apoptosis is an orchestrated cellular process important for maintaining the homeostasis between cell proliferation and cell death , and is also pivotal for the removal of diseased , damaged , and inflamed cells . apoptosis can be induced in two distinct pathways , both of which lead to the activation of effector caspases . the extrinsic pathway of apoptosis is initiated at the plasma membrane upon ligation of death receptors , leading to the subsequent activation of effector caspases . these caspases then execute the apoptotic pathway to trigger dna fragmentation and membrane blebbing , two hallmarks of apoptosis . the intrinsic pathway is initiated in response to intracellular stress , eventually leading to mitochondrial outer membrane permeabilization ( momp ) , releasing factors including cytochrome c and smac to activate downstream caspases . the b - cell lymphoma-2 ( bcl-2 ) family of proteins is central to the intrinsic pathway of apoptosis . the bcl-2 proteins share one of four bcl-2 homology ( bh ) domains , bh14 , and of those , the bh3 domain is critical for mediating interactions among the family members . the bcl-2 family of proteins can be grouped as either pro - apoptotic or anti - apoptotic . the pro - apoptotic group can be further divided as either multi - bh - domain proteins , including bax and bak , or as bh3-only proteins , such as bid , bad , bim , puma , and noxa . the anti - apoptotic group of bcl-2 proteins consists of bcl-2 , bcl - xl , bcl - w , mcl-1 , and bfl-1/a1 . two models account for how the various proteins interact to induce apoptosis ( fig .  1 ) . in the indirect activation model , the anti - apoptotic bcl-2 proteins physically interact and sequester bak or bax . upon apoptotic stimuli , bh3-only proteins release bak or bax by binding and neutralizing the anti - apoptotic bcl-2 proteins . in the direct activation model , anti - apoptotic bcl-2 proteins function by sequestering particular bh3-only proteins called bh3 activators , including bim , bid , and puma . upon apoptotic stimuli , bh3 sensitizers such as noxa or bad the two models converge when activated bak and bax oligomerize , leading to momp and subsequent apoptosis . therefore , the carefully regulated balance between pro - apoptotic and anti - apoptotic members of the bcl-2 family determine the survival of the cell . it has repeatedly been shown that the overexpression or loss of pro- or anti - apoptotic members of the bcl-2 proteins render cells correspondingly susceptible or resistant to apoptosis . therefore , deregulated apoptosis can be detrimental to the cell , tissue , or organ . the anti - apoptotic bcl-2 protein family members include bcl-2 , bcl - xl , bcl - w , mcl-1 , and bfl-1/a1 . in the indirect activation model , the anti - apoptotic proteins sequester bax and bak through physical interactions . the upregulation of bh3-only proteins will disrupt this interaction , releasing bax and bak to oligomerize , allowing mitochondrial outer membrane permeabilization ( momp ) , the release of key factors such as cytochrome c , and the induction of apoptosis . in the direct activation model , anti - apoptotic bcl-2 protein members sequester bh3 activators including puma , bid , and bim . bh3 sensitizers are able to disrupt this interaction , and the released activators directly activate bax and bak to allow momp and apoptosis . the anti - apoptotic group of bcl-2 family proteins is frequently found to be overexpressed in numerous cancers , causing both evasion of apoptosis and resistance to treatment . furthermore , since most anti - cancer drugs induce apoptosis through the intrinsic pathway , targeting the bcl-2 family of proteins with small molecule inhibitors ( smis ) is especially attractive clinically either in single agent therapy or combination treatment . while numerous compounds with anti - cancer activity have been shown to antagonize bcl-2 family of proteins , such as epigallocatenin-3-gallate ( egcg ) and chelerythrine , smis selective for the anti - apoptotic bcl-2 proteins have only recently been identified . the first bcl-2 selective smi , ha141 , was identified through a virtual screen in 2000 . due to the recent advances in crystallizing bcl - xl and its complex with the bak bh3 domain peptide , a hydrophobic pocket that interacts with other bh3 domains was identified and taken advantage of to aid drug design . the bcl-2 protein structure was then modeled and ha141 was discovered through a computer - aided screen . following this , the fluorescence polarization assay ( fpa ) was soon developed to allow experimental high - throughput screening for smis of anti - apoptotic family proteins . this assay uses fluorescein - conjugated bh3 peptides and recombinant anti - apoptotic bcl-2 proteins . using this method , several pan - bcl-2 inhibitors with moderate affinity were discovered , including bh31 , gossypol , and obatoclax . recently , structure - based design has also emerged to synthesize numerous high - affinity bcl-2 smis . using the nmr - guided fragment - based technology , low - affinity molecules , which bind to distinct sites on the hydrophobic groove of the bcl - xl protein , further rational design approaches have been successful at generating abt-737 based high - affinity bcl-2 smis , including compounds b-11 and compound 21 . moreover , the rational design of terphenyl - based bak - bh3 -helical proteomimetics resulted in the discovery of bh3-m6 , a pan - bcl-2 inhibitor that can inhibit both bcl - xl and mcl-1 to induce caspase dependent apoptosis . other developmental efforts have also been fruitful , resulting in the discovery of the selective mcl-1 antagonist maritoclax , and the high - affinity pan - bcl-2 inhibitor sabutoclax . currently , three bcl-2 inhibitors are undergoing clinical trials : abt-737 s orally bioavailable analog abt-263 ( navitoclax ) , at-101 [ ( -)-gossypol ] , and obatoclax ( gx15070 ) . these compounds include abt-737 and abt-263 , obatoclax , gossypol derivatives such as apogossypol , apogossypolone , and sabutoclax , other rational design - based compounds such as b-12 and bh3-m6 , and the recently identified maritoclax . however , the selectivity and affinities of these compounds for the anti - apoptotic bcl-2 family members differ greatly among the different compounds as well as between different assays ( fig .  2 ) . the difficulty in obtaining precise values for the affinity of bcl-2 smis can be attributed to the affinity differences among the peptides used for the fpa . for example , abt-737 s apparent affinity was 4-fold lower for bcl-2 , while 50-fold higher for bcl - w , when using a bid - peptide compared with using a bim - peptide . therefore interpretations of affinity data for bcl-2 smis should be taken with a grain of salt . green indicates relatively high affinity for the protein , orange indicates relatively low affinity for the protein , and red indicates no affinity for the protein was detected . the colors assigned were based on the drug s affinities relative to the affinity of abt-737 against bcl-2 , as well as on the relative affinities between the drug s targets . the selectivity of the bcl-2 smis for the anti - apoptotic members differ as well . while agents such as obatoclax and at-101 are classified as pan - bcl-2 inhibitors due to its moderate affinity for all anti - apoptotic members , abt-737 and its analog abt-263 are selective for bcl-2 , bcl - xl , and bcl - w as they are modeled after the bh3-only protein bad . it is also the most potent of the inhibitors so far possessing affinities in the sub - nanomolar range . a-385358 was rationally designed to be almost 100-fold more selective for bcl - xl over bcl-2 , and is the most selective inhibitor for bcl - xl to date . a-385358 was able to more potently kill bcl - xl - dependent compared with bcl-2-dependent lymphoma cells . maritoclax , also known as marinopyrrole a , was recently discovered as a mcl-1-specific inhibitor through the screening of small compound libraries . maritoclax is a natural product from a marine - derived species of streptomyces which binds to mcl-1 with comparable affinity as obatoclax ; however , unlike obatoclax , maritoclax does not bind to bcl - xl . maritoclax induces mcl-1 proteasomal degradation and shows similar or greater in vitro efficacy toward mcl-1 dependent cells as obatoclax . however , in cells dependent on bcl-2 or bcl - xl for survival , maritoclax demonstrates only marginal activity . importantly , unlike obatoclax , which induces cell death indiscriminately in healthy peripheral blood mononuclear cells ( pbmcs ) and large granular lymphocyte leukemia ( lgll ) , maritoclax is significantly less effective against healthy pbmcs compared with lgll cells . this suggests that maritoclax may be safer for treatment compared with pan - bcl-2 inhibitors . however , maritoclax induces cell death selectively in mcl-1 dependent cells in a bax / bak dependent manner , indicating that maritoclax acts through the intrinsic pathway of apoptosis . moreover , there is a positive correlation between the sensitivity to maritoclax and mcl-1 expression in primary lgll cells . lastly , actin is known to bind to and sequester the pro - apoptotic bh3-only protein bmf , which is released to sensitize the cell to apoptosis upon actin depolymerization . since bmf interacts with not only mcl-1 but also bcl-2 and bcl - xl , liberation of bmf from actin should induce cell death in both mcl-1 and bcl-2/bcl - xl dependent cells . however , given that maritoclax only kills mcl-1 dependent cells , it is unlikely that maritoclax induces apoptosis in a mechanism mediated by actin . since it is difficult for smis with micromolar affinities to reach