs work through a novel mechanism relying on the presence of TP, and RIPTACs bind to the TP and recruit the EP, promoting the ternary complex formation. The formation of this ternary complex can induce or enhance the PPIs between the TP and EP. Furthermore, the ternary complex can block the function of the EP, resulting in selective cell death in TP-expressing cells while sparing cells in the absence of the TP.

In conclusion, RIPTAC represents a groundbreaking drug discovery strategy that may provide targeted therapies, especially for cancers, by mitigating the side effects and assisting in overcoming mutation-triggered drug resistance. Importantly, RIPTACs may provide new therapeutic alternatives for clinically unavailable patients, including those with rare diseases. RIPTACs may be developed toward viable therapeutics as second- or third-line drugs, or orphan drugs. The discovery of AR RIPTACs and their positive preclinical results, especially the favorable PK properties, partially alleviating the concerns for the druggability of RIPTACs, make this strategy encouraging and particularly attractive. While the early results are promising, it is important to note that the development of RIPTACs is still in its initial stages, and some challenges lie ahead to address. Overall, the development of RIPTACs as potentially viable therapeutics for a range of diseases is an exciting prospect, and we anticipate seeing more RIPTACs in the drug discovery pipeline in the future.

A short teaser.

RIPTACs selectively kill cancer cells expressing the Target Protein (TP), significantly mitigating the side effects. This review summarizes the advances of this groundbreaking strategy, and discusses challenges, opportunities and future perspectives toward viable therapeutics.

Data availability

No data was used for the research described in the article.