Bcl-2-related proteins as drug targets

The Bcl-2 family of proteins provide the most unambiguous link between mitochondrial functions and apoptosis, as their only (or principal) functions appear to be as regulators of this cell death pathway. Rational drug design to manipulate the functions of these proteins has been hampered by the lack of a clear understanding of a biochemical or molecular function, with disruption of intra-family protein-protein interactions as the only known, but daunting, objective. There has been substantial progress in this task using molecular modeling and drug leads. The prospects are also good for development of chemical tools for functional analysis of the Bcl-2 proteins.     

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

MicroRNAs (miRNAs) can regulate cell survival and death by targeting apoptosis-related gene expression. miR-210 is one of the most hypoxia-sensitive miRNAs. In this study, we evaluated the roles of miR-210 in hypoxia-induced insults to neural cells. Treatment of neuro-2a cells with oxygen/glucose deprivation (OGD) induced cell apoptosis in a time-dependent manner. In parallel, OGD time-dependently increased cellular miR-210 levels. Knocking down miR-210 expression using specific antisenses significantly attenuated OGD-induced neural apoptosis. Concurrently, OGD increased hypoxia-inducible factor (HIF)-1α mRNA and protein syntheses. Pretreatment with YC-1, an inhibitor of HIF-1α, reduced OGD-caused cell death. Sequentially, OGD specifically decreased antiapoptotic Bcl-2 mRNA and protein levels in neuro-2a cells. A search by a bioinformatic approach revealed that miR-210-specific binding elements exist in the 3′-untranslated region of Bcl-2 mRNA. Application of miR-210 antisenses simultaneously alleviated OGD-involved inhibition of Bcl-2 mRNA expression. In comparison, overexpression of miR-210 synergistically diminished OGD-caused inhibition of Bcl-2 mRNA expression and consequently induced greater cellular insults. Taken together, this study shows that OGD can induce miR-210 expression through activating HIF-1α. And miR-210 can mediate hypoxia-induced neural apoptosis by targeting Bcl-2.     

Microparticles: targets and tools in cardiovascular disease

Cells communicate with other cells not only via direct cell-cell contact and the production of signaling molecules but also through release of microparticles (MPs). MPs are small vesicles released from stimulated and/or apoptotic cells. They harbor membrane proteins that are characteristic of the original parent cell and intracellular components involved in cell signaling. MPs are considered to be both biomarkers and effectors of cell signaling that maintain and/or initiate cell dysfunction. Thus, MPs can evoke endothelial dysfunction by decreasing nitric oxide (NO) production and promoting vascular inflammation which favor the prothrombotic state in atherosclerosis. Novel pharmacological approaches targeting MP production or properties could be used to treat cardiovascular pathologies. Paradoxically, another useful approach might be to employ engineered MPs with modified compositions as therapeutic agents to correct cardiovascular pathologies. This review is focused on the mechanisms of MP formation and their effects on target cells under physiological or pathophysiological conditions.     

Bcl-2 family members as molecular targets in cancer therapy

Escape from apoptosis is often a hallmark of cancer cells, and is associated to chemotherapy resistance or tumor relapse. Proteins from the Bcl-2 family are the key regulators of the intrinsic pathway of apoptosis, controlling the point-of no-return and setting the threshold to engage the death machinery in response to a chemical damage. Therefore, Bcl-2 proteins have emerged as an attractive target to develop novel anticancer drugs. Current pharmacological approaches are focused on the use of peptides, small inhibitory molecules or antisense oligonucleotides to neutralize antiapoptotic Bcl-2 proteins, lowering the threshold and facilitating apoptosis of cancer cells. We discuss here recent advances in the development of Bcl-2 targeted anticancer therapies.     

