The Enterococcus faecalis cytolysin: a novel toxin active against eukaryotic and prokaryotic cells

The enterococcal cytolysin, a two-peptide lytic system, is a divergent relative of a large family of toxins and bacteriocins secreted by pathogenic and non-pathogenic Gram-positive bacteria. This family includes the lantibiotics and streptolysin S. The enterococcal cytolysin is of interest because its activities enhance enterococcal virulence in infection models and, in epidemiological studies, it has been associated with patient mortality. The cytolysin is lethal for a broad range of prokaryotic and eukaryotic cells, and this activity requires two non-identical, post-translationally modified peptides. The smaller of the two peptides also plays a role in a quorum-sensing autoinduction of the cytolysin operon. As a trait that is present in particularly virulent strains of Enterococcus faecalis, including strains that are resistant to multiple antibiotics, it serves as a model for testing the value of developing new virulence-targeting therapeutics. Further, because of the interest in small membrane active peptides as therapeutics themselves, studies of the molecular structure/activity relationships for the cytolysin peptides are providing insights into the physical basis for prokaryotic versus eukaryotic cell targeting.     

Linking genomics to immunotherapy by reverse immunology--'immunomics' in the new millennium

The disclosure of the human genome sequence and rapid advances in genomic expression profiling have revolutionized our knowledge about molecular changes in malignant diseases. Rapidly growing gene expression databases and improvements in bioinformatics tools set the stage for new approaches using large-scale molecular information to develop specific therapeutics in cancer. On one hand, the ability to detect clusters of genes differentially expressed in normal and malignant tissue may lead to widely applicable targeting of defined molecular structures. On the other hand, analyzing the 'molecular fingerprint' of an individual tumor raises the possibility of developing customized therapeutics. One approach to use the emerging new datasets for the development of novel therapeutics is to identify genes that are specifically expressed in tumors as targets for immune intervention. This review will focus on the process from in silico analysis of expression databases and screening of potential candidate genes by bioinformatics to the in vitro and in vivo analysis to determine the immunogenicity of candidate tumor antigens. Basic biological principles of 'reverse immunology' as well as technical advantages and difficulties will be addressed.     

Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity

Botulinum neurotoxins (BoNTs) are among the most lethal biological substances to have been weaponized and are listed as biodefense category A agents. Currently, no small molecule (non-peptidic) therapeutics exist to counter this threat; hence, identifying and developing compounds that inhibit BoNTs is a high priority. In the present study, a high-throughput assay was used to identify small molecules that inhibit the metalloprotease activity of BoNT serotype A light chain (BoNT/A LC). All inhibitors were further verified using a HPLC-based assay. Conformational analyses of these compounds, in conjunction with molecular docking studies, were used to predict structural features that contribute to inhibitor binding and potency. Based on these results, a common pharmacophore for BoNT/A LC inhibitors is proposed. This is the first study to report small molecules (non-peptidics) that inhibit BoNT/A LC metalloprotease activity in the low microM range.     

Anti-inflammatory Compounds Parthenolide and Bay 11-7082 Are Direct Inhibitors of the Inflammasome

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1β and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-κB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-κB inhibitors revealed that the synthetic IκB kinase-β inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-κB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.     

Interaction of the putative anticancer alkaloid chelerythrine with nucleic acids: biophysical perspectives

Alkaloids represent an important group of molecules that have immense pharmacological potential. Benzophenanthridine alkaloids are one such class of alkaloids known for their myriad pharmacological activities that include potential anticancer activities. Chelerythrine is a premier member of the benzophenanthridine family of the isoquinoline group. This alkaloid is endowed with excellent medicinal properties and exhibits antibacterial, antimicrobial and anti-inflammatory properties. The molecular basis of its therapeutic activity is considered due to its nucleic acid binding capabilities. This review focuses on consolidating the current status on the nucleic acid binding properties of chelerythrine that is essential for the rational design and development of this alkaloid as a potential drug. This work reviews the interaction of chelerythrine with different natural and synthetic nucleic acids like double- and single-stranded DNAs, heat-denatured DNA, quadruplex DNA, double- and single-stranded RNA, tRNA and triplex and quadruplex RNA. The review emphasizes on the mode, specificity, conformational aspects and energetics of the binding that is particularly helpful for developing nucleic acid targeted therapeutics. The fundamental results discussed in this review will greatly benefit drug development for many diseases and serve as a database for the design of futuristic benzophenanthridine-based therapeutics.     

