Bcl-2 Antagonists: A Proof of Concept for CLL Therapy

Abstract

Defective apoptosis is a fundamental hallmark feature of CLL biology and is a major target of cancer therapy development. High levels of Bcl-2 family anti-apoptotic proteins are considered primarily responsible for inhibiting apoptosis in CLL cells. While several approaches were considered to selectively inhibit Bcl-2 family anti-apoptotic proteins, the discovery that gossypol binds and antagonizes anti-apoptotic effect of Bcl-2 family proteins was a major breakthrough in identifying specific Bcl-2 antagonists. The concept of mimicking BH3 domain emphasized the importance of Bcl-2 family-targeted therapy that can modulate the function of anti-apoptotic proteins. Although parent compound gossypol did not sustain in the clinic, its structural modifications led to the development of additional analogues that demonstrated improved efficacy and reduced toxicity in preclinical and clinical investigations. Proof of concept of this hypothesis was demonstrated by structure based BH3 mimetic ABT-737 that has shown greater cytotoxicity towards CLL cells both in pre-clinical models and clinical trials. Its oral compound ABT-263 has demonstrated the substantial susceptibility of chronic lymphocytic leukemia cells through Bcl-2 inhibition. Collectively, results of a Phase I Study of Navitoclax (ABT-263) in patients with relapsed or refractory disease warrants Bcl-2 as a valid therapeutic target in CLL. Importantly, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Mcl-1, Bcl-2 and Bcl-XL.

Introduction

For several decades, plant products have been an excellent source of anti-cancer agents. They have been in use as a single agent or in combination with other chemotherapeutic drugs for the treatment of cancers, including liquid and solid tumors (as reviewed in [1]). Vinca alkaloids such as vinblastine and vincristine obtained from plant Madagascar periwinkle, Catharanthus roseus (formerly known as Vinca rosea, Apocynaceae) were the first natural products advanced into clinical use for the treatment of cancer [1]. The isolation of paclitaxel (Taxol) from the bark of the Pacific Yew tree, Taxusbrevifolia Nutt belonging to the family Taxaceae, is another evidence of the success in natural product drug discovery [2, 3]. Camptothecin, isolated from the Chinese ornamental tree Camptotheca acuminate Decne (Nyssaceae) known in China as tree of joy, was advanced to clinical trials by NCI, but was dropped because of severe bladder toxicity. Etoposide and teniposide are two semi-synthetic derivatives of epipodophyllotoxin, an isomer of podophyllotoxin isolated from the roots of Podophyllum species, Podophyllum peltatum Linnaeus and Podophyllumemodi Wallich (Berberidaceae) [4] and are used in the treatment of lymphomas and other cancers [5]. Homoharringtonine obtained from the Chinese tree Cephalotaxus harringtonia var. drupacea (Sieb and Zucc.) (Cephalotaxaceae), is another plant-derived product in clinical use [6]. A racemic mixture of harringtonine and homoharringtonine has been used successfully for the treatment of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) [7]. Flavopiridol is a synthetic flavone, derived from the plant alkaloid rohitukine, which was isolated from Dysoxylum binectariferum Hook. f. (Meliaceae)[8] and tested in phase I and II clinical trials against a broad range of tumors [9]. Synthetic agent roscovitine which is derived from natural product olomucine, originally isolated from Raphanus sativus L. (Brassicaceae), is in Phase II clinical trials in Europe [10]. Combretastatin A-4 isolated from the bark of the South African tree Combretum caffrum (Eckl. &Zeyh.) Kuntze (Combretaceae) [11], is active against solid and hematological malignancies. Together, natural products have proven useful by themselves as anti-cancer agents or have been a great source of synthetic or semisynthetic derivatives for preclinical investigations and/or clinical trials.

