Divide and conquer: subproteomic approaches toward gastric cancer biomarker and drug target discovery

The discovery of biomarkers for early detection and treatment for gastric cancer are two important gaps that proteomics have the potential to fill. Advancements in mass spectrometry, sample preparation and separation strategies are crucial to proteomics-based discoveries and subsequent translations from bench to bedside. A great number of studies exploiting various subproteomic approaches have emerged for higher-resolution analysis (compared with shotgun proteomics) that permit interrogation of different post-translational and subcellular compartmentalized forms of the same proteins as determinants of disease phenotypes. This is a unique and key strength of proteomics over genomics. In this review, the salient features, competitive edges and pitfalls of various subproteomic approaches are discussed. We also highlight valuable insights from several subproteomic studies that have increased our understanding of the molecular etiology of gastric cancer and the findings that led to the discovery of potential biomarkers/drug targets that were otherwise not revealed by conventional shotgun expression proteomics.     

Perspective: a program to improve protein biomarker discovery for cancer

Biomarkers for cancer risk, early detection, prognosis, and therapeutic response promise to revolutionize cancer management. Protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology. An effective program to discover protein biomarkers using existing technology will require team science, an integrated informatics platform, identification and quantitation of candidate biomarkers in disease tissue, mouse models of disease, standardized reagents for analyzing candidate biomarkers in bodily fluids, and implementation of automation. Technology improvements for better fractionation of the proteome, selection of specific biomarkers from complex mixtures, and multiplexed assay of biomarkers would greatly enhance progress.     

Targeting protein homodimerization: a novel drug discovery system

To identify a novel class of antibiotics, we have developed a high-throughput genetic system for targeting the homodimerization (HD system) of histidine kinase (HK), which is essential for a bacterial signal transduction system (two-component system, TCS). By using the HD system, we screened a chemical library and identified a compound, I-8-15 (1-dodecyl-2-isopropylimidazole), that specifically inhibited the dimerization of HK encoded by the YycG gene of Staphylococcus aureus and induced concomitant bacterial cell death. I-8-15 also showed antibacterial activity against MRSA (methicillin-resistant S. aureus) and VRE (vancomycin-resistant Enterococcus faecalis) with MICs at 25 and 50 microg/ml, respectively.     

A tool for biomarker discovery in the urinary proteome: a manually curated human and animal urine protein biomarker database

Urine is an important source of biomarkers. A single proteomics assay can identify hundreds of differentially expressed proteins between disease and control samples; however, the ability to select biomarker candidates with the most promise for further validation study remains difficult. A bioinformatics tool that allows accurate and convenient comparison of all of the existing related studies can markedly aid the development of this area. In this study, we constructed the Urinary Protein Biomarker (UPB) database to collect existing studies of urinary protein biomarkers from published literature. To ensure the quality of data collection, all literature was manually curated. The website (http://122.70.220.102/biomarker) allows users to browse the database by disease categories and search by protein IDs in bulk. Researchers can easily determine whether a biomarker candidate has already been identified by another group for the same disease or for other diseases, which allows for the confidence and disease specificity of their biomarker candidate to be evaluated. Additionally, the pathophysiological processes of the diseases can be studied using our database with the hypothesis that diseases that share biomarkers may have the same pathophysiological processes. Because of the natural relationship between urinary proteins and the urinary system, this database may be especially suitable for studying the pathogenesis of urological diseases. Currently, the database contains 553 and 275 records compiled from 174 and 31 publications of human and animal studies, respectively. We found that biomarkers identified by different proteomic methods had a poor overlap with each other. The differences between sample preparation and separation methods, mass spectrometers, and data analysis algorithms may be influencing factors. Biomarkers identified from animal models also overlapped poorly with those from human samples, but the overlap rate was not lower than that of human proteomics studies. Therefore, it is not clear how well the animal models mimic human diseases.     

Application of proteomics to graft-versus-host disease: from biomarker discovery to potential clinical applications

Introduction: Graft-versus-host disease (GVHD) is a frequent and potentially life-threatening complication that occurs in many patients who undergo hematopoietic stem cell transplantation. In an effort to develop blood and tissue-based biochemical assays for GVHD diagnosis, high throughput proteomic platforms have been widely utilized for the identification and validation of disease biomarkers for both acute and chronic GVHD.

Areas covered: This article reviews biomarker research findings on acute and chronic GVHD ascertained by studying peripheral blood, urine and saliva that gives biological information on systemic or localized disease. While the primary focus of GVHD biomarker discovery has been on identification and validation of prognostic and predictive biomarkers that might allow stratification of disease risk, molecular biomarkers that might aid patient diagnosis and/or response to treatment have also been reported.

Expert commentary: Unbiased as well as targeted proteomic studies of acute and chronic GVHD have identified some distinguishing features of the two diseases especially the role of certain immune cell populations. A combination of patient risk stratification using panels of biomarkers and the application of novel targeted therapeutics should help to reduce the burden of GVHD, and hence improve the quality of life for patients following hematopoietic stem cell transplantation.     

