Bax BH3 Peptides Research Articles

Synthesis, characterization, biological applications, and molecular docking studies of some transition metal complexes with azo dye ligand derived from 5-methyl imidazole

Metal complexes for Pd (II) and Pt (IV) ions were prepared and spectral identified using a new heterocyclic azo dye ligand 2-[2′-(3,4-dimethyl carboxy phenyl)azo]-5-methyl imidazole. In an alkaline ethanolic solution, a diazonium chloride salt solution of 2-amino-3,4-dimethyl benzoic acid and 5-methyl imidazole was reacted. Various spectral techniques, such as elemental analysis, (1HNMR , mass, FT-IR, UV–Vis.) spectroscopy, thermal analysis and FE-SEM-EDX, have been used to investigate azo dye ligand and chelate complex structures. Due to the presence of Cl outside the coordination sphere, the Pt(IV)-complex is the electrolyte because it contains conductive species, while the Pd(II) complex is not an electrolyte and does not contain conductive species. Based on the results, the ligand (N, N, O) behaves as a tridentate and coordinates with the metal ion via N-atom of the azomethine group (C = N) of the heterocyclic imidazole molecule, N-atom of the azo group (N = N) which are the furthest from imidazole ring, and O-atom of the benzoic acid ring. DPPH radical scavenging assays were also carried out on synthesized compounds to determine their antioxidant activities. A DPPH radical scavenging assay was used to measure the antioxidant activity of produced compounds and to compare them with ascorbic acid, a well-known antioxidant. Accordingly, the MTT cytotoxicity assay was used to assess the anti-cancer and toxicity of Pt(IV)-Complex against a breast cancer cell line (MCF-7). Molecular docking studies were performed on the new ligand and its metal complexes. It has been shown that the biological activity of compounds against breast cancer receptors was demonstrated by their interaction with BCL-2 and a BAX BH3 peptide from the RCSB protein data bank, PDB ID: 2XA0.

The antitumor activity of Bax BH3 peptide delivered by gold nanoparticles

Cancer has long remained one of the primary causes of disease and death globally, and it continues to pose a significant threat to human health. Surgery, radiotherapy, and chemotherapy are traditional treatments for cancer which are still widely used. However, while both surgery and radiotherapy remain acceptably effective in addressing a variety of primary tumors, neither possesses therapeutic potential for unknown metastatic lesions which may exist elsewhere in the patient’s body. Conversely, while systemic chemotherapy does have the potential for therapeutic efficacy on both primary and metastatic lesions alike, drug targeting is often poor, side effects are typically high, and treatment efficacy is still often lacking. Nanomedicine presents a promising solution to the above issues. For example, nanocarriers can be used to increase drug selectivity and targeting, increasing efficacy and decreasing side effects. Based on the role of the lethal domain of Bcl-2 family proteins, which play roles in apoptosis, the BH3-only protein, a section of BH3 peptide with a sequence of Asp-Ala-Ser-Thr-Lys-Lys-Leu-Ser-Glu-Cys-Leu-Arg-Arg-Ile-Gly-Asp-Glu-Leu-Asp-Ser. However, treatments based on soluble BH3 peptides, as with those based on other biologically active macromolecules, exhibit low cell membrane permeability, poor stability against proteolysis, and low endosomal escape rates. In this paper, attempt to address these issues by developing a variety of BH3@gold nanoparticle drug systems with different coating ratios. We demonstrate, high loading efficiency and, excellent anti-tumor effects in vitro, including inhibition of proliferation and migration in the human lung adenocarcinoma cell line, A549. Our results present a new possibility for anticancer peptide drugs in the future.

Intracellular Delivery of Active Proteins by Polyphosphazene Polymers.

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

Cell-Permeable Bak BH3 Peptide Induces Chemosensitization of Hematologic Malignant Cells.

