Patent landscape of inhibitors and PROTACs of the anti-apoptotic BCL-2 family proteins

Abstract

Introduction:

The anti-apoptotic BCL-2 family proteins, such as BCL-2, BCL-XL, and MCL-1, are excellent cancer therapeutic targets. The FDA approval of BCL-2 selective inhibitor venetoclax in 2016 validated the strategy of targeting these proteins with BH3 mimetic small molecule inhibitors.

Areas covered:

This review provides an overview of the patent literature between 2016 and 2021 covering inhibitors and PROTACs of the anti-apoptotic BCL-2 proteins.

Expert opinion:

Since the FDA approval of venetoclax, tremendous efforts have been made to develop its analogues with improved drug properties. These activities will likely result in new drugs in coming years. Significant progress on MCL-1 inhibitors has also been made, with multiple compounds entering clinical trials. However, MCL-1 inhibition could cause on-target toxicity to normal tissues especially the heart. Similar issue exists with BCL-XL inhibitors, which cause on-target platelet toxicity. To overcome this issue, several strategies have been applied, including prodrug, dendrimer-based drug delivery, antibody-drug conjugate (ADC), and proteolysis targeting chimera (PROTAC); and amazingly, each of these approaches has resulted in a drug candidate entering clinical trials. We envision technologies like ADC and PROTAC could also be utilized to increase the therapeutic index of MCL-1 inhibitors.

1. Introduction

Apoptosis is a highly regulated cell death process through which the human body maintains tissue homeostasis and prevents cancer [1,2]. There are two distinct but convergent apoptotic pathways, the extrinsic pathway, mediated by the activation of cell surface “death receptors”, and the intrinsic pathway, activated by various exogenous and endogenous stimuli that lead to mitochondrial damage [3]. The B-cell lymphoma-2 (BCL-2) protein family, consisting of anti- and pro-apoptotic members, plays a crucial role in regulating the intrinsic apoptotic pathway. Anti-apoptotic proteins, including BCL-2, BCL-XL, MCL-1, BCL-W, and A1/BFL-1, are characterized by the presence of four BCL-2 homology (BH) domains, BH1, BH2, BH3, and BH4. Their anti-apoptotic role is mainly through BH1, BH2, and BH3 mediated interaction with pro-apoptotic Bcl-family proteins. The N-terminal BH4 domain binds proteins that do not belong to the BCL-2 family and involves in regulating the non-canonical activities of BCL-2 and BCL-XL [4]. Pro-apoptotic BCL-2 proteins include two varieties, multi-domain (BH1, BH2, BH3, and BH4) proteins BAX and BAK and a large group of BH3-only proteins, such as BIM, BID, BAD, NOXA, PUMA, and BMF. BAX and BAK can form pores in the mitochondrial outer membrane (MOM) through homo-oligomerization, causing the permeabilization of the MOM and cytochrome c release, and subsequent activation of the caspase cascade to trigger apoptosis. Anti-apoptotic BCL-2 proteins bind to the α-helical BH3 domain of BAX and BAK and prevent them from forming oligomers. BH3-only proteins bind to anti-apoptotic proteins to free up BAX and BAK or activate them via direct interactions. Thus, the protein-protein interactions (PPIs) among these three groups of proteins determine cell life and death (Figure 1) [2].

Figure 1.

Intrinsic apoptotic signalling pathway regulated by BCL-2 family of proteins.

Many cancers resist apoptosis and chemo-/targeted therapies through upregulating one or more anti-apoptotic BCL-2 proteins [5,6]. Therefore, inhibition of the PPIs between anti- and pro-apoptotic BCL-2 proteins to directly induce apoptosis in cancer cells is a highly attractive anticancer strategy [7–9]. Starting from the first potent BCL-2/BCL-XL dual inhibitor ABT-737 (Figure 2), developed through a fragment-based drug discovery using NMR approach [10], and the follow-up orally bioavailable analogues ABT-263 (navitoclax) (Figure 2) [11,12], many “BH3 mimetic” small-molecule inhibitors (SMIs) of the major anti-apoptotic proteins BCL-2, BCL-XL, and MCL-1 have been developed [6,13–19]. Since the approval of BCL-2 selective inhibitor ABT-199 (venetoclax) (Figure 2) by the FDA in 2016 for the treatment of chronic lymphocytic leukemia (CLL) [6,20,21], which ultimately validated the therapeutic strategy of targeting BCL-2 proteins, we witnessed accelerated drug discovery and development activities in this field. Numerous drug candidates targeting BCL-2, BCL-XL, or MCL-1 have entered clinical trials (Table 1) [18,19], notably also including two new therapeutic modalities, antibody-drug conjugate (ADC) mirzotamab clezutoclax (ABBV-155) (NCT03595059) [22] and proteolysis targeting chimera (PROTAC) DT2216 (NCT04886622) (Table 1) [23,24]. In this review, we provide a brief overview of the SMIs and PROTACs of BCL-2, BCL-XL, and MCL-1 reported in patent filings since 2016.

Figure 2.

Structures of ABT-737, ABT-263, and ABT-199

Table 1.

Summary of clinical stage drug candidates that target anti-apoptotic BCL-2 family proteins