Proteomics-driven cancer biomarker discovery: looking to the future

Availability of a suite of biomarkers for early detection, stratification into distinct subtypes, and monitoring progression or response to therapy promises significant improvements in clinical outcomes for cancer patients. However, despite the recent progress in proteomics technologies based on mass spectrometry (MS), discovery of novel clinical assessment tools has been slow. This is, partly due to the inherent difficulties in working with blood as the biospecimen for candidate discovery. A better understanding of the limitations of blood for comparative protein profiling and a better appreciation of the advantages of cancer tissue or cancer cell secretomes have the potential to greatly enhance the progress.     

Personalized medicine: revolutionizing drug discovery and patient care.

Advances in human genome research are opening the door to a new paradigm for practising medicine that promises to transform healthcare. Personalized medicine, the use of marker-assisted diagnosis and targeted therapies derived from an individual's molecular profile, will impact the way drugs are developed and medicine is practiced. Knowledge of the molecular basis of disease will lead to novel target identification, toxicogenomic markers to screen compounds and improved selection of clinical trial patients, which will fundamentally change the pharmaceutical industry. The traditional linear process of drug discovery and development will be replaced by an integrated and heuristic approach. In addition, patient care will be revolutionized through the use of novel molecular predisposition, screening, diagnostic, prognostic, pharmacogenomic and monitoring markers. Although numerous challenges will need to be met to make personalized medicine a reality, with time, this approach will replace the traditional trial-and-error practice of medicine.     

HER-2: a biomarker at the crossroads of breast cancer immunotherapy and molecular medicine

The oncoprotein encoded by the HER-2 oncogene is a member of the HER family of receptor tyrosine kinases and is actually the first successfully exploited target molecule in new biomolecular therapies of solid tumors. The association of HER-2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor-specific therapy. In addition, HER-2 overexpression fosters its immunogenicity, as shown by the frequency of B and T cell-mediated responses against this oncoprotein in cancer patients, and it is being investigated as a promising molecule for either passive and active immunotherapy strategies. This review summarizes a number of immune intervention approaches that target HER-2 in breast cancer.     

Systems modeling: a pathway to drug discovery

Systems modeling is emerging as a valuable tool in therapeutics. This is seen by the increasing use of clinically relevant computational models and a rise in systems biology companies working with the pharmaceutical industry. Systems models have helped understand the effects of pharmacological intervention at receptor, intracellular and intercellular communication stages of cell signaling. For instance, angiogenesis models at the ligand-receptor interaction level have suggested explanations for the failure of therapies for cardiovascular disease. Intracellular models of myeloma signaling have been used to explore alternative drug targets and treatment schedules. Finally, modeling has suggested novel approaches to treating disorders of intercellular communication, such as diabetes. Systems modeling can thus fill an important niche in therapeutics by making drug discovery a faster and more systematic process.     

Biomolecular NMR: a chaperone to drug discovery

Biomolecular NMR now contributes routinely to every step in the development of new chemical entities ahead of clinical trials. The versatility of NMR--from detection of ligand binding over a wide range of affinities and a wide range of drug targets with its wealth of molecular information, to metabolomic profiling, both ex vivo and in vivo--has paved the way for broadly distributed applications in academia and the pharmaceutical industry. Proteomics and initial target selection both benefit from NMR: screenings by NMR identify lead compounds capable of inhibiting protein-protein interactions, still one of the most difficult development tasks in drug discovery. NMR hardware improvements have given access to the microgram domain of phytochemistry, which should lead to the discovery of novel bioactive natural compounds. Steering medicinal chemists through the lead optimisation process by providing detailed information about protein-ligand interactions has led to impressive success in the development of novel drugs. The study of biofluid composition--metabonomics--provides information about pharmacokinetics and helps toxicological safety assessment in animal model systems. In vivo, magnetic resonance spectroscopy interrogates metabolite distributions in living cells and tissues with increasing precision, which significantly impacts the development of anticancer or neurological disorder therapeutics. An overview of different steps in recent drug discovery is presented to illuminate the links with the most recent advances in NMR methodology.     

Real-time molecular and cellular analysis: the new frontier of drug discovery

The pharmaceutical industry is currently facing the challenge of maintaining increased efficiency and productivity while contending with a deluge of genomic and high-throughput screening data. To ease the bottlenecks at target validation and lead optimization, the industry must look to the living cell, the ultimate target of all drugs, as a source of new biological knowledge. This new 'cell-centric' perspective must integrate reagents that report on the state of molecular processes within the cell, automated detection and analysis of these processes, and cellular knowledge, building into a single platform.     

From drug to protein: using yeast genetics for high-throughput target discovery

The budding yeast Saccharomyces cerevisiae has long been an effective eukaryotic model system for understanding basic cellular processes. The genetic tractability and ease of manipulation in the laboratory make yeast well suited for large-scale chemical and genetic screens. Several recent studies describing the use of yeast genetics for high-throughput drug target identification are discussed in this review.     

Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions. In agreement with a previous photo-cross linking analysis, YidC did not show any high-affinity interactions with cytosolic or periplasmic proteins. These three examples illustrate the utility of nanoscale lipid bilayers to identify the soluble peripheral partners of proteins intergrated in the lipid bilayer.     

Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein–lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein–lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches.