Robust lanthanide-based assays for the detection of anti-apoptotic Bcl-2-family protein antagonists

Anti-apoptotic Bcl-2-family proteins (Bcl-2, Bcl-x(L), Bfl-1, Mcl-1, Bcl-W and Bcl-B) have been recently validated as drug discovery targets for cancer, owed to their ability to confer tumor resistance to chemotherapy or radiation. The anti-apoptotic activity of Bcl-2 proteins is due to their ability to heterodimerize with their pro-apoptotic counterparts (proteins such as Bad, Bim or Bid) via a conserved peptide region termed BH3. Thus, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Here, we report on the development and evaluation of two novel Lanthanide-based assays that are formatted for high-throughput screening of small molecules capable of antagonizing BH3-Bcl-2 interactions. The assay conditions, robustness and reproducibility (Z' factors) are described. These assays represent useful tools to enable further studies in the search for novel, safe and effective anti-cancer agents targeting Bcl-2-family proteins.     

High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.     

Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: characteristics of broad-spectrum protein binding and its effects on anti-tumor activity

On the basis of the comparison of the structure of the Bim BH3: Bcl-x(L) complex and that of the ABT-737: Bcl-x(L) complex, a series of class A compounds were designed. These compounds had the basic skeleton of ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim BH3's broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B compounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most of these class B compounds showed better binding affinity to the target proteins than the class A compounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple tumor cell lines known to express Bcl-x(L), Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spectrum binding to Bcl-x(L), Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum properties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth of many tumor cells. Finally, our study provides a series of lead compounds that merit further research into anti-cancer therapeutics.     

Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-μl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (～10%) of each type per well.     

A surface groove essential for viral Bcl-2 function during chronic infection in vivo

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the gamma-herpesvirus 68 (gammaHV68) Bcl-2 family protein (gammaHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the gammaHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type gammaHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of gammaHV68 from latency and efficient persistent gammaHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection.     

Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax and Bak

A novel human member of the Bcl-2 family was identified, Bcl-B, which is closest in amino acid sequence homology to the Boo (Diva) protein. The Bcl-B protein contains four Bcl-2 homology (BH) domains (BH1, BH2, BH3, BH4) and a predicted carboxyl-terminal transmembrane (TM) domain. The BCL-B mRNA is widely expressed in adult human tissues. The Bcl-B protein binds Bcl-2, Bcl-X(L), and Bax but not Bak. In transient transfection assays, Bcl-B suppresses apoptosis induced by Bax but not Bak. Deletion of the TM domain of Bcl-B impairs its association with intracellular organelles and diminishes its anti-apoptotic function. Bcl-B thus displays a unique pattern of selectivity for binding and regulating the function of other members of the Bcl-2 family.     

Bcl-w(edding) with mitochondria

Bcl-2-family members (Bcl-2, Bax, Bcl-w and Bcl-x(L)) are crucial integrators of signals for cell survival and death; the pro- or antiapoptotic activities of these proteins are regulated by their subcellular localization. Bcl-2 directly inserts into the membranes, where it acts; however, Bax requires a stimulus-dependent translocation from an inactive cytosolic to an active membrane-inserted state. Recently, a novel mechanism is described for the survival factor Bcl-w, which is active while weakly associated with mitochondria. In apoptotic cells, a BH3-only protein neutralizes the survival activity of Bcl-w by binding to its "hydrophobic pockets", thereby releasing its C-terminal domain and allowing its insertion into the membrane. Here, we discuss the importance of this finding for a better understanding of the action mode of Bcl-w and other Bcl-2-family members.     

Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.     

Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.     

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-xL

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the α-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques—yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis—to elucidate specificity determinants for binding to Bcl-x_Lversus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x_L selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x_L, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x_L-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x_L binders.     

Identification of novel estrogen receptor alpha antagonists

We have identified novel estrogen receptor alpha (ERalpha) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERalpha ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERalpha LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-beta-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-beta-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.     

Structure-based design of rhodanine-based acylsulfonamide derivatives as antagonists of the anti-apoptotic Bcl-2 protein

A series of novel rhodanine-based acylsulfonamide derivatives were designed, synthesized, and evaluated as small-molecule inhibitors of anti-apoptotic Bcl-2 protein. These compounds exhibit potent antiproliferative activity in three human tumor cell lines (Hep G2, PC-3 and B16-F10). Among them, the most potent compounds 10 and 11 bind to Bcl-2 with a K(i) of 20 and 25 nM, respectively. Docking studies demonstrated that these two compounds orient similarly at the binding site of Bcl-2, and the calculated binding affinities (Glide XP score) of compound 10 is more negative than that of compound 11. The binding interactions of compounds with high binding affinity to Bcl-2 protein were analyzed.     