physiologically relevant concentrations in vivo , such compounds are often deemed to be useful only as experimental tools . however , some compounds with moderate affinity , such as obatoclax and at-101 , have already demonstrated efficacy against cancer in clinical trials . therefore , it is possible that these agents may act as non - selective bcl-2 inhibitors , killing cancer cells through multiple pathways . in an important study by vogler et al . , it was shown that between obatoclax , gossypol , and abt-737 , only abt-737 was unable to kill bax / bak double knockout cells . since bax / bak oligomerization is necessary for bcl-2 mediated apoptosis , the ability of obatoclax and gossypol to induce cell death in these cells signifies its actions on other cellular pathways . it was similarly shown that obatoclax does not induce cytochrome c release from isolated mitochondria at physiologically relevant levels . given the inhibitory effect of bcl-2 on beclin-1 , the mammalian homolog of the autophagy related gene 6 ( atg6 ) , bcl-2 smis often induce autophagy by disrupting bcl-2 and beclin-1 interactions . however , numerous reports have demonstrated beclin-1 independent induction of autophagy by gossypol and obatoclax , possibly causing autophagic cell death independent of bcl-2 interactions . other reports have also shown obatoclax and gossypol to possess activities unrelated to bcl-2 which may contribute to cell killing , such as reactive oxygen species generation or anion transport . therefore , abt-263 is possibly the only compound in clinical trials to selectively induce bcl-2 dependent cell death . all three bcl-2 smis in clinical trials , abt-263 , at-101 , and obatoclax , demonstrate remarkable preclinical efficacy in a variety of cell systems ( figure  3 ) . notably , these bcl-2 smis kill chemoresistant cell lines as efficaciously as susceptible cell lines , demonstrating a distinct mechanism of action and clinical promise . the potency of bcl-2 smis currently in clinical trials against various cancer cells in culture . green indicates high potency for the cancer type with an average ec50 value below 1 m , orange indicates a moderate potency with an average ec50 value between 1 and 10 m , and red indicates a low potency with an average ec50 value above 10 m . the white numbers inside the box signify the number of experiments examined to obtain the average . all three bcl-2 smis currently in clinical trials have shown to potentiate and synergize with chemotherapy and radiation in vitro . interestingly , mitogen - activated protein kinase ( mapk ) pathway inhibitors , such as the epidermal growth factor receptor ( egfr ) inhibitor gefitinib and erlotinib or the mek inhibitor uo126 synergize with bcl-2 smis . while the mapk pathway is frequently upregulated in cancers and is therapeutic target by itself , synergy between bcl-2 inhibitors and mapk pathway inhibitors may actually be due to crosstalk between the two pathways . it has been shown that egfr activation leads to stat3 activation , which in turn increases the expression of bcl-2 and bcl - xl . therefore , the inhibition of the mapk pathway reduces the expression of bcl-2 and bcl - xl , while bcl-2 inhibitors physically antagonize their anti - apoptotic actions to create a synergistic effect . recently , abt-737 has also been shown to possess immunosuppressive properties , possessing activity on lymphocytes and mast cells . due to the selectivity of abt-737 on specific bcl-2 family protein members as abt-737 disrupts bcl-2/bim interactions more readily compared with bcl - xl / bim , bcl - xl overexpressing cancer cells can be resistant to abt-737 treatment . the primary pathway for abt-737 resistance appears to be mcl-1 upregulation , as it is not targeted by the compound . it was repeatedly observed in a large number of cancer cell lines that mcl-1 expression levels inversely correlate with abt-737 susceptibility . human b - cell lymphoma cell lines derived from long - term exposure to abt-737 were resistant to its treatment through a significant increase in mcl-1 transcription and protein levels . the use of flavopiridol , a cyclin - dependent kinase 9 ( cdk9 ) inhibitor shown to downregulate mcl-1 , was able to restore abt-737 sensitivity . furthermore , melanoma , which is typically resistant to abt-737 treatment , can be rendered susceptible to the compound through genetic or non - selective pharmacologic inhibition of mcl-1 in multiple reports . obatoclax has demonstrated significant antagonistic activity against mcl-1 , and has been used experimentally to demonstrate mcl-1 mediated abt-737 resistance as well . as the induction of the mapk pathway also upregulates mcl-1 , the synergy between abt-737 and mapk pathway inhibitors can be due to its downregulation of mcl-1 . following the development of the first selective mcl-1 