Cotton plant and gossypol

Gossypol is a polyphenolic aldehyde derived from the cotton plant (Gossypium hirsutum L. Family Malvaceae, Fig. 1). It was originally discovered by Longmore and was later structurally elucidated by Adams and Edwards [12, 13]. Chemically it is 2-2′ bis(formyl-1,6,7-trihydroxy-5-isopropyl-3-methyl)-naphathalene. Gossypol inherently displayed a broad spectrum of physiochemical and biological properties such as insecticidal activity, anti-oxidant property, anti-fertility property and anti-cancer activity [14–16]. It also exhibited cytotoxic effect against various carcinoma cell lines both in vitro and in vivo settings [17–20]. Extensive investigations on gossypol had revealed its diversified mechanisms of action, which include inhibitory role on enzyme LDH [21], protein kinase C activity [22], DNA synthesis inhibition [23], regulation on cell cycle proteins Rb and cyclin D1 [24], cellular proliferation [25], ROS independent mitochondrial pathway of apoptosis [26], execution of extrinsic cell death pathway through up-regulation of Fas/Fas ligand [27], Bax or Bax/Bak independent activation of apoptosis [28], suppression of NF-κB activity [29] and induction of autophagy [30, 31]. In early clinical trials, racemic gossypol administration to patients with various cancers demonstrated that gossypol was well tolerated with minimal clinical efficacy [32–34].

Figure 1.

Cotton plant photographs; obtained from online website.

Gossypol as a BH3 mimetic

Over-expression of anti-apoptotic B-cell lymphocyte/leukemia-2 (Bcl-2) family proteins is common in many human cancers and is a major target of cancer therapy development [35]. Besides all the investigations on gossypol, the discovery that gossypol binds and antagonizes anti-apoptotic effect of Bcl-2 family proteins and induces apoptosis in cancer cells was a major breakthrough in modulating the function of Bcl-2 [36]. On the basis of in vitro displacement assays with the fluorescein-labeled BH3 peptide, Kitada et al demonstrated that gossypol directly interacts with Bcl-X_L and is able to displace BH3 peptides with an IC₅₀ of 0.5 μM [36] (Fig. 2). Given that Bcl-X_L is highly expressed in several hematological malignancies, gossypol was able to overcome the apoptotic resistance mediated by Bcl-X_L in CML [37] as well as in CLL. In vitro study on primary CLL lymphocytes demonstrated that gossypol at micromolar levels induced caspase independent, AIF-mediated apoptosis in all samples tested irrespective of the disease stage or prognostic markers (Fig. 3A)[38]. Further investigations illustrated that gossypol is the only compound that inhibited major anti-apoptotic proteins such as Bcl-2, Bcl-X_L, Mcl-1, Bcl-B with IC₅₀ values of 0.28, 3.03, 1.75, 0.36 respectively, as well as Bcl-W (1.4 μM) whose prime function is to sustain developing sperm cells [39–43].

Figure 2.

Surface representation of Bcl-x_L with the docked structure of Gossypol obtained by FlexX. The surface is depicted according to cavity depth (blue, surface exposed; yellow, buried) representation. Reprinted (adapted) with permission from (J. Med. Chem., 2003, 46 (20), pp 4259–4264). Copyright (2003) American Chemical Society.

Figure 3.

A. Percentage of cell death in CLL primary cells from 9 individual patients treated with gossypol for the indicated time. This research was originally published in Blood. Balakrishnan et al. Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells, Blood, 2008; 112(5):1971–80.© The American Society of Hematology. B. AT-101 circumvents the stromal mediated CLL cell survival. CLL lymphocytes from patients (n = 12) were cultured either in suspension medium (C) in suspension medium with 20 μM AT-101 (C + AT), or with stromal cells in the absence (C + S) or presence of AT-101 (C + S + AT) and the apoptosis was measured after 24 hours (72 hours for samples from patients 31 and 32) by annexin-binding assay. This research was originally published in Blood. Balakrishnan et al. AT-101 induces apoptosis in CLL B cells and overcomes stromal cell-mediated Mcl-1 induction and drug resistance. Blood, 2009;113(1):149–53.© The American Society of Hematology.

