Disease-associated Targets Research Articles

Advances in targeted therapeutic agents

Importance of the field: The number of disease-associated protein targets has significantly increased over the past decade due to advances in molecular and cellular biology technologies, human genetic mapping efforts and information gathered from the human genome project. The identification of gene products that appear to be involved in supporting the underlying cause of disease has offered the biopharmaceutical industry an opportunity to develop compounds that can specifically target these molecules to improve therapeutic responses and lower the risk of unwanted side effects that are commonly seen in traditional small chemical-based medicines.Areas covered in this review: An overview of targeted drug therapies is presented in this review. We include a review of the various classes of targeted therapeutic agents, the types of disease-associated molecules being targeted by these agents and the challenges currently being encountered for the successful development of these various platforms for the treatment of disease.What the reader will gain: An understanding of the current targeted therapy landscape, the discovery and selection of disease-specific gene products that are being targeted, and an overview of targeted therapies in preclinical and clinical studies. A description of the various targeted therapeutic platforms, target selection criteria and examples of each are discussed in order to provide the reader with the current status of the field and emerging areas of targeted therapy discovery and development.Take home message: Novel medications are in demand for the treatment of serious medical conditions including cancer, autoimmune, infectious and metabolic diseases. Targeted therapies offer a way to develop very specific treatments for serious medical conditions while concomitantly resulting in little to no off-target toxicity. Targeted therapies provide an opportunity to develop personalized medicines with superior treatment modalities for the patient and a better quality of life.

Tissue-Specific Target Analysis of Disease-Associated MicroRNAs in Human Signaling Pathways

MicroRNAs are a large class of post-transcriptional regulators that bind to the 3′ untranslated region of messenger RNAs. They play a critical role in many cellular processes and have been linked to the control of signal transduction pathways. Recent studies indicate that microRNAs can function as tumor suppressors or even as oncogenes when aberrantly expressed. For more general insights of disease-associated microRNAs, we analyzed their impact on human signaling pathways from two perspectives. On a global scale, we found a core set of signaling pathways with enriched tissue-specific microRNA targets across diseases. The function of these pathways reflects the affinity of microRNAs to regulate cellular processes associated with apoptosis, proliferation or development. Comparing cancer and non-cancer related microRNAs, we found no significant differences between both groups. To unveil the interaction and regulation of microRNAs on signaling pathways locally, we analyzed the cellular location and process type of disease-associated microRNA targets and proteins. While disease-associated proteins are highly enriched in extracellular components of the pathway, microRNA targets are preferentially located in the nucleus. Moreover, targets of disease-associated microRNAs preferentially exhibit an inhibitory effect within the pathways in contrast to disease proteins. Our analysis provides systematic insights into the interaction of disease-associated microRNAs and signaling pathways and uncovers differences in cellular locations and process types of microRNA targets and disease-associated proteins.

A status report on RNAi therapeutics

Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice.

RNA Interference Technologies and Therapeutics

RNA interference (RNAi) is a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNAs or blocking translation. In recent years, the use of RNAi for therapeutic applications has gained considerable momentum. It has been suggested that most of the novel disease-associated targets that have been identified are not ‘druggable’ with conventional approaches. However, any disease-causing gene and any cell type or tissue can potentially be targeted with RNAi.This review focuses on the current knowledge of RNAi mechanisms and the safety issues associated with its potential use in a therapeutic setting. Some of the most important aspects to consider when working towards the application of RNAi-based products in a clinical setting have been related to achieving high efficacies and enhanced stability profiles through a careful design of the nucleic acid sequence and the introduction of chemical modifications, but most of all, to developing improved delivery systems, both viral and non-viral. These new delivery systems allow for these products to reach the desired target cells, tissues or organs in a highly specific manner and after administration of the lowest possible doses. Various routes of application and target locations are currently being addressed in order to develop effective delivery systems for different targets and pathologies, including infectious pathologies, genetic pathologies and diseases associated with dysregulation of endogenous microRNAs. As with any new technology, several challenges and important aspects to be considered have risen on the road to clinical intervention, e.g. correct design of preclinical toxicology studies, regulatory concerns, and intellectual property protection. The main advantages related to the use of RNAi-based products in a clinical setting, and the latest clinical and preclinical studies using these compounds, are reviewed.