inhibitor , maritoclax was immediately shown to be efficacious as a single agent in abt-737 resistant , mcl-1 dependent cell lines , and was strongly synergistic with abt-737 . overexpression of noxa in multiple cancer systems have shown to restore drug susceptibility that is similar to mcl-1 downregulation , mechanistically demonstrating that sequestration of pro - apoptotic bcl-2 proteins by mcl-1 can mediate drug resistance . recently it was also found that mcl-1 can also be phosphorylated in response to abt-737 to increase its interaction with bim to prevent apoptosis . abt-737 was shown to be extremely efficacious as a single agent in sclc mouse xenograft models , as the compound was able to induce complete regression of tumors and prevent its regrowth for at least 2 months after the cessation of therapy . abt-737 by itself has also been shown to be effective in xenograft models for glioblastoma , multiple myeloma , acute myeloid leukemia , chronic myeloid leukemia , acute lymphoblastic leukemia , among others . furthermore , the pan - bcl-2 inhibitor at-101 was shown to be efficacious in prostate cancer xenografts in different cell line models , resulting in tumor growth reductions . in another study , however , at-101 was ineffective in a similar prostate cancer xenograft model , but was active in potentiating the effects of docetaxel . abt-263 ( navitoclax ) , which is being developed by abbott laboratories and genentech , was shown to be the most efficacious against chronic lymphocytic leukemia ( cll ) in phase i trials , with all 7 patients in one trial and 9 out of 21 patients with relapsed or refractory disease experiencing a partial response with median progression - free survival of more than a year . it was observed in these patients that mcl-1 expression inversely correlated with response to drug , matching previous preclinical observations . the recently published phase ii clinical trial data for abt-263 indicate that it lacks efficacy in patients with relapsed small cell lung carcinoma ( sclc ) , as only 1 out of 39 patients experienced a partial response , and 36 patients withdrew due to disease progression or adverse events . currently multiple clinical trials are still also underway to assess the efficacy of abt-263 in combination treatments . since bcl - xl is important for platelet survival , thrombocytopenia was observed across all clinical trials for abt-263 . however , the platelet decreases were all observed to be transient and reversible upon treatment termination , and has not been associated with clinically significant bleeding . other hematological toxicities including neutropenia have also been observed , and could present significant problems when treating patients with hematological malignancies with an already compromised bone marrow reserve . at-101 is in the pipeline for ascenta therapeutics and has only demonstrated moderate efficacy in one phase i / ii clinical trial against castration - resistant prostate cancer . in this trial , two of 24 patients experienced a partial decrease in prostate - specific antigen release , while no objective partial responses were observed . however , 4 individuals unexpectedly developed small intestinal obstructions possibly related to the drug , causing discontinuation of treatment . in a following larger clinical trial in combination with docetaxel and prednisone , at-101 did not manage to meet efficacy endpoints both as a single agent against nor in combination with topotecan or docetaxel / prednisone against sclc . gastrointestinal toxicities were observed across all clinical trials with single - agent trials , but were not increased in combination treatments . unlike abt-263 , obatoclax demonstrated moderate to no efficacy against hematological malignancies in phase i trials . however , in these clinical trials where obatoclax was either infused for one hour or three hours , significant neural adverse events were observed , including somnolence , euphoria , and ataxia . it was observed that a three - hour infusion was necessary to reduce the occurrence of toxic events . in a phase i trial , obatoclax demonstrated significant efficacy against sclc as a single agent , where 2 of 8 patients with sclc had partial response and 3 experienced stable disease . moreover , another phase i clinical trial in combination with carboplatin and etoposide against extensive - stage sclc was conducted . however , obatoclax in combination with topotecan against relapsed sclc did not meet efficacy endpoints . another phase ii trial also indicates obatoclax to be ineffective toward myelofibrosis , despite the disease s dependence on the bcl-2 family anti - apoptotic proteins . as clinical trial data for bcl-2 smis continue to be published , we will likely gain more insight on the efficacy of these inhibitors against cancer . so far , clinical