Recent Advances in PROTACs for Drug Targeted Protein Research

Hematologic malignancy (HM) is a malignant tumor originating from the hematopoietic system. Clinically, there are three common types of leukemia, multiple myeloma, and malignant lymphoma [87]. Some target proteins in hematological malignancies, such as BCL-XL, MDM2, STAT3, and MALT1, are also difficult to be drugged. The emergence of PROTAC technology brings hope for the treatment of such diseases. Herein, PROTAC molecules targeting hematological malignancies in recent years are introduced.

2.2.1. Leukemia

Leukemia is the most prevalent cancerous blood malignancy, and inhibitors targeting leukemia-related proteins are not effective in the treatment of leukemia. PROTAC technology may solve this dilemma.

The most prevalent acute leukemia in adults is acute myeloid leukemia (AML) [88], which is distinguished by aberrant proliferation and weakened differentiation ability of hematopoietic precursor cells, resulting in a large number of immature leukocytes in the bone marrow, peripheral blood, and even other tissues aggregate, while the number of other normal blood cells decreases dramatically [89]. Tripartite motif-containing protein 24 (TRIM24), also known as transcriptional intermediary factor 1α (TIF1α), is a multi-domain protein involved in the transcriptional regulation of the androgen receptor (AR) and other nuclear receptors [90]. A potential prostate cancer therapy target is TRIM24, which has been implicated in various tumorigenesis and disease progression [91,92,93,94,95,96]. Bromodomain inhibitors are far from enough as an anticancer strategy. Bradner’s team synthesized PROTAC 11 ( ) based on the TRIM24 bromodomain inhibitor IACS-9571 and the ligand of VHL. PROTAC 11 recruits VHL to cause the effective and selective degradation of TRIM24 in human acute myeloid leukemia MOLM-13 cells [42]. This study proposes TRIM24 as a fresh target for acute myeloid leukemia creation of drugs, providing a new avenue for “undruggable targets”.

MDM2 is an E3 ubiquitin ligase that promotes the degradation of the p53 tumor suppressor gene [97,98]. Wild-type p53 protein is inactivated by overexpressed MDM2 due to its reverse regulation [99]. Therefore, MDM2 is an attractive target for a new therapy for AML specifically for the treatment of MDM2-overexpressing but TP53 wild-type AML. Because p53 stabilization upregulates MDM2 protein levels, which limits the clinical efficacy of MDM2 inhibitors [100], Talpaz Moshe et al. developed an MDM2 PROTAC degrader that binds to and targets MDM2 for degradation, eliminating the inhibition of p53, thereby inducing apoptosis in leukemia cells [101]. Unfortunately, the structure of this compound has not been disclosed. Another team also reported PROTAC 12 ( ), a small-molecule degrader targeting MDM2, which effectively induces the rapid degradation of MDM2 in human leukemia cells at a concentration of <1 nm, which can inhibit the growth of B-cell acute lymphoblastic leukemia (B-ALL) RS4-11 cells and human acute myeloid leukemia cell MV4-11 at low nanomolar concentration and induce tumor regression in the RS4-11 xenotransplantation model [43].

FMS-like tyrosine kinase 3 (FLT-3) is a therapeutic target in AML, FLT-3 frequently occurring mutation of an internal tandem duplication (ITD) in the juxta-membrane domain [102,103]. Regarding the limited clinical efficacy of FLT-3 inhibitors [104,105], the Crews group converted the FLT-3 inhibitor quizartinib into the PROTAC molecule PROTAC 13 ( ), which induced MV4-11 cells at low nanomolar concentrations and degradation of FLT-3 ITD mutants in MOLM-14 cells. The study showed that PROTAC was able to inhibit cell growth more effectively than the inhibitor alone, and they also demonstrated that the compound was able to induce FLT-3 ITD degradation in vivo. This suggests that the degradation of FLT-3 ITD may provide a useful approach for therapeutic intervention in AML [44].

Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family, which responds to a variety of cytokines, growth factors, and other signals and activates the expression of downstream genes [106]. Dysregulation of STAT3 contributes to a variety of human cancers as well as many human illnesses. Therefore, STAT3 has long been considered a therapeutic target for diseases such as cancer. Unfortunately, small-molecule drugs targeting STAT3 are difficult to find due to poor specificity and other reasons [107]. In order to solve this problem, Shaomeng Wang’s research group used PROTAC technology to design a small molecule PROTAC 14 ( ) that can specifically degrade STAT3 in cancer cells. Apoptosis slows the development of a fraction of acute myeloid leukemia and anaplastic large cell lymphoma cell lines, and at well-tolerated dosages, the chemical completely and permanently eradicates tumors in a number of xenotransplantation mice models. Therefore, a possible cancer treatment method is to degrade the STAT3 protein [45].