Compound	Structure	Target	Sponsor	Clinical Trial Status
S55746		BCL-2	Servier	Phase 1 (NCT02920697) Completed
BGB-11417	Not yet disclosed	BCL-2	Beigene	Phase 1 (NCT04277637; NCT04883957) Phase 1/2 (NCT04771130; NCT04973605)
S65487/VOB560		BCL-2	Servier/Novartis	Phase 1 (NCT03755154; NCT04702425) Phase 1/2 (NCT04742101)
LOXO-338	Not yet disclosed	BCL-2	Eli Lilly	Phase 1 (NCT05024045)
LP-108	Not yet disclosed	BCL-2	Guangzhou Lupeng Pharmaceutical	Phase 1 (NCT04139434; NCT04356846)
APG-2575		BCL-2	Ascentage Pharma	Multiple Phase 1 and Phase 2 trials
ZN-d5	Not yet disclosed	BCL-2	Zentalis Pharmaceuticals	Phase 1/2 (NCT05199337) Phase 1 (NCT04854174)
TQB3909	Not yet disclosed	BCL-2	Chia Tai Tianqing Pharmaceutical	Phase 1 (NCT04975204, not yet recruiting)
ABBV-623	Not yet disclosed	BCL-2	Abbvie	Phase 1 (NCT04804254)
ABT-263 (navitoclax)		BCL-2 & BCL-XL	AbbVie	Phase 3 (NCT04472598)
APG-1252		BCL-2 & BCL-XL (Prodrug)	Ascentage Pharma	Phase 1 (NCT04001777; NCT04893759) Phase 1/2 (NCT04210037; NCT04354727; NCT05186012)
AZD0466		BCL-2 & BCL-XL (dendrimer conjugate)	AstraZeneca	Phase 1/2 (NCT04865419) Phase 1/2 (NCT05205161, not yet recruiting)
LP-118	Not yet disclosed	BCL-2 & BCL-XL	Guangzhou Lupeng Pharmaceutical/Newwave Pharmaceutical	Phase 1 (NCT05025358; NCT04771572)
ABBV-155 (mirzotamab clezutoclax)	Not yet disclosed	BCL-XL (ADC)	Abbvie	Phase 1 (NCT03595059)
S64315/MIK665		MCL-1	Servier/Novartis	Phase 1 (NCT03672695; NCT04702425) Phase 1/2 (NCT04629443)
AMG-176 (tapotoclax)		MCL-1	Amgen	Phase 1 (NCT05209152, not yet recruiting) Phase 1 (NCT02675452)
AMG-397 (murizatoclax)		MCL-1	Amgen	Phase 1 (NCT03465540, terminated)
AZD5991		MCL-1	AstraZeneca	Phase 1/2 (NCT032188683, suspended)
GS-9716	Not yet disclosed	MCL-1	Gilead Sciences	Phase 1 (NCT05006794)
PRT1419	Not yet disclosed	MCL-1	Prelude Therapeutics	Phase 1 (NCT05107856; NCT04837677; NCT04543305)
ABBV-467	Not yet disclosed	MCL-1	Abbvie	Phase 1 (NCT04178902, terminated)
DT2216		BCL-XL (degrader)	Dialectic Therapeutics	Phase 1 (NCT04886622)

2. Patent review of small-molecule BCL-2 inhibitors

BCL-2 is the founding member of the BCL-2 family proteins, originally discovered at the breakpoint of t(14;18) chromosome translocation in human follicular lymphomas in 1985 [25,26]. Its pro-survival role was first reported in 1988 [27]. A number of hematological malignancies are found to be dependent on this protein for survival [28]. ABT-737 is the first reported potent inhibitor of BCL-2, and also inhibits BCL-XL and Bcl-w [10]. Navitoclax is an orally bioavailable analogue of ABT-737, and has been evaluated in various clinical trials but most have been terminated due to the rapid induction of thrombocytopenia, an on-target toxicity due to its inhibition of BCL-XL [11,29–32]. This is because platelets are dependent on BCL-XL for maintain their viability [33,34]. ABT-199 was subsequently designed to have high selectivity for BCL-2 over BCL-XL thus spare the platelet toxicity [20]. In 2016, the FDA granted approval to ABT-199 for the treatment of adult CLL with 17p deletion [21], and subsequent approvals for the combination therapy with low intensity chemotherapy in 2018 for acute myeloid leukemia (AML) [35]. Currently, eight BCL-2 selective inhibitors are in clinical trials, including APG-2575 (lisaftoclax) [36], BGB-11417 [37], VOB560 (S65487) [38], LOXO-338 [39], ZN-d5 [40], LP-108, TQB3909, and ABBV-623; and S55746 [41] has completed Phase 1 trial. Therefore, it is not surprising that many patents have been disclosed around these clinical-stage drugs. Several mechanisms of acquired drug resistance to venetoclax have been identified [42–44]. One involves point mutations in BCL-2, including G101V and D103Y mutations, that significantly reduce the binding affinity of venetoclax to BCL-2 [43,44]. We expect at least some of the clinical stage BCL-2 inhibitors are able to target these BCL-2 mutants.

2.1. University of Michigan/Ascentage Pharma

APG-2575 was disclosed in a patent (WO2018/027097) published in 2018 by the University of Michigan [45]. Sixty-six BCL-2 inhibitors with the general formula A were disclosed (Figure 3). Binding affinities for BCL-2, BCL-XL, and MCL-l were tested using fluorescence polarization (FP) assays. Cell viability assays was conducted in BCL-2 dependent cell lines RS4;11 and Molm13. APG-2575 displayed slight selectivity for BCL-2 over BCL-XL (IC₅₀ = 2 nM and 5.9 nM, respectively), and with IC₅₀ > 5000 nM against MCL-1 in fluorescein labeled Flu-BIM, Flu-BAK, and Flu-BID displacement FP assay. Cytotoxicity of APG-2575 in RS4;11 and Molm13 cells was found to be IC₅₀ = 5.5 nM and 6.4 nM respectively. Pharmacokinetic (PK) properties were found to be comparable to that of ABT-199. In the in vivo studies the compound exhibited maximum tumor growth inhibition with 100 mg/kg/week treatment in a RS4;11 xenograft model. Interestingly, compound 1 (Figure 3), the enantiomer of APG-2575, had better selectivity for BCL-2 over BCL-XL (IC₅₀ = 1.3 nM and 14.8 nM, respectively) and higher cytotoxicity in RS4;11 and Molm13 cells (IC₅₀ = 2.8 nM and 1.8 nM, respectively).

Figure 3.

Structures of general formula A, APG-2575, and compound 1 (University of Michigan/Ascentage Pharma)

2.2. BeiGene

In 2019, BeiGene published a patent (WO2019/210828) where over 350 BCL-2 inhibitors with the general formula B were disclosed (Figure 4) [46]. Protein inhibition against BCL-2 and BCL-XL was tested by using FP and/or TR-FRET assays. Cytotoxicity was tested in RS4;11 and MOLT-4 (BCL-XL-dependent) cell lines. One of the most potent compounds 2 exhibited > 1000-fold selectivity for BCL-2 over BCL-XL (IC₅₀ = 0.015 and 18 nM, respectively). Compound 2 also potently and selectively inhibited BCL-2-dependent RS4;11 growth over BCL-XL-dependent MOLT-4 cells (IC₅₀ = 0.41 nM and 2520 nM, respectively). Compound 2 is superior to venetoclax in a number of efficacy studies in mouse xenograft tumor models (WO2021/110102) [47]. In another patent published in 2021 (US2021/0269433), BeiGene disclosed hundreds more BCL-2 inhibitors based on the same general formula B [48]. Selected compounds were also tested for their binding affinity to drug-resistant mutant BCL-2-G101V protein. One of the most potent compounds 3 (Figure 4) exhibited potent and selective inhibition of BCL-2 (IC₅₀ = 0.063 and 27 nM for BCL-2 and BCL-XL, respectively), and IC₅₀ of 0.38 nM and 957 nM in killing RS4;11 and MOLT-4 cells, respectively. It also displayed a binding IC₅₀ of 0.25 nM against BCL-2-G101V.