trials show promise for abt-263 against cll , and obatoclax in combination treatments against sclc . however , numerous clinical trials have demonstrated a lack of efficacy where it was expected , such as at-101 against prostate cancer . a higher affinity pan bcl-2 inhibitor sabutoclax ( bi-97c1 ) has recently been developed , and may yield clinical promise as it possesses nanomolar affinities for all of mcl-1 , bcl-2 , and bcl - xl proteins , and induces cell death in a bax / bak - dependent manner . moreover , it has demonstrated both in vitro and in vivo xenograft model efficacy against bcl-2 dependent and mcl-1 dependent prostate cancer cell lines . currently , research efforts have began focusing on different dosing regimens using bcl-2 smis to improve efficacy . metronomic dosing , the uninterrupted use of a low dose of compound , have been suggested for bcl-2 inhibitors including at-101 and tw-37 . it is hypothesized that the metronomic dosing of bcl-2 inhibitors is able to more effectively induce growth arrest by targeting the tumor microenvironment . sequential dosing of multiple therapies also shows preclinical promise . the sequential use of bortezomib and ha141 was more efficacious than both single agent and combination treatment regimens in in vitro models of multiple myeloma . sequential dosing may be particularly useful for abt-263 , as it can not be administered for extended periods of time due to its severe toxicity on platelets . moreover , both preclinical and clinical data have indicated the importance of mcl-1 mediated abt-263 resistance . as maritoclax is strongly synergistic with abt-737 , the sequential use of abt-263 with mcl-1 selective inhibitors may be particularly useful both to prevent the development of resistant cell populations and to allow the recovery of platelets in patients . high - affinity pan bcl-2 inhibitors could cause much toxicity in the patient , as numerous healthy cells depend on the maintenance of anti - apoptotic bcl-2 family members for survival . thus selective bcl-2 smis demonstrate a distinct advantage in drug safety , although selectivity can also lead to resistance , as was seen in the case of abt-263 and abt-737 . as simultaneous treatment of patients with multiple bcl-2 inhibitors is imprudent due to toxicity reasons , it may be more sensible to use sequential treatments in order to minimize undesired side effects . it is known that the downregulation of mcl-1 itself is efficacious against numerous cancer cells to mediate both cell killing and drug sensitivity . importantly , experiments show that multiple cancers , including multiple myeloma and acute myeloid leukemia , are highly dependent on mcl-1 to sustain growth . as abt-737 for example , downregulation of mcl-1 is able to completely kill acute myeloid leukemia cells in both cell culture and in vivo mouse models . following optimization , more mcl-1 inhibitors with higher selectivity and affinity are likely to be generated for clinical use . the bcl-2 family proteins play a significant role in maintaining survival for a large number of cancers . smis for the anti - apoptotic proteins have already demonstrated that cancer cells can be killed through this distinct mechanism of action despite their resistance status to previous therapies . these inhibitors have demonstrated efficacy both as single agents and in combination therapy to improve patient outcome . however , selectivity , toxicity , resistance , and bioavailability remain a challenge for many of these compounds . further efforts should focus on reducing undesired toxicities from these drugs through dosing or combination therapies , as well as chemical advances in synthesizing more selective bcl-2 inhibitors .
deregulated apoptosis is a hallmark of cancer , and the b - cell lymphoma-2 ( bcl-2 ) family of proteins is pivotal to mediating the intrinsic pathway of this process . 
 recent advances have yielded both pan - bcl-2 small molecule inhibitors ( smis ) that inhibit both the bcl-2 and the mcl-1 arm of the bcl-2 family anti - apoptotic proteins , as well as selective smis to differentially target the two arms . of these smis , abt-263 ( navitoclax ) , at-101 
 [ ( -)-gossypol ] , and obatoclax ( gx15 - 070 ) are currently in clinical trials for multiple cancers . 
 while pan - bcl-2 inhibitors such as at-101 and obatoclax can be more toxic for inhibiting all members of the anti - apoptotic bcl-2 family of proteins , resistance can quickly develop for abt-263 , a selective bcl-2 inhibitor . in this article 
 , we discuss the current status of bcl-2 family smis in preclinical and clinical development . as 
 mcl-1 upregulation is a major mechanism of abt-263 resistance , mcl-1-specific inhibitors are expected to be efficacious both in combination / sequential treatments and as a single agent against cancers resistant to abt-263 .