Inhibitors of the anti-apoptotic Bcl-2 proteins: a patent review

INTRODUCTION: The B-cell lymphoma-2 (Bcl-2) family of proteins is central to the regulation of apoptosis, which is vital for proper tissue development and cellular homeostasis. Anti-apoptotic proteins, members of the Bcl-2 family, are an important survival factor for many cancers and their overexpression has been associated with tumor initiation, progression, and resistance to current anticancer therapies. Therefore, strategies seeking to antagonize the function of Bcl-2 anti-apoptotic proteins have been extensively studied for developing a novel cancer therapy. AREAS COVERED: This review covers research and patent literature of the last 15 years dealing with the discovery and development of inhibitors of the Bcl-2 protein family. EXPERT OPINION: The feasibility of disrupting protein-protein interactions between anti-apoptotic and pro-apoptotic proteins, members of the Bcl-2 family, using peptidomimetics and small-molecule inhibitors has been successfully established. Three small-molecule inhibitors have entered human clinical trials, which will allow the evaluation of this potential therapeutic approach in cancer patients. It will be important to gain a better understanding of pan and selective Bcl-2 inhibitors in order to facilitate future drug design efforts.     

Acylpyrogallols as inhibitors of antiapoptotic Bcl-2 proteins

A series of acylpyrogallols were designed, synthesized, and evaluated as small-molecule inhibitors of antiapoptotic Bcl-2 proteins. The most potent compound 9 (TM-179) binds to Bcl-2 with an IC50 of 170 nM and to Mcl-1 with a Ki of 37 nM. Compound 9 potently inhibits cell growth and induces apoptosis in human breast and prostate cancer cell lines.     

ApoG2 inhibits antiapoptotic Bcl-2 family proteins and induces mitochondria-dependent apoptosis in human lymphoma U937 cells

Lymphoma is one of the most common types of hematological malignancies and proteins from the Bcl-2 family are highly expressed in human lymphomas. Apogossypolone (ApoG2), the most potent gossypol derivative, has been classified as a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins. Here, we assessed the in-vitro cytotoxicity of ApoG2 on human U937 lymphoma cells, and explored the underlying intracellular molecular mechanisms of ApoG2. Using the WST-8 assay, we found that ApoG2 inhibited growth of U937 cells in a dose-dependent and time-dependent manner, and the IC50 values were 30.08, 14.81, and 9.26 mumol/l for 24, 48, and 72 h treatments, respectively. ApoG2 also induced apoptosis in U937 cells, as noted through changes in morphological characteristics, including cellular internucleosomal DNA fragmentation and the appearance of a sub-G1 apoptotic peak. Treatment with ApoG2 downregulated Bcl-xL and Mcl-1 protein expression and blocked the binding of Bcl-2 with Bax protein. Furthermore, ApoG2 led to an abundant release of cytochrome c from mitochondria and a five-fold increase in the activity of caspase-3 and caspase-9. Taken together, our results suggest that ApoG2 could effectively suppress the growth of human lymphoma cell line U937 through the inhibition of the antiapoptotic Bcl-2 family proteins and the induction of mitochondria-dependent apoptotic cell death.     

Nuclear proteins: promising targets for cancer drugs

Recent progress in cancer drug therapy has recognized that the nucleus of the eukaryotic cell is an active site for many cellular processes important to the development of cancer. Many of these processes take place in specialized compartments of the nucleus. One of such sub-nuclear compartments is the promyelocytic leukemia nuclear body (PML NB). In acute promyelocytic leukemia (APL), PML forms a fusion protein with the retinoic acid receptor (RAR) alpha as a result of chromosomal translocation. This PML-RAR alpha fusion protein is responsible for the proliferative and de-differentiated phenotype of the leukemic cells and is the target of all-trans retinoic acid (ATRA). Another example of the specialized sub-nuclear compartments important in the targeting of cancer is the nucleolus. Recently, it has been proposed that the nucleolus serves as a stress sensor for the cell, and the molecular mechanism underlying this proposal has been discovered. Moreover, many anti-cancer drugs target specific protein-protein interactions within the nucleus. We will discuss current development surrounding two such target proteins: the hypoxia-inducible factor 1 alpha (HIF-1alpha) and FKBP25. Furthermore, chromatin structure, which is affected by modifications of core histones, has become a target of anti-cancer drugs. In this review, we will emphasize the significance of nuclear proteins as promising targets for cancer drug therapy by discussing a few key ideas, in three broad categories of specialized sub-nuclear compartments, protein-protein interactions, and the modifications of the chromatin structure.     