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C H3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcγ receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcγ receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Molecular profiling of a rat model of colitis: Validation of known inflammatory genes and identification of novel disease-associated targets

Inflammatory bowel disease (IBD) is a disease of unknown etiology characterized by acute and chronic relapsing inflammation. The most suitable animal model for studying this disease is still under debate. Microarray transcript profiling has the potential to illuminate the molecular processes that are involved in both the human condition and animal models. To identify differentially expressed genes in the 2,4,6-trinitrobenzene sulfonic acid (TNBS) model of experimental colitis and compare gene expression profiles with that reported in patients. Colitis was induced by TNBS administration (30 mg in 50% ethanol) in female Sprague-Dawley rats. Controls received the vehicle. Seventy-two hours later, the animals were killed, the colons were removed and scored for damage, and total RNA was isolated. Gene expression levels were analyzed after hybridizing experimental and control cDNA to PIQOR Toxicology Rat Microarrays containing 1,252 genes. Genes with 2-fold or more higher or 0.5-fold or less lower expression levels were selected as significantly differentially expressed. Results were validated using real-time reverse-transcription polymerase chain reaction (RT-PCR). We observed increased expression of genes that have previously been shown to be up-regulated in IBD patients, including chemokines/cytokines, extracellular matrix/remodeling genes, transcription factors and tumor necrosis factor family members. Using real-time RT-PCR, we validated 9 of 10 critical genes identified by DNA microarray. Fibulin 2 and lysyl oxidase are among some of the novel genes not previously associated with IBD that could potentially be related to the pathogenesis of this condition. We provide evidence supporting the TNBS colitis model as an appropriate tool to study the pathology of IBD and identify molecular targets with clinical relevance.

From tissue phenotype to proteotype: Sensitive protein identification in microdissected tumor tissue

Correlation of disease phenotype with protein profile (proteotype) is a significant challenge for biomedical research. The main obstacles have been the need to insure sufficient quantities of pure protein sample, the reproducibility of protein display, and rapid and accurate protein identification. We present a modified approach that combines enhanced detection sensitivity with tissue microdissection from frozen primary renal cancer tissues of different histological subtypes, followed by 2D gel analysis and protein identification with MALDI mass spectrometry. We obtained reliable and highly consistent results in phenotypically similar tumors of each individual subtype by performing strict morphological control of the analyzed tumor cells without physical or chemical alteration of the frozen tissue samples. By application of non-oxidizing silver staining, proteins were resolved and identified with high levels of specificity and sensitivity. This new combination of techniques allows not only for sensitive identification of specific protein patterns that correspond to a histological tumor phenotype, but also for identification of specific disease-associated protein targets.

A General Technique To Rank Protein−Ligand Binding Affinities and Determine Allosteric versus Direct Binding Site Competition in Compound Mixtures

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.

Optical imaging fiber-based single live cell arrays: a high-density cell assay platform.

A high-density, ordered array containing thousands of microwells is fabricated on an optical imaging fiber. Each individually addressable microwell is used to accommodate a single living cell. A charged coupled device (CCD) detector is employed to monitor and spatially resolve the fluorescence signals obtained from each individual cell, allowing simultaneous monitoring of cellular responses of all the cells in the array using reporter genes (lacZ, EGFP, ECFP, DsRed) or fluorescent indicators. Yeast and bacteria cell arrays were fabricated and used to perform multiplexed cell assays with resolution at the single-cell level. Monitoring gene expression in single yeast cells carrying a two-hybrid system was used to detect in vivo protein-protein interactions. The single-cell array technology provides a new platform for monitoring the unique multiple responses of large populations of individual cells from different strains or cell lines. The rich data acquired by the cell array has the potential to be employed as a new tool for cell biology research as well as to improve cell-based high-throughput screening (HTS) applications, such as the validation of new disease-associated cellular targets and the early-stage evaluation of potential drug candidates.