Figure 4.

Structures of general formula B and compounds 2, 3, 4, 5, and 6 (BeiGene)

Additional analogues were disclosed in patent WO2021/083135 [49]. One representative compound 4 (Figure 4) from this patent exhibited a binding IC₅₀ of 0.027 nM against BCL-2-WT and 1.2 nM against BCL-2-G101V. In cell viability assays, compound 4 displayed IC₅₀s of 0.34 nM, 1.1 nM, and 31.9 nM against RS4;11, BaF3 with BCL-2 WT, and BaF3 with BCL-2-G101V, respectively. In another follow-up patent (WO2021/208963) of this series [50], a representative analogue 5 (Figure 4) exhibited similar binding affinity to BCL-2-WT and BCL-2-G101V, and high potency in killing both RS4;11 cells and RS4;11 cells with BCL-2-G101V knock-in (IC₅₀ = 0.05 nM and 0.11 nM, respectively). As the data displayed for compound 6 (Figure 4), many compounds in this series were also highly potently in binding to BCL-2-D103Y mutant and showed no difference when compared with their binding affinity for BCL-2-WT.

2.3. Centaurus Biopharma

In a 2018 patent (WO2018/041284) published by Centaurus disclosed 44 BCL-2 selective compounds based on the general formula C (Figure 5) [51], which are close analogues of venetoclax. A number of these compounds are more potent than venetoclax based on an FP assay using FITC-Bim as a probe. The best compound 7 had an IC₅₀ of 0.36 nM for BCL-2 and 50.2 nM for BCL-XL. In addition, compound 7 selectively inhibited BCL-2-dependent DOHH2 cell growth over BCL-XL-dependent MOLT-4 cells (IC₅₀ = 14 nM and > 1,000 nM, respectively).

Figure 5.

Structures of general formula C and compound 7 (Centaurus Biopharma)

2.4. Prelude Therapeutics

In 2021, Prelude Therapeutics published a patent (US2021/0346405) on the development of BCL-2 inhibitors with 107 analogues disclosed [52]. The general chemical structure D, features a reversed N-acyl sulfonamide moiety compared to venetoclax, is illustrated in Figure 6. BCL-2 binding affinities were determined with an HTRF assay using FAM-BAD as a probe. Compounds were categorized according to their binding affinity. A representative compound 8 was found to have an IC₅₀ < 10 nM.

Figure 6.

Structures of general formula D and compound 8 (Prelude Therapeutics)

2.5. Fochon Pharmaceuticals

In 2021, Fochon Pharmaceuticals published a patent (WO2021/223736) where hundreds of BCL-2 inhibitors with the general structure E were disclosed (Figure 7) [53]. The compounds were screened for their cytotoxicity in RS4;11, DoHH2, and Toledo cell lines. To test activities against drug-resistant BCL-2 mutations, selected compounds were tested in RS4;11 cells with BCL-2-G101V or BCL-2-F104V mutant stably expressed. A number of compounds exhibited potent activity against all these cell lines. Compounds 9 and 10 are two representative molecules (Figure 7). The company’s clinical stage BCL-2 inhibitor FCN-338 has been licensed to Eli Lilly and currently in Phase 1 clinical trials as LOXO-338 (Table 1).

Figure 7.

Structures of general formula E and compounds 9 and 10 (Fochon Pharmaceuticals)

2.6. Chia Tai Tianqing Pharmaceutical Group

Patent WO2020/238785 filed by Chia Tai Tianqing Pharmaceutical published a series of venetoclax analogues with the general structure F (Figure 8) [54]. A representative compound 11 inhibited BCL-2 with an IC₅₀ of 1.49 nM and RS4;11 cell growth with an IC₅₀ of 1.9 nM. Compound 11 was also effective in inhibiting tumor growth in a RS4;11 xenograft mouse model.

Figure 8.

Structures of general formula F and inhibitor 11 (Chia Tai Tianqing)

2.7. Shenzhen TargetRx

In 2019, Shenzhen TargetRx published a series of “deuterated” venetoclax analogues with various degree of deuteration on venetoclax in a patent disclosure (WO2019/001383) [55]. As shown in the general formula G (Figure 9), the deuterium replacement can be anywhere on venetoclax. All the listed compounds showed similar target binding (BCL-2/BCL-XL) and cellular (RS4;11) activity to venetoclax. The representative compound 12 displayed higher stability in an in vitro assay using mouse liver microsome compared to venetoclax (t_1/2: 307 min for 12, 81 min for venetoclax; Cl_int 4.5 μL/min/mg for 12, 17.1 μL/min/mg for venetoclax). Compared to venetoclax, compound 12 also showed lower clearance in a PK study in rats, resulting a ~1.5-fold increase of plasma drug concentration.

Figure 9.

Structures of general formula G and compound 12 (Shenzhen TargetRx)

2.8. Guangzhou Lupeng Pharmaceutical & Newwave Pharmaceutical

In 2017, Newwave pharmaceutical published a patent (WO2017/132474) where 43 BCL-2 inhibitors were disclosed with the general formula H (Figure 10) [56]. Binding affinities against BCL-2 were tested for selected compounds through an FP assay. One of the most potent compounds 13 was found to have potent BCL-2 binding with an IC₅₀ of < 10 nM. The same compound exhibited an IC₅₀ of < 15 nM in inhibiting RS4;11 cell growth whereas the IC₅₀ against BCL-XL/BCL-2 dual dependent H146 cells was found to be > 2000 nM, which indicates that the compound is BCL-2 selective. In two follow-up patents (WO2020/041406 and WO2020/140005), additional compounds were disclosed [57,58]. One of the most potent compounds 14 (Figure 10) in WO2020/140005 exhibited an IC₅₀ of 0.45 nM in BCL-2 binding affinity. One representative compound 15 (Figure 10) from patent WO2020/041406 exhibited an IC₅₀ of 0.3 nM against RS4;11 cells along with an IC₅₀ of 0.01 nM in BCL-2 binding affinity.

Figure 10.

Structures of general formula H and compounds 13, 14, and 15 (Guangzhou Lupeng)

3. Patent review of small-molecule MCL-1 inhibitors

The importance of MCL-1 in promoting cancer cell survival and drug resistance to chemo-/radio-therapies, targeted therapies, and BCL-2/BCL-XL inhibitors is well-documented [59–70], highlighting the potential of targeting MCL-1 as an effective cancer therapeutic strategy. However, similar to targeting BCL-2/BCL-XL, the long and shallow BH3-binding pocket on the surface of MCL-1, as well as the strong PPI between MCL-1 and the BH3 motifs of the pro-apoptotic BCL-2 proteins, has made the development of potent and selective MCL-1 inhibitors with suitable drug-like properties highly challenging [17,71]. Similar to the development of BCL-2/BCL-XL inhibitors, initial discovery of BH3 mimetic MCL-1 inhibitors were also conducted at Abbott Laboratories/Abbvie with the first patent publication in 2007 (WO2007/008627) [72,73]. Abbvie subsequently reported A1210477 (Figure 11), a highly potent MCL-1 inhibitor (Ki = 0.45 nM, TR-FRET) with > 280-fold selectivity over BCL-2 and BCL-XL [74,75]. However, S63845 (Figure 11), developed by Servier and Vernalis, is the first reported MCL-1 inhibitor with efficacy in tumor models [76]. S63845 has high binding affinity and selectivity for MCL-1 (Kd = 0.19 nM, SPR) over BCL-2 and BCL-XL (Ki > 10 μM, FP). In the last five years, many MCL-1 inhibitors have been reported and many of them have been tested in clinical trials (Table 1). Notable ones include VU661013 [77–79], AMG-176 [80], AMG-397 [81], AZD5991 [82,83], S64315/MIK665 [84,85], PRT1419 [86], ABBV-467, and GS-9716. Detailed analysis of these compounds can be found in some recent review articles [17,71,87]. Associated with these preclinical and clinical drug candidates are tremendous amount of activities in the patent literature over the last decade, which have been comprehensively reviewed in 2017 and 2019 [88,89]. This review serves as a follow-up with a focus on recent patent publications. We also reanalyze some patents since 2016 in connection with some of the recently disclosed structures of drug candidates.

Figure 11.

Structures of A1210477, S63845, and VU661013

3.1. Les Laboratories Servier & Vernalis (R&D) Limited

Clinical stage drug candidate S64315/MIK665 (Table 1) (NCT04629443, NCT03672695, NCT04702425), co-developed by Novartis and Servier, was originally disclosed in a patent (WO2015/097123) published in 2015 by Les Laboratories Servier & Vernalis (R&D) [90]. Over 800 compounds were disclosed with the general formula I in this patent (Figure 12). In 2016, the company published four more patents covering similar chemical spaces [91–94]. As reported in a follow-up publication [85], S64315 was developed through structure guided optimization of lead compound 17 [84], which in turn was derived from fragment screening hit 16 (Figure 12) [95]. S64315 exhibited high binding affinity to MCL-1 with a Ki of 0.029 nM and high cytotoxicity against H929 cells with an IC₅₀ of 1.7 nM in the presence of 10% FCS, and achieved tumor regression in MCL-1 dependent cancer models. Modifications on the 4-fluorophenyl group of S64315, such as replacing it with a 3-chlorophenyl group (compound 18, Figure 12), were found to be well-tolerated. The co-crystal structure of 18 in complex with MCL-1 (PDB code: 6YBG) revealed an induced binding pocket by the phenyl substituent on the thiophene ring that can tolerate bigger substituents (Figure 12) [85].

Figure 12.

Structures of general formula I, MIK665, compounds 16, 17 and 18, and co-crystal structure of 18 in complex with MCL-1 (Les Laboratories Servier & Vernalis)

3.2. Abbvie

In 2019, Abbvie published a series of four patents (WO2019/035899, WO2019/035911, WO2019/035914, and WO2019/035927) that cover compounds based on the general formula J (Figure 13) [96–99]. Totally 540 macrocyclic MCL-1 inhibitors were disclosed. MCL-1 binding affinities were determined via a TR-FRET assay with or without 10% human serum (HS). Many compounds showed binding affinities at picomolar levels. Cell viability was tested in AMO-1 and H929 cell lines. Many compounds exhibited sub-nanomolar IC₅₀. In vivo efficacy studies of selected compounds were performed in an AMO-1 xenograft model. For example, TGI_max % (maximum tumor growth inhibition) and TGD % (tumor growth delay) were found to be 99% and 235% with 25 mg/kg/day IP dosing of compound 19. As indicated in the co-crystal structure of 18:MCL-1 (Figure 13), design of 19 appears to be inspired by the Servier compounds S63845/S64315, where two ends of the molecule were cleverly connected to form a seventeen-membered ring. Patent WO2019/035927 focused on introducing polyoxygenated moiety to the previous compounds [99]. One of the most potent compounds 20 (Figure 13) had improved MCL-1 binding affinity and cellular activity but equally effective in inhibiting tumor growth in the AMO-1 xenograft model when compared with 19. A similar series of analogues represented by compound 21 was disclosed in WO2019/035911 [97]. Although MCL-1 binding affinity and cellular activity of 21 was lower than those of 20, it achieved in vivo efficacy in the same tumor model at a lower dose (89% TGI with 6.25 mg/kg/day), indicating a better PK property.

Figure 13.

Structures of general formula J, MIK665, compounds 19, 20 and 21, and co-crystal structure of 18 in complex with MCL-1 (Abbvie)

3.3. Amgen

Amgen filed a series of patent applications (WO2016/033486, WO2017/147410, WO2018/183418, WO2019/046150, WO2019/036575, and WO2019/173181) [100–105] with the disclosure of thousands of compounds based on general formula K (Figure 14). Binding affinities of the compounds to MCL-1 were determined through a HTRF Bim displacement assay. Cell viability assay was determined in a human multiple myeloma cell line (OPM2) in a 10% FBS environment. AMG-176, the first MCL-1 inhibitor enter clinical trials, was disclosed in patent application WO2016/033486 [100], followed by a publication in Cancer Discovery in late 2018 [80]. AMG-176 exhibited picomolar binding affinity to MCL-1 (Ki = 80 pM), high selectivity over BCL-2 (> 11,000-fold) and BCL-XL (> 8,000-fold), good cellular activity (IC₅₀ = 240 nM, OPM2), and in vivo efficacy in multiple xenograft models [80]. AMG-176 has been in phase I clinical trial (NCT02675452) since 2016 for evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics in patients with relapsed or refractory multiple myeloma or relapsed or refractory acute myeloid leukemia. AMG-397, the second MCL-1 inhibitor from Amgen and the first orally administrated MCL-1 inhibitor entered clinical trial, was disclosed in patent application WO2018183418 [102]. Compared to AMG-176, AMG-397 exhibited improved binding affinity to MCL-1 (Ki = 12 pM) and cellular activity (IC₅₀ = 70 nM, OPM2). AMG-397 also displayed excellent in vivo efficacy with oral administration. For example, in an OPM-2 xenograft mouse model, 9 of 10 mice were tumor free at the end of the study with 50 mg/kg, once or twice weekly dosing of the drug [87]. However, clinical studies of AMG-397 has been terminated [106].