Hematologic malignancies such as leukemias and lymphomas are among the leading causes of pediatric cancer death worldwide, and although survival rates have improved with conventional treatments, the development of drug-resistant cancer cells may lead to patient relapse and limited possibilities of a cure. Drug-resistant cancer cells in these hematologic neoplasms are induced by overexpression of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein families, such as Bcl-XL, Bcl-2, and Mcl-1. We have previously shown that peptides from the BH3 domain of the proapoptotic Bax protein that also belongs to the Bcl-2 family may antagonize the antiapoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. Furthermore, cell-permeable Bax BH3 peptides also elicit antitumor activity and extend survival in a murine xenograft model of human B non-Hodgkin's lymphoma. However, the activity of the BH3 peptides of the proapoptotic Bak protein of the Bcl-2 family against these hematologic malignant cells requires further characterization. In this study, we report the ability of the cell-permeable Bak BH3 peptide to restore apoptosis and induce chemosensitization of acute lymphoblastic leukemia and non-Hodgkin's lymphoma cell lines, and this event is enhanced with the coadministration of cell-permeable Bax BH3 peptide and represents an attractive approach to improve the patient outcomes with relapsed or refractory hematological malignant cells.

Live Attenuated Salmonella enterica Expressing and Releasing Cell-Permeable Bax BH3 Peptide Through the MisL Autotransporter System Elicits Antitumor Activity in a Murine Xenograft Model of Human B Non-hodgkin's Lymphoma.

The survival of patients with non-Hodgkin's lymphoma (NHL) has substantially improved with current treatments. Nevertheless, the appearance of drug-resistant cancer cells leads to patient relapse. It is therefore necessary to find new antitumor therapies that can completely eradicate transformed cells. Chemotherapy-resistant cancer cells are characterized by the overexpression of members of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family, such as Bcl-XL, Bcl-2, and Mcl-1. We have recently shown that peptides derived from the BH3 domain of the pro-apoptotic Bax protein may antagonize the anti-apoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. In this study, we investigated the feasibility of releasing this peptide into the tumor microenvironment using live attenuated Salmonella enterica, which has proven to be an ally in cancer therapy due to its high affinity for tumor tissue, its ability to activate the innate and adaptive antitumor immune responses, and its potential use as a delivery system of heterologous molecules. Thus, we expressed and released the cell-permeable Bax BH3 peptide from the surface of Salmonella enterica serovar Typhimurium SL3261 through the MisL autotransporter system. We demonstrated that this recombinant bacterium significantly decreased the viability and increased the apoptosis of Ramos cells, a human B NHL cell line. Indeed, the intravenous administration of this recombinant Salmonella enterica elicited antitumor activity and extended survival in a xenograft NHL murine model. This antitumor activity was mediated by apoptosis and an inflammatory response. Our approach may represent an eventual alternative to treat relapsing or refractory NHL.

Mathematical Modeling of Linear Docking. II. Estimating the Effect of Point Mutations on the Affinity between Protein Molecules

A new method enabling the qualitative determination of the dissociation constant of peptides to full-length proteins and the estimation of the effect of point mutations in peptides on the stability of a formed complex with whole proteins was presented. Based on the developed approach, a qualitative correlation was revealed between the obtained results and the dissociation constant using the formation of a biocomplex of the Bmf, Puma, Bad, Hrk, Bax, Bik, Noxa, Bid, Bim, and Bak BH3-peptides and the Bcl-xl protein and a biocomplex of the Bax BH3-peptides and the Bcl-2 protein with consideration for the replacement of amino acid residues as an example.

After Embedding in Membranes Antiapoptotic Bcl-XL Protein Binds Both Bcl-2 Homology Region 3 and Helix 1 of Proapoptotic Bax Protein to Inhibit Apoptotic Mitochondrial Permeabilization

Bcl-XL binds to Bax, inhibiting Bax oligomerization required for mitochondrial outer membrane permeabilization (MOMP) during apoptosis. How Bcl-XL binds to Bax in the membrane is not known. Here, we investigated the structural organization of Bcl-XL·Bax complexes formed in the MOM, including the binding interface and membrane topology, using site-specific cross-linking, compartment-specific labeling, and computational modeling. We found that one heterodimer interface is formed by a specific interaction between the Bcl-2 homology 1-3 (BH1-3) groove of Bcl-XL and the BH3 helix of Bax, as defined previously by the crystal structure of a truncated Bcl-XL protein and a Bax BH3 peptide (Protein Data Bank entry 3PL7). We also discovered a novel interface in the heterodimer formed by equivalent interactions between the helix 1 regions of Bcl-XL and Bax when their helical axes are oriented either in parallel or antiparallel. The two interfaces are located on the cytosolic side of the MOM, whereas helix 9 of Bcl-XL is embedded in the membrane together with helices 5, 6, and 9 of Bax. Formation of the helix 1·helix 1 interface partially depends on the formation of the groove·BH3 interface because point mutations in the latter interface and the addition of ABT-737, a groove-binding BH3 mimetic, blocked the formation of both interfaces. The mutations and ABT-737 also prevented Bcl-XL from inhibiting Bax oligomerization and subsequent MOMP, suggesting that the structural organization in which interactions at both interfaces contribute to the overall stability and functionality of the complex represents antiapoptotic Bcl-XL·Bax complexes in the MOM.