Despite the broad spectrum of functionalities, toxic properties associated with hypokalemia and its effect on Bcl-W leading to male infertility limited gossypol’s application in clinical use. Though gossypol is unlikely candidate for clinic, it represented a lead compound for generation of a new class of antineoplastic agents [44]. Several efforts were undertaken to detoxify the compound by altering its structure to produce analogues with improved efficacy and/or reduced toxicity. Structural modification of gossypol guided by a model of multidimensional nuclear magnetic resonance based structural analysis led to the development of additional analogues of gossypol with improved efficacy. The rest of this review is devoted to discuss various gossypol derivatives and their efficacy in preclinical and clinical investigations.

AT-101

Gossypol naturally exists as a mixture of two enantiomers (+) and (−) that exhibit different biological activity. It was notable that (−)-gossypol also called AT-101, developed by Ascenta Pharmaceuticals, was approximately twice as active as racemic gossypol with added oral bio-availability. Similar to gossypol, AT-101 contains two reactive aldehyde groups and also binds to the BH3 motif of all major anti-apoptotic proteins, with better affinity (e.g., 0.32, 0.48, and 0.18 μM for Bcl-2, Bcl-X_L, and Mcl-1, respectively) [45]. Studies evaluating the therapeutic response of AT-101 with respect to the effect of protective stromal cells demonstrated that AT-101 can completely overcome stroma mediated protectivity in CLL primary cells (Fig. 3B) [46].

There was a differential mechanism for survival advantage provided by two distinct microenvironments; enhanced cell survival was mediated by Mcl-1 protein induction in bone marrow microenvironment, while nurse like cells (a representative lymph node microenvironment) protected CLL cells via augmentation of Bfl-1 protein [46–49]. Targeting Bcl-2 family proteins with AT-101 markedly enhanced the therapeutic effects of several chemotherapeutic agents such as cyclophosphamide and rituximab both in vitro and in vivo models of B-cell lymphomas [50]. A recent report states that AT-101 not only triggers Bax activation but also induces mitochondrial SMAC release to enhance Bax-mediated cellular apoptosis [51]. In clinical study of AT-101 with topotecan in relapsed and refractory small-cell lung cancer [52] was not active but showed promise in the double blind placebo controlled randomized phase II study of AT-101 plus docetaxel in non-small cell lung carcinoma patients [53].

Apogossypol

Given that gossypol and AT-101 have toxicity problems likely due to two reactive aldehyde groups at 8, 8′-positions on the naphthalene rings, a semi-synthetic derivative apogossypol, was synthesized lacking two aldehyde groups, with enhanced activity and reduced toxicity [41, 54, 55]. Apogossypol and gossypol are shown to exhibit similar oral and intravenous pharmacokinetic profiles as well as in vitro stability, although apogossypol demonstrated a slower clearance rate, larger AUC (area under curve), and better microsomal stability [55, 56] suggesting favorable pharmacokinetics for this analogue.

Because gossypol enantiomers displayed differential pro-apoptotic activities, atropisomers of apogossypol were synthesized, evaluated and compared with racemic apogossypol for cellular activity [54]. 5, 5′ substituted ketone and amide apogossypol derivatives such as BI-79D10, compound 8r and BI-97C1 (sabutoclax) were synthesized. Each compound was subsequently tested for its ability to inhibit Bcl-X_L in an in vitro fluorescence polarization competition assay as well as for its pro-apoptotic activity in human cancer cell lines. The potent compound BI-79D10 was shown to bind to Bcl-X_L, Bcl-2, and Mcl-1 with IC₅₀ values of 190, 360, and 520 nmol/L, respectively. It inhibited cell growth in the human lung cancer cell line with an EC₅₀ value of 680 nmol/Land induced apoptosis in human lymphoma cell line. This compound had improved plasma and microsomal stability relative to apogossypol and showed little cytotoxicity against Bax/Bak-/- mouse embryonic fibroblast cells [57]. Compound 8r inhibited the binding of BH3 peptides to Bcl-X_L, Bcl-2, Mcl-1, and Bfl-1 with IC₅₀ values of 0.76, 0.32, 0.28, and 0.73 μM, respectively. This compound also potently inhibited cell growth of human lung cancer and human B-cell lymphoma cell lines in vitro and displayed efficacy in transgenic mice in which Bcl-2 is overexpressed in splenic B-cells. Similar to BI-79D10, 84 is also stable and appears to be a promising drug lead [58].