Figure 14.

Structures of general formula K, AMG-176, and AMG-397 (Amgen)

3.4. AstraZeneca

AstraZeneca’s AZD5991 has been in phase I clinical trial (NCT03218683) since 2017 for patients with hematological malignancies. The study is recently suspended, pending further evaluation of safety related information. The journey for the development of AZD5991 started with the investigation of two reported compounds 21 and 22 (Figure 15) by Abbott Laboratories (WO2008/131000) [107]. It was found that 21 bounds to the BH3 binding domain in MCL-1 in a 2:1 ratio when they form co-crystal structures (PDB: 6FS2) [82]. The first 21 molecule binds with higher binding affinity, with the 2-carboxylic acid group forming an ionic interaction with Arg263 of MCL-1 and the naphthyl group buried in an induced-fit pocket. The second 21 molecule remains in the proximity of the first molecule and binds with lower binding affinity. C6 of the 2-tolyl group of the first molecule is 3.5 Å away from the methyl group of the 2-tolyl group of the second molecule. Similar phenomenon was observed with 22. However, the MCL-1 binding affinity of the bis-compound 23, formed by connecting two molecules of 22 with a suitable linker, was lower than expected (IC₅₀ = 0.77 μM for 23 vs. 0.29 μM for 22, TR-FRET assay). En route to the synthesis of 23, compound 24 was isolated as a byproduct and found to have improved potency (IC₅₀ = 0.042 μM). Co-crystal structure of compound 24 with MCL-1 (PDB ID: 6FS1) revealed that the inhibitor remained in a “U” shape and the 5-methyl group on pyrazole is 3.6 Å away from C3 of the naphthyl group. These two positions were thus connected to generate compound 25 (WO2017/182625) [108]. R-isomer of 25, later named as AZD5991 and disclosed in patent application WO2018/178227 [109], was found to be more active than the S-form (IC₅₀ = 0.7 nM vs 6.3 μM for MCL-1 binding). In another patent (WO2018/178226) filed by the company in 2018, they disclosed 42 compounds with the general formula L (Figure 15) [110].

Figure 15.

Structures of general formula L, compounds 21, 22, 23, 24, and 25, AZD5991, and co-crystal structures of 21 and 24 in complexes MCL-1 (AstraZeneca)

3.5. Janssen Pharmaceuticals

Janssen disclosed a series of macrocyclic MCL-1 inhibitors in 2020 and 2021 (WO2020/254471, WO2021/165370, WO2021/099579, and WO2021/099580) based on two closely related general formulas M1 and M2 (Figure 16) [111–114]. These compounds are structurally similar to AstraZeneca’s compounds in Figure 15, especially with formula M2, while in formula M1, the ring is formed via connecting the nitrogen atom of the indole group and the pyrazole substituent is moved from C7 to C4. Activities of the compounds were determined using caspases 3/7 activity assay (Caspase-Glo) in MOLP8 cell line. One of the most active compounds from WO2020/254471 was 26 (Figure 16), which exhibit an AC₅₀ of 50 nM in the Caspase-Glo assay. A representative compound 27 in the follow up patent publication WO2021/165370 displayed increased potency in the same assay (AC₅₀ = 8.8 nM). Compound 27 also exhibited high binding affinity to MCL-1 with a Ki of 0.026 nM, determined by an HTRF assay using a BIM probe. One of the most potent compounds disclosed in WO2021/099580 was compound 28 (Figure 16), which exhibited a Ki of 0.04 nM but weak cellular activity with an AC₅₀ of 400 nM in the Caspase-Glo assay. MCL-1 binding affinity of most of the compounds disclosed in WO2021/099579 was improved from the previous patent. One representative potent example 29 (Figure 16) exhibited binding Ki of 0.03 nM and Caspase-Glo AC₅₀ of 23 nM. These compounds are pure atropisomers but their absolute stereochemistry was not determined in these patent disclosures.

Figure 16.

Structures of general formulas M1, M2, and N, and compounds 28, 29 and 30 (Janssen)

In another patent publication WO2021/005043, Janssen disclosed 42 macrocyclic MCL-1 inhibitors with general formula N (Figure 16) [115]. In these molecules, the cyclobutane ring as in Amgen’s compounds was replaced by an azetidine ring. Compound 30 was one of the most potent with a binding affinity Ki of 0.09 nM (HTRF) and AC₅₀ of 120 nm (Caspase-Glo). The structure of 30 is very similar to AMG-397 where the notable difference is the azetidine ring and a fluoro-substituent on the olefin (Figure 16).

3.6. Gilead Sciences

In 2019, Gilead Science published a patent (WO2019/222112) with the general formula O where 464 MCL-1 inhibitor examples were disclosed (Figure 17) [116]. As we have previously seen, the N-acyl sulfonamide group of AMG-176/AMG-397 plays a crucial role in binding through forming a salt bridge with Arg263 of MCL-1 (PDB:6UDV). In this patent, the sulfonamide group of the macrocyclic molecules was bioisosterically replaced with an N-acyl sulfonoimidamide. One of the most potent compounds 31 in this disclosure exhibited a Mcl-l binding affinity IC₅₀ of 0.024 nM in an AlphaLISA assay and a cellular EC₅₀ of 13.34 nM in SKBR3 cells in a cell viability assay. In a follow-up patent publication (WO2021/096860), they disclosed 172 MCL-1 inhibitor structures of general formula P (Figure 17) [117]. The most potent compound 32 exhibited a MCL-1 binding affinity IC₅₀ of 0.048 nM and a cellular EC₅₀ of 6.0 nM in SKBR3 Cells.

Figure 17.