Unlatched BAX Pairs for Death

Self-interacting BAX proteins permeabilize outer mitochondrial membranes to trigger apoptotic cell death. Czabotar et al. present two revealing structures of BAX dimers: one dimer has an activator BH3 helix bound into its canonical cleft, and the other dimer exposes a planar hydrophobic face potentially critical for membrane interactions.

Bax Forms an Oligomer via Separate, Yet Interdependent, Surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1-3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1-3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1-3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.

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 γ-herpesvirus 68 (γHV68) Bcl-2 family protein (γHV68 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 γHV68 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 γHV68 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 γHV68 from latency and efficient persistent γHV68 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.

Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides

Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl-2 and Bcl-XL inhibit apoptosis by binding to proapoptotic proteins such as Bax, thereby preventing chemotherapy-induced or radiation-induced release of cytochrome c from mitochondria and subsequent activation of the caspase protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL function in tumor cells have focused on developing agents to inhibit the interactions of these proteins with proapoptotic proteins. Peptides derived from the BH3 domains of proapoptotic proteins have been shown to disrupt the interactions of Bcl-2 and Bcl-XL with key binding partners in cell-free reactions and to promote cellular apoptosis. However, less is known about the targets of BH3 peptides in intact cells as well as the sequence, length, and conformational requirements for peptide biological activity. In this report, we show that cell-permeable Bax BH3 peptides physically disrupt Bax/Bcl-2 heterodimerization in intact cells and that this disruption correlates with peptide-induced cell death. A point-mutant, control peptide that failed to disrupt intracellular Bax/Bcl-2 interactions also failed to promote apoptosis. To determine important sequence, length, and structural requirements for peptide activity, we generated and systematically analyzed the biological activities of 17 Bax BH3 peptide variants. Peptides were quantitatively examined for their ability to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization in vitro and to promote cytochrome c release from mitochondria isolated from Jurkat, HL-60, U937, and PC-3 cells. Our results define 15 amino acids as the minimal length required for Bax BH3 peptide biological activity and show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH2 terminal to the core. In addition, circular dichroism spectroscopy revealed that high alpha-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimization of Bax BH3 peptide as a therapeutic anticancer agent.

Divergence of Genbank and human tumor Bcl-2 sequences and implications for binding affinity to key apoptotic proteins

Heterodimerization of antiapoptotic and pro-apoptotic Bcl-2 family of proteins provides an important mechanism for apoptosis regulation. Knowledge about key amino acids in the binding groove of native Bcl-2 contributing to this interaction will greatly facilitate the design of Bcl-2-specific inhibitors. There are two different Bcl-2 sequences, M13994 and M14745, in Genbank. Chimeric proteins Bcl-2(1) and Bcl-2(2) derived from the above sequences, although similar in structure, showed different binding affinities to Bak and Bad BH3 peptides (Petros et al., 2001). In this study, we show that the Bcl-2(1) sequence in normal and tumor human tissue samples differs from M13994 and M14745, and contains P59, T96, R110, S117 and G237. The actual sequence in the binding pocket matches the Bcl-2-Ig fusion sequence X06487, originally identified in a t(14:18) translocation of the Bcl-2 gene, associated with follicular lymphoma. The possible effects of the observed amino acid differences compared to M13994 and M14745 were investigated by combining structural data with fluorescence anisotropy. G110R substitution confers on Bcl-2(1) substantially increased binding affinity to Bak, Bad and Bax BH3 peptides, demonstrating that R110 is a key contributor to the BH3 binding affinity of Bcl-2. Although NMR structure did not predict R110 involvement in binding to these BH3 peptides, fluorescence anisotropy data clearly points to a critical role for this residue in binding to pro-apoptotic Bcl-2 family members.