A new analogue, BI-97C1, an optically pure and most potent diastereoisomer of compound 8r, inhibited the binding of BH3 peptides to Bcl-X_L, Bcl-2, Mcl-1, and Bfl-1 with IC₅₀ values of 0.31, 0.32, 0.20, and 0.62 μM, respectively. The compound potently inhibited cell growth of human prostate cancer, lung cancer, and lymphoma cell lines with EC₅₀ values of 0.13, 0.56, and 0.049 μM, respectively. This analog displayed in vivo efficacy in transgenic mice models and also demonstrated superior single-agent antitumor efficacy in a prostate cancer xenograft model. In prostate cancer cells, BI-97C1 (sabutoclax) sensitized MDA7/IL-24 mediated toxicity as well as exerted significantly improved therapeutic response in colorectal cancer patients in combination with AD5/3-MDA7 [59, 60]. Together, apogossypol and its analogues have demonstrated efficacy in preclinical studies, but they have not yet made to clinical trials.

Gossypolone and apogossypolone

Gossypolone, a major metabolite of AT-101, was synthesized without hydroxyl group upon oxidation reaction. It displayed similar cytotoxic effects as AT-101, with greater water solubility, lower toxicity with anti-proliferative activity against human cancer cell lines [61, 62]. Apogossypolone also known as ApoG2 is an analogue of gossypolone with three- to six-fold more potency than the parent compound (-)-gossypol. It was synthesized by the removal of hydroxyl and two reactive aldehyde groups to improve stability and reduce toxicity [63]. ApoG2 demonstrated a higher binding affinity to its targets (Ki, 35, 660, and 25 nM for Bcl-2, Bcl-X_L, and Mcl-1, respectively) [64] suggesting to have high inhibitory constants for Mcl-1 and Bcl-2, but not for Bcl-X_L and induced apoptosis in number of cancer cell lines by blocking binding of Bim and Bcl-2. Preclinical studies of apogossypolone in follicular lymphoma exhibited growth inhibitory effects in vitro and in vivo in SCID xenograft models through activation of intrinsic and extrinsic caspases and release of AIF [65, 66]. In CLL, Bax/Bak was required for apogossypolone induced cell death[67]. Head to head comparison of apogossypolone with gossypol revealed that ApoG2 was more stable and better tolerated by mice than was racemic gossypol, with no toxicity on peripheral blood lymphocytes [68]. ApoG2 has also been shown to potently disturb the proliferation of nasopharyngeal carcinoma cells by suppressing the c-Myc signaling pathway [69]. In vivo, it induced regression in several tumor xenograft models and its maximum tolerated dose (MTD) was comparatively higher than the MTD of AT-101. Given that 5,5′ substituted apogossypol derivatives displayed improved in vitro and in vivo activities compared to apogossypol, derivatives of 5,5′-substituted apogossypolone were synthesized by replacing their isopropyl groups with alkyl, ketone and amide groups, which resulted in compounds with improved biological activities [70].

Concept - to - clinic

The mechanistic approach of identifying novel agents to induce pan-inhibition of anti-apoptotic proteins opened a new avenue of successfully treating diseases that inherit high levels of Bcl-2 family proteins. Although natural product gossypol and its derivatives were not effective by themselves in the clinic, the concept of employing BH3 mimetics to modulate the function of Bcl-2 has been quite successful. Two novel Bcl-2 inhibitors obatoclax and ABT-737 have represented the proof-of- principle of this approach in clinic.