Structures of general formulas O and P, and compounds 31 and 32 (Gilead)

3.7. Ascentage Pharma

Ascentage Pharma published a patent (WO2020/151738) in 2020 with the general formula Q where more than 100 macrocyclic MCL-1 inhibitor examples were disclosed (Figure 18) [118]. MCL-1 binding affinities of the compounds were determined through an FP assay and cellular activity was tested in OPM2 and H929 cell lines. One of the most potent compounds 33 exhibited a Ki of 5.5 nM in binding along with IC₅₀s of 48 nM and 23 nM in OPM2 and H929 cells, respectively. In a subsequent patent application (WO2020/147802) with the general formula R, they disclosed another series of macrocyclic MCL-1 inhibitors (Figure 18) [119]. Pharmacokinetic profile of seven selected compounds were compared with that of AMG-176. Compound 34 exhibited higher plasma drug concentration.

Figure 18.

Structures of general formulas Q and R, and compounds 33 and 34 (Ascentage Pharma)

3.8. Prelude Therapeutics

Prelude Therapeutics published two patent applications in 2020. In one patent (WO2020/123994) they disclosed several chemical entities as MCL-1 inhibitors with the general formula S (Figure 19) [120]. One of the most potent compounds 35 exhibited a Ki of 0.7 nM in a cell free MCL-1/Bim displacement assay. The thieno[2,3-d]pyrimidine core of S64315 was replaced with a pyrrolo[2,1-f][1,2,4]triazine to afford 35. The other patent by the company (WO2020/097577) disclosed 315 macrocyclic MCL-1 inhibitors with the general formula T (Figure 19) [121]. Several compounds exhibited Ki < 1 nM in binding affinity for MCL-1 and IC₅₀ < 500 nM in inhibiting H929 cell viability.

Figure 19.

Structures of general formulas S and T, and compounds 35 and 36 (Prelude Therapeutics)

3.9. California Institute of Technology

A patent (WO2021/067827) disclosed by California Institute of Technology included 126 examples with the general formula U (Figure 20) [122]. The thieno[2,3-d]pyrimidine core of S64315 was replaced with an isothiazolo[5,4-c]pyridine to generate this series of compounds. Binding affinities for MCL-1, BCL-2, and BCL-XL were determined by a biotinylated peptide ligand displacement assay. Cell growth inhibition assay was performed on two groups of cell lines, “sensitive” and “resistant”, categorized based on whether their growth was inhibited by AMG-176 or MIK665. Two representative compounds 37 and 38 (Figure 20) exhibited good potency and selectivity in binding to MCL-1 and were potent against “sensitive” cells. However, no quantitative data were given in the patent application.

Figure 20.

Structures of general formula U and compounds 37 and 38 (California Institute of Technology)

3.10. Others

Zeno Management published two patents (WO2020/185606 and WO2021/126316) with the general formula V where around 80 macrocyclic MCL-1 inhibitors were disclosed (Figure 21) [123,124]. These compounds are structurally similar to AZD5991. Several compounds, such as 39 and 40 (Figure 21), in these patents exhibited IC₅₀ < 10 nM in MCL-1 binding based on an HTRF assay along with IC₅₀ < 100 nM in a CellTiter-Glo luminescent cell viability assay in H929 cells. Similar compounds have also been disclosed by Unity Biotechnology (WO2021/092053) with the general formula W [125] and Shanghai Hengrui (WO2020/063792) with the general formula X [126] (Figure 21). In Unity’s patent, several compounds exhibited Ki < 10 pM binding affinity for MCL-1 and over > 10,000-fold selectivity over BCL-XL and BCL-2 based on an AlphaLISA assay. One of the most potent compounds 42 in Hengrui’s patent exhibited a Ki of 0.16 nM in a Bim displacement assay and potent cell growth inhibition in both AMO-1 and MV-4-11 cell lines (IC₅₀ = 26 nM for both).

Figure 21.

Structures of general formulas V, W, and X, and compounds 39, 40, 41, and 42

4. Patent review of PROTACs targeting BCL-2 family proteins

Conceptualized by the Crews and Deshaies laboratories in 2001 [127], targeted protein degradation (TPD) by PROTACs has evolved into a paradigm shifting technology in drug discovery and development [128,129]. PROTACs are heterobifunctional molecules consist of three parts: an E3-binding ubiquitin ligase recruiting ligand, a ligand that binds to the target protein of interest (POI), and a linker unit that connects the two. PROTACs recruit the POI to the E3 ligase complex, promote proximity-induced ubiquitination and subsequent proteasomal degradation of the POI. The PROTAC molecule then becomes available for the next rounds of action (Figure 22). Given the catalytic nature of their mode of action, PROTACs offer a variety of potential advantages over SMIs [128,129], notably the more pronounced and longer-lasting disruption of protein function and PK-PD decoupling---in vivo protein degradation extends beyond the pharmacokinetic profile. PROTACs also have the ability to address both the enzymatic and non-enzymatic functions of proteins because of the complete target removal. For proteins that lack a functional binding site, PROTACs can be built on non-functional binders for effective degradation, which expands the “druggable” target space. In addition, it has also been demonstrated that target depletion selectivity by PROTACs can substantially exceed the binding selectivity of their corresponding parent target protein ligands. Moreover, because PROTACs rely on E3 ligases to induce protein degradation, it is possible for them to achieve cell type/tissue selectivity if they recruit an E3 ligase that is differentially expressed [130]. A variety of POIs have been successfully targeted by PROTACs and to date, more than a dozen PROTAC molecule have entered clinical trials, highlighting the promise of this technology [24]. Associated with these drug development activities are a high volume of patent disclosures in the PROTAC field in the last five years. A recent review by Benowitz et al had an extensive coverage on the patent space of PROTACs [131]. This section of the review will focus on patent filings that cover PROTACs targeting BCL-2 family of proteins.

Figure 22.

