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The natural product gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture and was identified as an active compound in a cell-based high-throughput screening assay for activators of caspases, proteases involved in apoptosis. Using the antiapoptotic Bcl-2 family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescence polarization assay. Analysis of competition for BH3 peptide binding revealed that GA inhibits all six human Bcl-2 family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited [concentrations required for 50% inhibition (IC50), in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogues of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax−/−bak−/− cells in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of antiapoptotic Bcl-2 family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells. [Mol Cancer Ther 2008;7(6):1639–46]

Supplementary Fig. from Gambogic acid is an antagonist of antiapoptotic Bcl-2 family proteins

In this paper, I explore how seamless embedding of four mathematical concepts: counting, ratio, distribution, and statistical significance, into a unit on inheritance enables students to gain a deeper insight into biological phenomena than a qualitative only approach achieves. The topic of Mendelian inheritance is used to illustrate this proposal. An introductory activity is presented that separates the specialized genetics vocabulary from the inheritance process, allowing the initial mathematical relationship to be developed and subsequently followed by the language requirements.

// Martin C. Pearce 1 , John T. Gamble 1, 2 , Prasad R. Kopparapu 1 , Edmond F. O'Donnell 1 , Monica J. Mueller 1 , Hyo Sang Jang 1 , Julie A. Greenwood 2 , Arnold C. Satterthwait 3 , Robert L. Tanguay 4, 5, 6 , Xiao-Kun Zhang 3 and Siva Kumar Kolluri 1, 4, 5, 6 1 Cancer Research Laboratory, Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331, USA 2 Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon 97331, USA 3 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92031, USA 4 Department of Environmental & Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331, USA 5 Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA 6 Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA Correspondence to: Siva Kumar Kolluri, email: siva.kolluri@oregonstate.edu Keywords: chemoresistance; Bcl-2; NuBCP-9; paclitaxel; lung cancer Received: February 19, 2018 Accepted: April 24, 2018 Published: May 25, 2018 ABSTRACT Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form “inflammasomes” that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1β and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1β secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1β production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

El fragmento carboxilo terminal, rico en cisteínas, de la proteína 1 de superficie del merozoito (MSP-1) desempeña un papel importante en la invasión del merozoito de la malaria al eritrocito. Anticuerpos contra esta región de MSP-l inhiben la invasión.

NLRP1 (NLR family, pyrin domain-containing 1) is a contributor to innate immunity involved in intracellular sensing of pathogens, as well as danger signals related to cell injury. NLRP1 is one of the core components of caspase-1-activating platforms termed “inflammasomes,” which are involved in proteolytic processing of interleukin-1β (IL-1β) and in cell death. We previously discovered that anti-apoptotic proteins Bcl-2 and Bcl-X L bind to and inhibit NLRP1 in cells. Using an in vitro reconstituted system employing purified recombinant proteins, we studied the mechanism by which Bcl-2 and Bcl-X L inhibit NLRP1. Bcl-2 and Bcl-X L inhibited caspase-1 activation induced by NLRP1 in a concentration-dependent manner, with K i ≈ 10 nM. Bcl-2 and Bcl-X L were also determined to inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and Bcl-X L was demonstrated to interfere with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-X L , which are located between the first and second α-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-X L inhibit NLRP1.

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax-/-cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.

B cell-activating factor belonging to the TNF family receptor (BAFF-R), a member of the TNFR superfamily, plays a role in autoimmunity after ligation with BAFF ligand (also called TALL-1, BLyS, THANK, or zTNF4). BAFF/BAFF-R interactions are critical for B cell regulation, and signaling from this ligand-receptor complex results in NF-κB activation. Most TNFRs transmit signals intracellularly by recruitment of adaptor proteins called TNFR-associated factors (TRAFs). However, BAFF-R binds only one TRAF adaptor, TRAF3, and this interaction negatively regulates activation of NF-κB. In this study, we report the crystal structure of a 24-residue fragment of the cytoplasmic portion of BAFF-R bound in complex with TRAF3. The recognition motif 162PVPAT166 in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTβR, but is presented in a completely different structural framework. This region of BAFF-R assumes an open conformation with two extended strands opposed at right angles that each make contacts with TRAF3. The recognition motif is located in the N-terminal arm and intermolecular contacts mediate TRAF recognition. In the C-terminal arm, key stabilizing contacts are made, including critical hydrogen bonds with Gln379 in TRAF3 that define the molecular basis for selective binding of BAFF-R solely to this member of the TRAF family. A dynamic conformational adjustment of Tyr377 in TRAF3 occurs forming a new intermolecular contact with BAFF-R that stabilizes the complex. The structure of the complex provides a molecular explanation for binding affinities and selective protein interactions in TNFR-TRAF interactions.

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.