Obatoclax

Obatoclax (GX015-070; Geminx) is a hydrophobic molecule that was specifically designed to inhibit all relevant anti-apoptotic members of the Bcl-2 family (IC₅₀ of Bcl-2, Bcl-X_L, Mcl-1, was 1.11, 4.69, 2.9 μM respectively)[39]. Preclinical studies with single agent obatoclax demonstrated that GX015-070 antagonizes Mcl-1 and overcomes Mcl-1-mediated resistance to apoptosis [71]. Further studies revealed that obatoclax demonstrate a promising role in apoptosis induction in a variety of hematological malignancies such as multiple myeloma [72], AML [73], CLL [74, 75], MCL [76] and solid tumors like breast [77] and pancreatic cancers [78]. The mechanism of action of obatoclax was not limited to apoptosis as it was effective in inducing autophagy in several cancer cells [79, 80]. Since it is a pan Bcl-2 family inhibitor it has the advantage to be rationally combined with other targeted anti-cancer agents such as ABT-737 [73], HDAC inhibitors [79], sorafinib [81], TRAIL [82] and proteasome inhibitor, bortezomib.

Obatoclax is first in its class of BH3 mimetics to enter clinical trials. Multiple phase I and phase II trials evaluating safety profile and MTD in patients with refractory leukemia and myelodysplasia demonstrated tolerability of obatoclax by IV infusion [83]. Phase I study of single agent obatoclax in 26 heavily pretreated CLL patients at doses ranging from 3.5–14 mg/m² showed modest clinical activity through Bax mediated mechanism of apoptosis [84]. A phase II study of obatoclax in combination with topotecan in patients with relapsed SCLC demonstrated that this combination was not better in response rate than topotecan alone [85]. Clinical investigations in relapsed or refractory classical Hodgkin’s lymphoma demonstrated that obatoclax has limited clinical activity [86]. Together these results imply that a need for less toxic and better targeted Bcl-2 antagonists in the clinic is pressing.

ABT-737

ABT-737 is a cell permeant small molecule synthesized to bind to the hydrophobic BH3 binding groove of anti-apoptotic proteins with higher affinity (K_i ≤ 1 nM) to Bcl-X_L, Bcl-2 and Bcl-W, but not to proteins Bcl-B, Mcl-1 and Bfl-1 (K_i= 0.46 ± 0.11 μM, >1 μM and >1 μM, respectively). With plasma protein binding, this compound had lower affinity to Bcl-2 and Bcl-X_L (100 nM and 35 nM respectively) [87, 88]. Of all the putative BH3 mimetics in the field, ABT-737 was the only agent that was shown to specifically target Bcl-2 proteins by directly activating the cell death machinery via Bax/Bak activation [89]. ABT-737 reactivated program cell death in vitro and in vivo (xenograft models) both in solid tumors as well as hematological malignancies such as MCL [90], CML [91], AML [92], CLL [93, 94], MM [95, 96] and ALL [97, 98]; all of them express high levels of Bcl-2 family anti-apoptotic proteins. Overall, this compound predominantly induces apoptosis via intrinsic pathway by disrupting Bcl-2/Bax association [92] or dissociating Bim from Bcl-2 [99], thus typically representing the function of a BH3 only protein either by activating pro-apoptotic Bcl-2 proteins (Bak/Bax) or by inhibiting anti-apoptotic protein functions (Bcl-2/Bcl-X_L).

However, as described below, ABT-737 has some limitations. Unlike other pan Bcl-2 inhibitors, ABT-737 does not bind to other members of Bcl-2 family, such as Mcl-1 or Bfl-1 [100]. Seed analysis of off-target siRNAs revealed an essential role of Mcl-1 in resistance to ABT-737 [101]. Several mechanistic studies showed that targeting proteins that critically stabilizes Mcl-1 via inhibiting ubiqitination enhanced the sensitivity of ABT-737 [102, 103]. Accordingly, multiple strategies were employed to neutralize Mcl-1 and potentiate ABT-737 to cells for apoptosis such as combinations with obtaoclax, a pan Bcl-2 inhibitor, sorafinib, bortezomib [104], HDAC inhibitor [105] and numerous-other chemotherapeutic agents [106]. Combination with homoharringtonine, a protein translation inhibitor that reduces Mcl-1 protein [107], or co-administration of roscovitine [108], a CDK inhibitor that decreases Mcl-1 transcript and protein levels-demonstrated synergy with ABT-737.