Molecular basis of Bcl-X(L)-p53 interaction: insights from molecular dynamics simulations

Bcl-X(L), an antiapoptotic Bcl-2 family protein, plays a central role in the regulation of the apoptotic pathway. Heterodimerization of the antiapoptotic Bcl-2 family proteins with the proapoptotic family members such as Bad, Bak, Bim and Bid is a crucial step in the apoptotic regulation. In addition to these conventional binding partners, recent evidences reveal that the Bcl-2 family proteins also interact with noncanonical binding partners such as p53. Our previous NMR studies showed that Bcl-X(L): BH3 peptide and Bcl-X(L): SN15 peptide (a peptide derived from residues S15-N29 of p53) complex structures share similar modes of bindings. To further elucidate the molecular basis of the interactions, here we have employed molecular dynamics simulations coupled with MM/PBSA approach. Bcl-X(L) and other Bcl-2 family proteins have 4 hydrophobic pockets (p1-p4), which are occupied by four systematically spaced hydrophobic residues (h1-h4) of the proapoptotic Bad and Bak BH3 peptides. We observed that three conserved hydrophobic residues (F19, W23 and L26) of p53 (SN15) peptide anchor into three hydrophobic pockets (p2-p4) of Bcl-X(L) in a similar manner as BH3 peptide. Our results provide insights into the novel molecular recognition by Bcl-X(L) with p53.     

A new assay based on fluorescence resonance energy transfer to determine the binding affinity of Bcl-xL inhibitors

We developed a new assay of Bcl-xL inhibitors based on fluorescence resonance energy transfer that occurs between an AEDANS-labeled Bak-BH3 peptide and three tryptophans in the BH1 and BH2 domains of Bcl-xL. The method can tolerate up to 5% DMSO, and it was validated with several Bcl-xL inhibitors. It can be adapted to screen for compounds targeting other Bcl-2 family proteins.     

Discovery of novel biaryl sulfonamide based Mcl-1 inhibitors

Mcl-1 is an anti-apoptotic protein overexpressed in hematological malignancies and several human solid tumors. Small molecule inhibition of Mcl-1 would offer an effective therapy to Mcl-1 mediated resistance. Subsequently, it has been the target of extensive research in the pharmaceutical industry. The discovery of a novel class of Mcl-1 small molecule inhibitors is described beginning with a simple biaryl sulfonamide hit derived from a high through put screen. A medicinal chemistry effort aided by SBDD generated compounds capable of disrupting the Mcl-1/Bid protein-protein interaction in vitro. The crystal structure of the Mcl-1 bound ligand represents a unique binding mode to the BH3 binding pocket where binding affinity is achieved, in part, through a sulfonamide oxygen/Arg263 interaction. The work highlights the some of the key challenges in designing effective protein-protein inhibitors for the Bcl-2 class of proteins.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X_L bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

Design,synthesis and preliminary bioactivity studies of indomethacin derivatives as Bcl-2/Mcl-1 dual inhibitors

Bcl-2 family proteins, which divides into pro-apoptosis proteins and anti-apoptosis proteins, are involved in cell apoptosis progression. As numerous studies illustrated, targeting Bcl-2 family proteins is more and more attractive and practicable to cancer treatment. In this work, we designed and synthesized a series of indomethacin derivatives as new inhibitors for Bcl-2 family proteins. Our results of binding assay to Bcl-2 proteins, MTT assay and apoptotic assay indicated that some compounds had potent binding affinity to Bcl-2/Mcl-1 but not Bcl-X_L. Furthermore, compound 8j showed improved anti-proliferative activity than known Bcl-2 inhibitor WL-276.     

Structure-based discovery of a new class of Bcl-xL antagonists

Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are central regulators of the apoptotic pathway and due to their ability to confer tumor resistance to chemotherapy or radiation, have been recently validated as targets for cancer drug discovery. Since the crucial interaction between pro- and anti-apoptotic members occurs via a conserved region located on the surface of the protein, a viable way to inhibit the anti-death activity of Bcl-2 proteins is to design small molecule inhibitors that occupy this cavity. Here, we describe a structure-based approach that led to the identification of four small molecule inhibitors directed at the hydrophobic groove on the surface of the Bcl-2 family protein Bcl-xL. The compounds were characterized in a number of assays including in vitro binding using 15N-labeled protein, a displacement DELFIA assay, and a cell-based viability assay with human cancer cells.