作用于Bcl-2家族抗凋亡亚族蛋白的小分子抑制剂的研究进展

细胞的凋亡是维持机体平衡的重要因素。细胞凋亡由一系列细胞因子调控。Bcl-2蛋白家族是细胞凋亡的关键性调节因子。Bcl-2家族分为抗凋亡和促凋亡两个亚族，他们的相互作用对细胞凋亡信号传导起调控作用。很多肿瘤细胞高表达Bcl-2抗凋亡亚族成员Bcl-2/Bcl-xL。近年来，随着Bcl-2家族各成员的晶体结构相继阐明，人们开始寻找作用于Bcl-2家族抗凋亡亚族蛋白的小分子抑制剂。本文从药物设计角度对该方面的进展作一综述。     

In vivo veritas: Bcl-2 and Bcl-XLmediate tumor cell resistance to chemotherapy

Apoptosis is cellular suicide, the functional opposite of mitosis. It may play an important role in tissue growth control and removal of damaged and premalignant cells. The fact that diverse chemotherapeutic agents induce apoptosis, while they engage different intracellular targets and cause DNA damage, raises a concern that tumors resistant to chemotherapy are unable to initiate the apoptotic process. The anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-X_L, play an important role in the regulation of apoptotic cell death. Bcl-2 and Bcl-X_Lhave been reported to confer chemotherapy resistance in short-term survival assays in vitro. However, they failed to provide a long-term clonogenic survival advantage. Thus, the role of anti-apoptotic Bcl-2 and Bcl-X_Lon chemotherapy resistance in vivo remains unclear. In vivo, tumor cells receive survival signals from the extracellular microenvironment. Since the microenvironmental factors have been reported to modulate the expression and function of Bcl-2 family proteins, Bcl-2 and Bcl-X_Lmight be associated with the chemotherapy resistance in vivo through the influence of these factors. Consistent with this hypothesis, several investigators have recently reported that the sensitivity to chemotherapy in in vitro clonogenic assays did not correlate with that in in vivo tumor models. The lack of microenvironmental factors might cause the discrepancy between in vitro clonogenic growth and in vivo tumor growth. These results suggest that Bcl-2 and Bcl-X_Lcould contribute to chemotherapy resistance in vivo, along with already defined drug resistance mechanisms (i.e. P-glycoprotein, MRP). Therapies aimed at suppressing the expression and function of Bcl-2 and Bcl-X_Lor at intercepting microenvironmental factors might successfully overcome chemotherapy resistance.     

Methionine in and out of proteins: targets for drug design

The increasing need for new antibiotics to overcome rapidly developing resistance mechanisms observed in clinical isolates of Gram-positive and Gram-negative eubacteria has placed critical emphasis on the search for new antibacterial enzyme targets and the structural and mechanistic investigation of such targets. Among these potential targets, the enzymes responsible for integrating the amino acid methionine into proteins, along with its subsequent post-translational modification and repair, have emerged as promising candidates for the development of novel antibiotics. As well, there is increasing evidence for the importance of several of these enzymes in the development of anti-cancer, anti-parasitic, and anti-atherosclerotic drugs. Within the last three years, the crystal structures of all of these enzymes have been determined, which offers an unprecedented source of structural information for inhibitor design. The development of combinatorial chemistry and high throughput screening procedures has quickly provided several potent, specific inhibitors for a number of these enzymes, particularly the peptide deformylase, methionine aminopeptidase, and methionyl-tRNA synthetase enzymes. This review critically analyzes the future potential for inhibition of enzymes in this pathway, allowing for a pragmatic view of the success of inhibitor developments and highlighting areas in which further investigations are warranted.     