Mechanism of PROTAC mediated protein degradation

4.1. BioVentures, LLC and Dialectic Therapeutics

As mentioned above, BCL-XL is also a well-validated cancer target but the clinical application of BCL-XL inhibitors is limited by their on-target platelet toxicity. BCL-XL is the most common BCL-2 family member overexpressed in solid tumors, as well as in a fraction of leukemia and lymphoma cells [132]. Bioinformatics analyses also find a strong correlation between the levels of BCL-XL expression and resistance to a panel of standard chemotherapeutic drugs in 60 NCI tumor cell lines [133]. Highly potent and selective BCL-XL inhibitors, such as A-115546317 and its orally active analogue A-1331852 (Figure 23) have been developed; however, they can only be used as tool molecules due to the on-target thrombocytopenia toxicity [134–136]. BioVentures of the University of Arkansas for Medical Sciences disclosed two patents (WO2017/184995 and WO2019/144117) where various BCL-XL/BCL-2 selective or dual inhibitors were used as “warheads” to generate CRBN- or VHL-recruiting PROTACs [137,138]. Because both CRBN and VHL E3 ligases are minimally expressed in platelets, these PROTACs selectively inducing BCL-XL degradation in normal cells, senescent cells, and cancer cells but not in platelets, resulting in decreased toxicity to platelets [23,139–142]. Compound 43 (Figure 23) is a representative PROTAC in WO2017/184995. As shown in a follow-up publication, 43 exhibited a 22-fold increase in selectivity for MOLT-4 cells (BCL-XL-dependent) over human platelets compared to its warhead A-1155463. DT2216, a clinical stage drug candidate (Table 1), is a representative compound of the general formula Y (Figure 23) disclosed in WO2019/144117. DT2216 binds to both BCL-XL and BCL-2 (Ki = 12.82 nM and 1.82 nM, respectively); However, it only degrades BCL-XL based on western blot and proteomics studies [23]. Most significantly, DT2216 exhibited > 190-fold selectivity for MOLT-4 over human platelets and was much safer and effective than ABT-263 in several xenograft models [23].

Figure 23.

Structures of general formula Y, A-1155463, A-1331852, and PROTACs 43 and DT2216 (Dialectic Therapeutics)

4.2. University of Florida Research Foundation

In 2020, a patent (WO2020/163823) filed by the University of Florida disclosed a series of BCL-XL/BCL-2 targeting PROTACs [143]. Instead of attaching the linker to the solvent exposed region that exits from the P4 binding pocket of BCL-XL/BCL-2 as in previous patent filings, these new PROTACs utilized the attaching point on BCL-XL/BCL-2 ligands that sits outside of the P2 binding pocket. Cell viability of all the compounds were tested in BCL-XL dependent MOLT-4 and BCL-2 dependent RS4;11 cell lines. Several compounds exhibited IC₅₀ < 50 nM in both cell lines. More details of these compounds were published in two follow-up papers [144,145]. Most significantly, compound 44 (753b) (Figure 24) was found to potently degrade both BCL-XL and BCL-2, thus could potentially have broader anti-cancer spectrum compared to DT2216 [145]. In another patent disclosure (WO2021/146536), the carboxylic acid and the adamantane group of A-1331852 (Figure 23) were utilized as linker tethering points for the synthesis of PROTACs [146]. Both VHL and CRBN E3 ligands were used. Degraders were categorized according to their MOLT-4 potency and 14 degraders had EC₅₀ < 10 nM. One of the most potent PROTAC 45 is displayed in Figure 24.

Figure 24.

Structures of PROTACs 44 and 45 (University of Florida)

4.3. Recurium IP Holdings

In 2021, Recurium IP Holdings published two patents (WO2021/007307 and WO2021/222114) disclosed a series of BCL-XL PROTACs with the general formula Z (Figure 25) [147,148]. The chlorophenyl group of ABT-263 was replaced with a substituted bicyclo[1.1.1]pentanyl group and the resulting inhibitors were used as the warheads for the synthesis of the PROTACs. VHL and CRBN ligands were used along with different types of linkers for construct the PROTACs. Degradation of the selected PROTACs were tested in MOLT-4 cells. Two most potent compounds 46 and 47 (Figure 25) exhibited concentration dependent BCL-XL degradation. Almost complete degradation was found at 110 nM for both the compounds.

Figure 25.

Structures of general formula Z and PROTACs 46 and 47 (Recurium)

4.4. Shanghai Tech University and Fudan University

In a 2021 patent publication (WO2021/078301) by a team at Shanghai Tech University and Fudan University, hundreds of PROTACs were disclosed [149]. These PROTACs were derived from ABT-199, ABT-163, and ABT-199 analogue 48 (Figure 26). A number of analogues exhibited DC₅₀ < 10 nM in inducing BCL-XL degradation. Surprisingly, despite the weak binding to MCL-1 (Ki > 1,000 nM), many PROTACs potently induced MCL-1 degradation. Near complete MCL-1 degradation was observed for a number of PROTACs at 10 nM. However, it was not confirmed whether the MCL-1 degradation is a direct effect. It is also worth to note that MCL-1 is short lived with a half-life of < 1 hour in many cell types and its stability can be affected by multiple kinases/phosphatases [150]. In addition, the stability of MCL-1 can also be regulated by BH3-only protein Noxa through its C-terminal tail that contains a degron sequence [151]. One of the most potent PROTACs 49 (Figure 26) exhibited higher potency in various cell viability assays when compared with ABT-199 (IC₅₀ = 0.36 vs 7.79 nM in RS4;11; 2.18 vs 20.57 nM in MV4;11; 26.77 vs 6670 nM in MM1S; 6.14 vs 140 nM in Shh Light II cells).

Figure 26.

Structures of ABT-199, ABT-263, compound 48, and PROTAC 49 (Shanghai Tech University and Fudan University)

4.5. Others

Newwave Pharmaceutical published a patent (WO2021/066873) in 2021 where a number of BCL-2 inhibitor-based PROTACs were disclosed [152]. One representative VHL-recruiting PROTAC 50 (Figure 27) was selected for studies in a RS4;11 xenograft model but no data were given. A group in France disclosed a patent (WO2021/180966) that covers a series of MCL-1 PROTACs based on a MCL-1 inhibitor pyridoclax [153,154]. A representative compound 51 (Figure 27) exhibited moderate MCL-1 degradation at 1 μM. A group from Dalian University of Technology disclosed a patent (CN107382862) on a series of PROTACs molecules base on 1H-benco[de]isoquinoline-1,3(2H)-dione [155]. Two lead PROTACs, 52 and 53 (Figure 27) were reported in a follow-up publication by the same group [156]. Despite the highly similar structures, 52 selectively degrades MCL-1 (DC₅₀ = 0.7 μM) and 53 selectively degrades BCL-2 (DC₅₀ = 3.0 μM). However, it should be noted that the mechanism of action of the ligands for 51-53 has not been thoroughly validated; thus the degradation by these compounds might be the result of off-target effects.

Figure 27.