The cyclin-dependent kinase (CDK) family are serine/threonine kinases [108] that function in cell cycle regulation and transcription. Excessive activation of CDK proteins results in dysregulated cell proliferation that promotes tumor progression, and inhibition of some CDK family members has been shown to be a viable approach to cancer therapy [109]. But the design of selective small-molecule inhibitors is often hindered by similar ligand-binding pockets. Furthermore, current inhibitors cannot disrupt scaffold function [110]. To solve this problem, Gray’s team used the PROTAC strategy to describe a phthalimide-based degrader, PROTAC 15 ( ), since this degrader forms a different ternary complex with the E3 ligase CRBN; so, this degrader is specific and proteome-wide selective for CDK6. PROTAC 15 exploits the selective dependence of AML cells on CDK6 to rapidly degrade CDK6, enabling dynamic mapping of its direct role in coordinating signaling and gene control in AML [46].

BRD2, BRD3, BRD4, and BRDT are the key members of the bromodomain and extraterminal domain (BET) family of proteins, which are crucial for epigenetic control [111,112,113]. A large number of small molecule degraders targeting BRD4 have demonstrated therapeutic activity in preclinical models of AML and inflammatory diseases. In 2015, Bradner’s lab independently reported CRBN-dependent BET protein degradation of PROTAC molecules—thalidomide-based PROTAC 16 ( ) [47]. In 2017, Ciulli et al. described a PROTAC molecule PROTAC 17 ( ) related to VHL binders [15], the degrader can bind to BRD4 and VHL to mediate the degradation of BRD4. Due to the structural instability of CRBN binder thalidomide, the Rankovic lab discovered new CRBN binders with higher chemical stability and ligand efficiency in 2021 and designed a novel BET PROTAC molecule PROTAC 18 ( ) with higher efficiency. Their data showed that PROTAC 18 can inhibit the viability of human acute myeloid leukemia MV4-11 cells at picomolar concentrations (IC50 = 3 pM) [48]. Subsequently, Ciulli’s group designed a trivalent PROTAC molecule PROTAC 19 ( ) based on MZ1 and the BET inhibitor MT1 that can bind to two BRD4 proteins, which carries two binding domains for BET proteins and one for the E3 binding domain to which ubiquitin ligase binds. They found that PROTAC 19 was 300-fold more active in degrading BRD2 protein than the existing bivalent PROTAC molecules ARV-771 and MZ1. And PROTAC 19 can more effectively inhibit the viability of MV4-11 cells and induce apoptosis of prostate cancer cell line 22RV1 [49].

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy originating from immature T-cell precursors. A previous study found that T-ALL was dependent on BCL-XL [114]. The BCL-2 protein family member BCL-XL is essential for the survival of cancer cells [115,116,117]. However, BCL-XL-specific inhibitors targeted, dose-limiting platelet toxicity, leading to thrombocytopenia, which limits their application in acute leukemia [118]. Konopleva Marina Y investigated the preclinical efficacy of PROTAC7 in T-ALL cell lines in vitro and in living T-ALL patient-derived xenotransplantation (PDX) models. This study shows that T-ALL cells are highly sensitive to PROTAC 7 in vitro. In a living T-ALL PDX model, the use of PROTAC 7 in combination with chemotherapy can alleviate leukemia and prolong patient survival. In conclusion, PROTAC 7 targeting BCL-XL is an efficient and safe adjuvant therapy in T-ALL [119]. In another study, Zheng Guangrong et al. reported two PROTAC BCL-XL degraders, PROTAC 20 ( ) [50] and PROTAC 21 ( ) [51], which can act in a dose- and time-dependent manner. Degrades BCL-XL in MOLT-4 T-ALL cells with unique selectivity for MOLT-4 cells compared to traditional BCL-XL inhibitors, suggesting that T-ALL can be improved by converting the inhibitor to PROTAC treatment window.

Chronic myelogenous leukemia (CML) is most often caused by the loss of auto-inhibitory constraint of the c-ABL kinase domain in the oncogenic fusion protein BCR-ABL [120,121]. With the advent of BCR-ABL-targeted tyrosine kinase inhibitors (TKIs), CML has become a chronic but manageable disease, but due to the presence of persistent leukemia stem cells (LSCs), all CML patients must receive lifelong treatment [122,123,124]. Therefore, targeting BCR-ABL degradation may bring new benefits to CML patients. In 2017, the Crews group demonstrated PROTACs that bind to VHL and CRBN E3 ligases, respectively, to known kinase inhibitors (such as imatinib, bosutinib, and dasatinib). Western blot showed that the CRBN-based PROTAC molecule PROTAC 22 ( ) could degrade the BCR-ABL fusion protein and c-ABL-expressed receptors in K562 CML cells. When using the VHL binder, only dasatinib-based PROTACs observed degradation of c-ABL, and no degradation of BCL-ABL was observed for all VHL-based compounds. No degradation of c-ABL and BCL-ABL was observed with imatinib-based PROTACs [52]. In addition, Naito’s group developed BCR-ABL degraders PROTAC 23 ( ) [54] and PROTAC 24 ( ) [53] called SNIPER (ABL), which induced the degradation of BCR-ABL protein in BCR-ABL positive CML cells and inhibited the growth of chronic myeloid leukemia K562 cells. Taken together, the above-mentioned multiple studies suggest that the degradation of BCR-ABL is a potential strategy for the treatment of BCR-ABL-driven chronic myelogenous leukemia.