Applying genomics tools to identify therapeutic targets for asthma

Asthma is a chronic inflammatory disease of the airways, the susceptibility to which is strongly influenced by genetics. Genomics, the study of the human genome, is redefining the process for rapidly identifying novel therapeutic targets for asthma and other diseases. One approach is to search for polymorphisms in genes that increase susceptibility to the disease in order to identify genes and cellular pathways relevant to the disease process. In asthma, for example, regardless of the genetic factors that contribute to susceptibility, good drug targets could be found that affect epithelial integrity, allergic response, and the recruitment or activity of inflammatory cells. Such targets may consist of proteins that are specifically expressed in certain cell types, proteins whose expression is regulated during the disease process or proteins involved in the destructive process. This review discusses some of the genomics tools that can be used to identify new molecular targets, which in turn are useful in screening for novel compounds likely to affect diseases such as asthma.     

Applying genomics tools to identify therapeutic targets for asthma

Asthma is a chronic inflammatory disease of the airways, the susceptibility to which is strongly influenced by genetics. Genomics, the study of the human genome, is redefining the process for rapidly identifying novel therapeutic targets for asthma and other diseases. One approach is to search for polymorphisms in genes that increase susceptibility to the disease in order to identify genes and cellular pathways relevant to the disease process. In asthma, for example, regardless of the genetic factors that contribute to susceptibility, good drug targets could be found that affect epithelial integrity, allergic response, and the recruitment or activity of inflammatory cells. Such targets may consist of proteins that are specifically expressed in certain cell types, proteins whose expression is regulated during the disease process or proteins involved in the destructive process. This review discusses some of the genomics tools that can be used to identify new molecular targets, which in turn are useful in screening for novel compounds likely to affect diseases such as asthma.     

Rab proteins and endocytic trafficking: potential targets for therapeutic intervention

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo- and endocytic pathways. In their active forms, rab proteins serve in cargo selection and as scaffolds for the sequential assembly of effectors requisite for vesicle budding, cytoskeletal transport, and target membrane fusion. Rab protein function is in turn tightly regulated at the level of protein expression, localization, membrane association, and activation. Alterations in the rab GTPases and associated regulatory proteins or effectors have increasingly been implicated in causing human disease. Some diseases such as those resulting in bleeding and pigmentation disorders (Griscelli syndrome), mental retardation, neuropathy (Charcot-Marie-Tooth), kidney disease (tuberous sclerosis), and blindness (choroideremia) arise from direct loss of function mutations of rab GTPases or associated regulatory molecules. In contrast, in a number of cancers (prostate, liver, breast) as well as vascular, lung, and thyroid diseases, the overexpression of select rab GTPases have been tightly correlated with disease pathogenesis. Unique therapeutic opportunities lie ahead in developing strategies that target rab proteins and modulate the endocytic pathway.     

Isoniazid-induced apoptosis in HepG2 cells: Generation of oxidative stress and Bcl-2 down-regulation

Isoniazid (INH) is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. However it has a serious limitation of being hepatotoxic. Delineating the mechanism underlying INH-induced hepatotoxicity may be beneficial in devising ways to counteract its toxic manifestations. Studies in human hepatoma HepG2 cells have indicated that INH exposure causes induction of apoptosis. This study was aimed at identifying the key components/pathways of the INH-induced apoptotic pathway using HepG2 cells. HepG2 cells were exposed to increasing concentrations of INH (6.5, 13, 26, and 52?mM). Hydrogen peroxide (0.3?mM) served as positive control. After incubating for specific time intervals cells were harvested and evidences of cytotoxicity, oxidative stress, and apoptosis were sought. The findings indicated that INH exposure causes increased ROS generation along with alteration in levels of enzymatic antioxidants such as Superoxide dismutase, Catalase, and Glucose-6-Phosphate dehydrogenase. Altered Bcl-2/Bax content, cytochrome-c translocation, caspase activation, and DNA fragmentation emphasized involvement of apoptosis.