Engineering the variable region of therapeutic IgG antibodies

Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics.Key words: antibody therapeutics, variable region, engineering, affinity, pharmacokinetics, stability, immunogenicity     

Optimization algorithms for functional deimmunization of therapeutic proteins

Background  

To develop protein therapeutics from exogenous sources, it is necessary to mitigate the risks of eliciting an anti-biotherapeutic immune response. A key aspect of the response is the recognition and surface display by antigen-presenting cells of epitopes, short peptide fragments derived from the foreign protein. Thus, developing minimal-epitope variants represents a powerful approach to deimmunizing protein therapeutics. Critically, mutations selected to reduce immunogenicity must not interfere with the protein's therapeutic activity.     

Non-‘classical’ MEKs: A review of MEK3-7 inhibitors

The MAPK pathways are an enduring area of interest due to their essential roles in cell processes. Increased expression and activity can lead to a multitude of diseases, sparking research efforts in developing inhibitors against these kinases. Though great strides have been made in developing MEK1/2 inhibitors, there is a notable lack of chemical probes for MEK3-7, given their central role in stimuli response, cell growth, and development. This review summarizes the progress that has been made on developing small molecule probes for MEK3-7, the specific disease states in which they have been studied, and their potential to become novel therapeutics.     

综述与专论: 核酸适配体在分子医学中的应用

分子医学着眼于从疾病的分子层面出发,为个性化精准诊疗提供解决方案。然而,在众多疾病的诊疗中由于缺乏有力的分子识别工具,目前从分子水平上理解和研究疾病仍受到制约。核酸适配体是通过指数富集的配体系统进化（SELEX）技术在体外筛选得到的单链寡核苷酸,具有高选择性、高亲和力、易细胞内化、良好的组织渗透和快速的组织积累能力。近年来,由于其易合成、成本低、稳定性高且免疫原性低,核酸适配体作为分子工具应用于疾病的诊疗一体化受到广泛关注。本综述围绕分子医学中的核酸适配体,讨论了核酸适配体在疾病诊断中的应用,包括基于核酸适配体的肿瘤标志物发现、液体活检、分子成像。介绍了核酸适配体在癌症治疗中的应用包括基于核酸适配体的抑制剂、核酸适配体药物偶联物、纳米药物和核酸适配体介导的免疫治疗。最后对核酸适配体在临床诊疗和产业化面临的问题进行了讨论,包括基于应用场景的筛选方法、核酸适配体与靶标复合物结构、亲和力的机制以及核酸适配体在血液循环中的稳定性等方面。     

Histone Deacetylase (HDAC) 10 Suppresses Cervical Cancer Metastasis through Inhibition of Matrix Metalloproteinase (MMP) 2 and 9 Expression

Aberrant expression of histone deacetylases (HDACs) is associated with carcinogenesis. Some HDAC inhibitors are widely considered as promising anticancer therapeutics. A major obstacle for development of HDAC inhibitors as highly safe and effective anticancer therapeutics is that our current knowledge on the contributions of different HDACs in various cancer types remains scant. Here we report that the expression level of HDAC10 was significantly lower in patients exhibiting lymph node metastasis compared with that in patients lacking lymph node metastasis in human cervical squamous cell carcinoma. Forced expression of HDAC10 in cervical cancer cells significantly inhibited cell motility and invasiveness in vitro and metastasis in vivo. Mechanistically, HDAC10 suppresses expression of matrix metalloproteinase (MMP) 2 and 9 genes, which are known to be critical for cancer cell invasion and metastasis. At the molecular level, HDAC10 binds to MMP2 and -9 promoter regions, reduces the histone acetylation level, and inhibits the binding of RNA polymerase II to these regions. Furthermore, an HDAC10 mutant lacking histone deacetylase activity failed to mimic the functions of full-length protein. These results identify a critical role of HDAC10 in suppression of cervical cancer metastasis, underscoring the importance of developing isoform-specific HDAC inhibitors for treatment of certain cancer types such as cervical squamous cell carcinoma.     

Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies

Monoclonal antibodies are the fastest growing class of biologics in the pharmaceutical industry. The correlation between mAb glycosylation and aggregation has not been elucidated in detail, yet understanding the structure-stability relationship involving glycosylation is critical for developing successful drug formulations. We conducted studies of temperature-induced aggregation and compared the stability of both glycosylated and aglycosylated forms of a human IgG1. In parallel, we also performed molecular dynamics simulations of the glycosylated full antibody to gain an understanding of the polysaccharide surroundings at the molecular level. Aglycosylated mAbs are somewhat less stable and therefore aggregate more easily than the glycosylated form at the temperatures studied. Glycosylation seems to enhance solubility and stability of these therapeutics and thus might be important for long-term storage.     

Autoantibodies in cancer: prognostic biomarkers and immune activation

The development of proteomic technologies that display a wide variety of antigenic structures has led to the identification of autoantibodies to cancer-derived tumor antigens. These autoantibodies have been detected in sera from patients with multiple cancer types, and are being evaluated as biomarkers for early cancer detection. It is not known whether these antibodies also contribute to active immune surveillance or even tumorigenicity of developing tumors. Here, we review which tumor antigen-specific antibodies are prognostic biomarkers of cancer outcome, and emerging proteomic methods for the isolation and cloning of these antibodies for potential molecular diagnostics and therapeutics.     

Autoantibodies in cancer: prognostic biomarkers and immune activation

The development of proteomic technologies that display a wide variety of antigenic structures has led to the identification of autoantibodies to cancer-derived tumor antigens. These autoantibodies have been detected in sera from patients with multiple cancer types, and are being evaluated as biomarkers for early cancer detection. It is not known whether these antibodies also contribute to active immune surveillance or even tumorigenicity of developing tumors. Here, we review which tumor antigen-specific antibodies are prognostic biomarkers of cancer outcome, and emerging proteomic methods for the isolation and cloning of these antibodies for potential molecular diagnostics and therapeutics.     

Engineering the variable region of therapeutic IgG antibodies

Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics.     

Nitrones as therapeutics in age-related diseases

Age-related diseases deprive individuals of a higher quality of life and therefore therapeutics for their treatment provide significant potential. An overview of the observations of nitrones as potential therapeutics in several age-related diseases is presented. Treatment of acute ischemic stroke is one condition where a nitrone (NXY-059) is in late phase 3 clinical trials now. Also presented is a summary of the most recent work we have accomplished on the anticancer activity of the nitrones in a hepatocellular carcinoma. The mechanistic basis of action of these compounds in several animal models is not yet understood at the molecular levels; however, it does appear clear that their anti-inflammatory properties are central to their action, which is based on their ability to down-regulate exacerbated signal transduction processes.     

BRCA1-directed,enhanced and aberrant homologous recombination

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitinligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.     

Modulating molecular chaperone Hsp90 functions through reversible acetylation

The molecular chaperone protein Hsp90 is a key regulator of approximately 100 'client' proteins crucial for numerous cell signaling processes. Consequently, understanding the molecular underpinnings that regulate Hsp90 activity is an important biological endeavor. Exciting new results now suggest that, at least for nuclear receptor activity, Hsp90 function is directly regulated by histone deacetylase 6 (HDAC6). These observations have consequences for various biological processes and potentially important implications for the development of cancer therapeutics.     

Utilization of genomic signatures to direct use of primary chemotherapy

The success of treatment of cancer patients depends on matching the most effective therapeutic regimen with the characteristics of the individual patient, balancing benefit against risk of adverse events. The primary challenge in achieving this goal is the heterogeneity of the disease, recognizing that breast, lung, colon and other cancers are not single diseases but rather an array of disorders with distinct molecular mechanisms. Genomic analyses, and in particular gene expression profiling, has been shown to have the capacity to dissect this heterogeneity and afford opportunities to match therapies with the characteristics of the individual patient's tumor. Here we review the success in developing gene expression signatures that have the capability of predicting response to various commonly used and newly developing cancer therapeutics. We further discuss the challenges and the opportunities in utilizing these tools in present-day clinical practice.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.