TRAFs (tumor necrosis factor receptor [TNFR]-associated factors) bind to the cytoplasmic portion of liganded TNFRs and stimulate activation of NF-κB or JNK pathways. A modulator of TRAF signaling, TANK, serves as either an enhancer or an inhibitor of TRAF-mediated signaling pathways. The crystal structure of a region of TANK bound to TRAF3 has been determined and compared to a similar CD40/TRAF3 complex. TANK and CD40 bind to the same crevice on TRAF3. The recognition motif PxQxT is presented in a boomerang-like structure in TANK that is markedly different from the hairpin loop that forms in CD40 upon binding to TRAF3. Critical TANK contact residues were confirmed by mutagenesis to be required for binding to TRAF3 or TRAF2. Binding affinity, measured by isothermal titration calorimetry and competition assays, demonstrated that TANK competes with CD40 for the TRAF binding site.

Background: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. Results: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II β turn. Conclusions: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

We have recently identified a series of compounds which efficiently inhibit Anthrax lethal factor (LF) metallo-protease. Here we present further structure activity relationship and CoMFA (Comparative Molecular Field Analysis) studies on newly derived inhibitors. The obtained 3D QSAR model was subsequently compared with the X-ray structure of the complex between LF and a representative compound. Our studies form the basis for the rational design of additional compounds with improved activity and selectivity.

An antibody engineering strategy was employed to build high affinity ligands and antagonists of integrins alpha v beta 3 and alpha IIb beta 3. Previously, we inserted the integrin recognition motif, RGD, into the antigen binding site of a human antibody and selected the optimal flanking sequences from a phage-display library (Barbas, C. F., Languino, L. R., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007). The resulting antibody, Fab-9, blocked the function of integrin alpha v beta 3 but also bound to the ligand binding site of platelet integrin alpha IIb beta 3. In this report, the antibody engineering effort has been extended by 1) redesigning Fab-9 to achieve specificity for platelet integrin alpha IIb beta 3, 2) building non-RGD-containing antibodies that bind the ligand binding site of both beta 3-integrins, and 3) testing the hypothesis that peptides derived from complementarity determining regions (CDR) can be used to emulate the activity of the parent synthetic antibody. These goals were accomplished by subjecting the original antibody, Fab-9, to a "motif optimization" (MTF). A phage library was constructed in which the residues flanking the RGD motif in Fab-9 were maintained, but the RGDX sequence was randomized. This library was panned on purified alpha IIb beta 3 to identify high affinity binders. Four function-blocking antibodies lacking RGD, but with specificity for alpha IIb beta 3, were characterized. The antibody with the highest preference for alpha IIb beta 3, MTF-10, had an adhesion sequence of KGDN. This sequence is similar in primary structure to the active sequence within the disintegrin barbourin, which also antagonizes alpha IIb beta 3 (Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266, 9359-9362). MTF-10 had a 70-fold higher affinity for alpha IIb beta 3 than alpha v beta 3. Through our selection strategy, we also identified several antibodies that lack RGD but still blocked ligand binding to both integrins with high affinity. Therefore, the RGD sequence is not necessary for a high affinity interaction with the ligand binding site of beta 3-integrins. Further investigation showed that the activity of inhibitory antibodies could be emulated by synthetic peptides derived from the protein sequences of the antibody's HCDR3. CDR-derived peptides blocked ligand binding to integrins and maintained essentially the same specificity as the parent antibody.

Bcl-2 can be converted into a pro-apoptotic molecule by nuclear receptor Nur77. The development of Bcl-2 converters as anti-cancer therapeutics has been explored by us. We reported recently the identification of a Nur77-derived Bcl-2 converting peptide (NuBCP) and its enantiomer, which induce apoptosis of cancer cells in vitro and in animals. The apoptotic effect of NuBCP enantiomers and their activation of Bax are not inhibited but rather potentiated by Bcl-2. Using fluorescence polarization assays, we determined that NuBCP enantiomers bind both quantitatively and stoichiometrically to the Bcl-2 loop, which shares the characteristics of structurally adaptable regions with many cancer-associated signaling proteins. NuBCP-9 enantiomers act as molecular switches to dislodge the Bcl-2 BH4 motif exposing its BH3 motif. Mechanistically we demonstrated, using fluorescence quenching based liposome assays, that NuBCP-9-induced Bcl-2 conformational change not only neutralizes Bcl-2's inhibition of Bax-mediated membrane permeabilization but also exposes the Bcl-2's BH3 motif that in turn neutralizes Bcl-XL's inhibition of Bax like BH3 motif-derived peptides and compounds. Our results provide mechanistic insight into Bcl-2 conversion and identify a new direction for developing Bcl-2-based cancer therapeutics. (This work is in part supported by the grant GM062964 to J. Lin from the National Institute of Health.)

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with > or =50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with Kd 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.

The natural product gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture and was identified as an active compound in a cell-based high-throughput screening assay for activators of caspases, proteases involved in apoptosis. Using the antiapoptotic Bcl-2 family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescence polarization assay. Analysis of competition for BH3 peptide binding revealed that GA inhibits all six human Bcl-2 family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited [concentrations required for 50% inhibition (IC50)