Extensive studies on CLL primary cells showed nM efficacy of ABT-737 (EC₅₀ of 4.5 ± 2.2 nM) in displacing Bim from Bcl-2 to induce cell death suggesting Bcl-2 complexed to Bim is the critical target for ABT-737 in CLL [93]. Even though it was speculated that BH3 mimetics should function without p53, CLL primary cells with p53 deletion or dysfunction showed decreased sensitivity to ABT-737, while combination with nutlin showed synergy implying the significance of p53 in ABT-induced apoptosis [109]. Furthermore, ABT-737 also was shown to overcome the lymph node-mediated (CD40-stimulated) CLL cell survival via balancing the NOXA/Mcl1 axis [110]. Studies using gene-targeted mouse strains demonstrated that while ABT-737 avidly bind to Bcl-2, Bcl-X_L and Bcl-W in vitro, it was found that only Bcl-2 is its critical target in vivo suggesting that tumors exclusively over-expressing Bcl-2 are most likely to benefit [111].

ABT-263

A major limitation of ABT-737 is that it is not orally bioavailable. ABT-263 (Navitoclax) was synthesized with oral properties and first tested in xenograft model and was reported to disrupt Bcl-2/Bcl-X_L interactions with pro-death proteins (e.g., Bim), leading to the initiation of apoptosis within 2 hours post treatment [112]. Human cancer cells and a panel of SCLC xenograft models were tested for dose and schedule evaluation and combined with standard cytotoxic agents for induction of apoptosis [113, 114]. Phase I study of ABT-263 in patients with SCLC and other solid tumors demonstrated that navitoclax is safe and well tolerated, with dose-dependent thrombocytopenia as the major adverse effect [115]. Patients with relapsed or refractory lymphoid malignancies (n=55) were enrolled in a phase 1 dose-escalation study of safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity of ABT-263, demonstrating that Navitoclax has a novel mechanism of peripheral thrombocytopenia and T-cell lymphopenia, attributable to high-affinity inhibition of Bcl-X_L and Bcl-2, respectively[116]. A phase II study of single agentABT-263 to evaluate safety and toxicity, response rate, progression free and overall survival revealed that the baseline levels of biomarkers correlated with clinical benefit in SCLC patients [117]. Recent report obtained from clinical trials conducted in patients with relapsed or refractory CLL revealed that lymphocytosis was reduced by more than 50%. Importantly, clinical activity was indeed observed in patients with fludarabine-refractory disease, bulky adenopathy, and (17p) del subset of CLL. Consistent to preclinical data, low Mcl1 expression and high Bim: Mcl1or Bcl-2 ratios correlated with clinical response [118]. The data so far obtained from all clinical studies with ABT-263 have demonstrated favorable anti-leukemic activity in many cancers except it induces a rapid but reversible thrombocytopenia.

ABT-199

Navitoclax is a potent Bcl-X_L inhibitor, circulating platelet survival is dependent on Bcl-X_L, inhibiting Bcl-X_L also destroyed platelets leading to thrombocytopenia, a dose limiting side effect. So the goal was to develop an agent that inhibits mostly Bcl-2, a critical survival factor for cancer cells, while sparing Bcl-X_L that is important for survival of circulating platelets. ABT-199 is a reverse engineered version of ABT-263 designed to have high affinity for Bcl-2, but low affinity for Bcl-X_L. Given that Bcl-2 is a validated drug target for CLL, this provided a solid scientific rationale for using it in clinical trials for CLL. A Phase 1, open-label, multicenter study evaluating the safety and pharmacokinetics profile of ABT-199 in relapsed or refractory chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) is ongoing[119].

Conclusion and Future Approaches

For several years the hallmark of cancer treatment has been the use of traditional chemotherapy. These cytotoxic agents target rapidly dividing cells including certain normal cells thus lacking specificity and/or selectively. Although conventional chemotherapy remains the treatment of choice for many malignancies, targeted therapies are now a component of treatment for many types of cancers. In the past decade, of the new anticancer drugs approved by FDA, 15 have been targeted therapies, compared with only five traditional chemotherapeutic agents[120].

Given that CLL is replicationally quiescent disease and its prognosis exclusively depends upon the expression of Bcl-2 family anti-apoptotic proteins, agents that neutralize the anti-apoptotic properties of molecular targets could be a more defined choice of treatment for this disease. There have been several agents synthesized and tested for inhibition of Bcl-2 family anti-apoptotic proteins. Maritoclax was specifically designed to inhibit the protein Mcl-1, the major anti-apoptotic target of CLL [121]. ABT-199 was synthesized to have more inhibitory effect on Bcl-2 in comparison to Bcl-X_L. Bad-like BH3 mimetic ABT-263 or ABT-737 that lack ability to bind to Mcl1 is confirmed to be a true BH3 mimetic that induce Bax/Bak dependent apoptosis. Several putative BH3 mimetics such as GX15-070, TW37, gossypol and analogues were tested and disclosed as pan Bcl-2 family anti-apoptotic protein inhibitors (Table 1). Since Bcl-2 protein also interacts with autophagic protein such as BECLIN1, pharmacological BH3 mimetics competitively disrupt the inhibitory interaction between Beclin-1 and Bcl-2 or Bcl-X_L, thereby stimulating autophagy. Thus, BH3 mimetics have the ability to trigger both apoptotic and autophagic response machineries. Additionally, combining BH3 mimetic with chemotherapeutic agents sensitized cancer cells better than single agent alone. These statements provide strong evidence upon the notion that targeted therapeutics; particularly Bcl-2 antagonists have optimistic future in the clinic. It could add more strength to the therapeutic index if the agent is a derivative of natural product, which in turn spares normal cells. Promoting further investigations on combination with B-cell receptor kinase inhibitors that exhibit promising activity in preclinical and clinical trials (CAL-101, R406, BTK inhibitions) should infer additional insights on the mechanism of actions of these agents.

Table 1.

overview of Bcl-2 family antagonists

BH3 mimetic	TARGETS	PRECLINICAL	CLINICAL
Gossypol[20]	Bcl-2, XL, Mcl-1, Bcl-w, Bcl-b, BFL1	Yes	Yes
AT-101[52]	Bcl-2, XL, Mcl-1, Bcl-w, Bcl-b, BFL1	Yes	Yes
Apogossypol	Bcl-2, XL, Mcl-1, Bclw, Bclb	Yes	No
BI-79D10[57]	Bcl-2, XL, Mcl-1	Yes	No
Compound r[58]	Bcl-2, XL, Mcl-1, BFL-1	Yes =	No
BI-97C1[120] Sabutoclax	Bcl-2, XL, Mcl-1, BFL-1	Yes	No
Gossypolone[61]	Bcl-2, XL, Mcl-1, Bcl-w, Bcl-b	Yes	No
Apogossypolone (ApoG2)[65]	Bcl-2, XL, Mcl-1	Yes	No
Obatoclax[83]	Bcl-2, XL, Mcl-1, Bcl-w, Bcl-b, BFL-1	Yes	Yes
ABT-737[92]	Bcl-2, XL, Bcl-W	Yes	No
ABT-263[121] Navitoclax	Bcl-2, XL, Bcl-W	Yes	Yes
ABT-199	Bcl-2	Yes	Yes
Maritoclax[119]	Mcl-1	Yes	No
Bims2A [122]	Mcl-1	No	No
Mcl-1 SAHB[123]	Mcl-1	No	No
TW 37[45]	Bcl-2, XL, Mcl-1	Yes	No