Structures of PROTACs 50, 51, 52, 52, and dMCL1-2

5. Expert Opinion

Restoring apoptosis in cancer cells is an important strategy of treating cancer and overcoming drug resistance to various cancer therapies. Development of BH3 mimetic SMIs, that can block the PPIs between pro-apoptotic and anti-apoptotic BCL-2 family proteins thus activate the intrinsic apoptotic pathway in cancer cells, has been the major focus in pharmaceutical companies and academic labs in the last two decades. Due to the long and shallow surface of the BH3-binding groove of anti-apoptotic proteins BCL-2, BCL-XL, and MCL-1, as well as the high affinity of the native PPIs, the development of highly potent, selective, and drug-like SMIs for these proteins has been challenging. A breakthrough in the field was made by Abbott Laboratories (now known as Abbvie), who reported ABT-737, the first highly potent SMI of BCL-2 proteins discovered through an innovative NMR-based fragment screening coupled with structure guided optimization approach [10]. Continued effort by the company eventually led to the FDA approval of BCL-2 selective inhibitor venetoclax [21], which ultimately validated the strategy of targeting the anti-apoptotic BCL-2 family of proteins.

The impressive clinical success of venetoclax in leukemia treatment has triggered many follow-up drug discovery and development activities, resulting in a large number of patent disclosures and advancement of nine BCL-2 selective inhibitors into clinical trials. Despite its high molecular weight and very poor water solubility, venetoclax is orally bioavailable, which reflects the tremendous amount of medicinal chemistry efforts required to achieve these reasonable drug-like properties. However, the venetoclax oral tablets require a special formulation and have limited drug loading, a significant problem with patients who need high doses. To overcome this issue, AbbVie developed a phosphate prodrug of venetoclax, ABBV-167, which has significantly increased water solubility, reduced food effect, and enhanced bioavailability in healthy volunteers compared to venetoclax [157]. The issue may not be as big with some of the newly disclosed BCL-2 inhibitors that have improved PK properties and/or increased potency, which will lower the doses needed for the therapeutic effects.

Same as other targeted therapies, acquired drug resistance was observed after prolonged treatment with venetoclax [43,44]. Point mutations, including G101V and D103Y, that significantly reduce the affinity of BCL-2 for venetoclax, are associated with the drug resistance. As disclosed by BeiGene [50], newer BCL-2 inhibitors have been designed to counter these mutations. These inhibitors could be developed to treat ALL/AML patients resistant to venetoclax. Acquired resistance to venetoclax could also be caused by up regulation of other two major anti-apoptotic BCL-2 family proteins, BCL-XL and MCL-1 [158]. In addition, many cancers intrinsically resistant to venetoclax monotherapy because of their dependency/co-dependency on BCL-XL and/or MCL-1, especially in solid tumors [132,133]. Therefore, as reflected by the activities in patent filings and clinical trials, it is highly desirable to develop BCL-XL and MCL-1 inhibitors to significantly expand the clinical opportunity in cancer treatment.

Remarkable efforts have been made in the development of MCL-1 inhibitors, resulting in advancement of seven drug candidates into clinical trials (Table 1). However, several clinical trials have been suspended or terminated due to “safety signal for cardiac toxicity” or “strategic decision”. The observed clinical cardiac events are likely an on-target toxicity as conditional MCL-1 knockout in mice led to lethal cardiac failure [159,160]. Therefore, all the MCL-1 inhibitors currently under development could have limited therapeutic window. Data from the currently ongoing clinical trials will reveal more on the fate of this class of drugs. Drug combinations of MCL-1 inhibitors with targeted therapies or chemotherapies may improve safety and efficacy as they may reduce the dose of MCL-1 inhibitors. Novartis disclosed an alternative approach in two patents (WO2020/236817 and WO2020/236825) that cover MCL-1 inhibitor-based ADCs [161,162]. As these ADCs utilize antibodies that targeting antigens on tumors or cancer cells, they would likely have reduced toxicity to normal tissues. Recent reports, such as on compound 52 [156] and dMCL1-2 [163] (Figure 27), also demonstrated the degradability of Mcl-1 through the PROTAC technology. As shown in DT2216, it is possible to reduce cardiac toxicity if Mcl-1 PROTACs can be constructed to recruit E3 ligases that are selectively expressed in cancer cells.

The development of BCL-XL selective or BCL-2/BCL-XL dual inhibitors has been hampered by the on-target, dose-limiting platelet toxicity, which unlikely be overcome by optimizing BH3 mimetic SMIs. Several strategies have been implemented to improve the therapeutic window and are currently being tested in clinical studies. APG-1252 (Table 1) is a prodrug derived from a BCL-2/BCL-XL dual inhibitor and designed to minimize the drug exposure to platelets. The compound is currently being developed by Ascentage Pharma to treat patients with small cell lung cancer (SCLC) and other solid tumors [164]. Utilizing a dendrimer-based delivery technology, AstraZeneca has developed AZD-0466 for clinical studies [165]. The drug candidate has been shown to have an improved therapeutic index. In addition, AbbVie is developing ABBV-155, an ADC that is based on a selective BCL-XL inhibitor and targets B7H3 expressing cancer cells, for the treatment of advanced solid tumors [22]. And lastly, an innovative utilization of the emerging PROTAC technology to differentially degrade BCL-XL in cancer cells and platelets [23]. This strategy has also resulted in a clinical-stage drug candidate DT2216.

In conclusion, significant advancements have been made in the last five year in the development of molecules that target three major anti-apoptotic BCL-2 proteins, BCL-2, BCL-XL, and MCL-1. The impressive progress in this field is largely benefited from the implementation of a number of innovative drug discovery technologies developed in the past two decades, including fragment-based drug discovery, ADC, and PROTAC. Having so many clinical-stage drug candidates with diverse profiles in the pipeline, we envision near future success of the development of new drugs targeting the intrinsic apoptotic pathway, which will certainly expand valuable treatment options for cancer patients.

Article highlights.

• Brief discussion on the importance of targeting anti-apoptotic BCL-2 proteins as an anti-cancer strategy.

• Brief summary of the clinical-stage drug candidates that target three major anti-apoptotic BCL-2 proteins, BCL-2, BCL-XL, and MCL-1.

• Comprehensive patent analyses on the development of the small molecule inhibitors and PROTAC degraders of BCL-XL, BCL-2, and MCL-1 in the last five years.

• Brief discussion on the various drug discovery technologies implemented in these patent disclosures.

• Brief discussion on the issues facing each target and the potential solutions.