Therapeutic opportunities involving cellular oncogenes: novel approaches fostered by biotechnology

Biotechnological processes are having a major impact on many industrial sectors, including the pharmaceutical industry. The contributions of recombinant DNA and hybridoma technologies to modern therapeutics include production of natural and unnatural peptides, subunit vaccines, monoclonal antibodies and nucleic acid hybridization probes for in vitro and in vivo diagnostics and biological imaging, therapeutic monoclonal antibodies as tissue-specific delivery systems or as agents to confer passive immunity, production of therapeutic targets for rational drug design, and the use of cloned enzymes as stereospecific catalysts in large-scale production of small medicinal molecules. Biotechnological advances have led to the identification of a discrete set of genes, oncogenes, which may be essential contributing factors for a great variety and number of human cancers. In addition, biotechnological innovations are fostering the exploitation of oncogenes as novel therapeutic targets for cancer diagnosis, prognosis, and treatment. Because oncogenes are activated in transformation by either qualitative or quantitative mechanisms, however, different biotechnology-based therapeutic approaches are required for each class.     

Mechanism of tissue-selective drug action in the cardiovascular system

Analysis of the human genome project tells us that there may be as few as 3000 genes that are likely to be good drug targets. Although the number of targets is still very large, these data have been interpreted by some to mean that the pharmaceutical industry may someday run out of novel drug targets. Despite the doom and gloom of such analysis, there is considerable reason for optimism. Drugs may exhibit selectivity of action beyond that predicted by target expression alone. Drugs that act at a single molecular target may have very different pharmacology and, as a result, different therapeutic uses. Three well-characterized model systems are highlighted to illustrate this point. The first model system is exemplified by nifedipine and verapamil, both of which act on L-type calcium channels. Both drugs are used to treat hypertension, but only verapamil can be used to produce atrioventricular block in patients with atrial fibrillation. The second model system describes the therapeutic exploitation of unusual conditions that occur in the ischemic myocardium to produce drugs that are more effective for suppressing ischemia-induced arrhythmias. The third model system discusses the mechanisms through which phosphodiesterase-5 (PDE5) inhibitors act selectively to facilitate penile erection while having little effect in the non-penile vasculature that also expresses PDE5.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Targeting B cells for the treatment of rheumatoid arthritis

The role of B cells in rheumatoid arthritis (RA) has been debated for decades. However, recent clinical trial data indicating that depletion of B cells in RA patients is of therapeutic benefit has validated the importance of this cell type in the pathogenesis of the disease. Elucidation of the molecular basis of B cell development and activation has allowed the identification of a number of possible therapeutic targets that are appealing for drug development. This review discusses briefly a number of these molecules and the rationale for targeting them for the treatment of RA.     

Yeast killer toxins: from ecological significance to application

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.     

Strengthening the Case for Epilepsy Drug Development: Bridging Experiences from the Alzheimer’s Disease Field—An Opinion

Given the sheer number of drugs (over 20!) available for treatment of seizures, epilepsy can be considered one of the most successful areas in pharmaceutical development and especially for neuroscience. However, despite the large number of drug treatment options available for managing patients with epilepsy, there remains considerable unmet need. For example, the overall impact on seizure control has not been substantial with approximately 30% of patients remaining refractory or their seizures not adequately controlled. Also there is need for epilepsy prevention and for certain sub-populations with severe intractable epilepsy. High unmet need often drives new industry investment into therapeutic market opportunities, however the profound success of antiepileptic drugs has contributed to the hurdles for industry investment in new therapies for epilepsy. Furthermore, the payor environment has also changed with new challenges for evidence generation and demonstration of additive value above existing standard of care treatments. Challenges in translational science, in the clinical trial environment including cost and operational technical difficulty, and in the commercial environment have resulted in the pharmaceutical industry directing investments away from epilepsy into other therapeutic areas such as oncology and immunology as opportunities for higher probabilities of success and returns of investment. The neuroscience area in general is perceived a high risk area and a notable exception has been the active industry involvement in Alzheimer’s disease (AD), especially for therapeutics that could modify the course or prevent AD. AD is a very high risk area with no successful efficacious treatments found to date despite recent failures, there remains promise that therapies are forthcoming. The promise is fueled by a number of innovative factors that reduced R&D challenges in the AD field and contributed to a high level of drug development activity and investment. This paper addresses hurdles facing epilepsy drug discovery and development and focuses on some key solutions that could be eased to facilitate industry interest. Similarities in drug development challenges provide opportunities that bridge experiences and learnings from AD to epilepsy. Overall, the epilepsy field is probably in a good position for advancing into the next generation therapeutics of antiepileptic drugs targeted for increased efficacy in refractory epilepsy and for antiepileptogenesis.

     

Frontier of therapeutic antibody discovery:The challenges and how to face them

Therapeutic monoclonal antibodies have become an important class of modern medicines.The established technologies for therapeutic antibody discovery such as humanization of mouse antibodies,phage display of human antibody libraries and transgenic animals harboring human IgG genes have been practiced successfully so far,and many incremental improvements are being made constantly.These methodologies are responsible for currently marketed therapeutic antibodies and for the biopharma industry pipeline which are concentrated on only a few dozen targets.A key challenge for wider application of biotherapeutic approaches is the paucity of truly validated targets for biotherapeutic intervention.The efforts to expand the target space include taking the pathway approach to study the disease correlation.Since many new targets are multi-spanning and multimeric membrane proteins there is a need to develop more effective methods to generate antibodies against these difficult targets.The pharmaceutical properties of therapeutic antibodies are an active area for study concentrating on biophysical characteristics such as thermal stability and aggregation propensity.The immunogenicity of biotherapeutics in humans is a very complex issue and there are no truly predictive animal models to rely on.The in silico and T-cell response approaches identify the potential for immunogenicity;however,one needs contingency plans for emergence of antiproduct antibody response for clinical trials.     

Chemokine receptor antagonism as an approach to anti-inflammatory therapy: 'just right' or plain wrong?

Inflammation plays a pivotal role in exacerbating a wide array of human diseases. The chemokines are a group of proteins that control the movement and activation of the immune cells involved in all aspects of the inflammatory response. Recently, their cognate receptors have attracted considerable interest as therapeutic targets, in part because they are G-protein-coupled receptors, which have been antagonized successfully before by the pharmaceutical industry. Indeed, several companies have now reported the development of selective small-molecule chemokine receptor antagonists, and some of these compounds have even entered human Phase I clinical trials. Preclinical studies of the responsiveness of murine models of inflammation to either pharmacologic or genetic intervention have suggested that antagonism of some chemokine receptors may well prove to be a safe and efficacious approach to anti-inflammatory therapy.     

Proteases as drug targets

The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.     

Most recent developments in strategies to reduce the progression of structural changes in osteoarthritis: today and tomorrow

Osteoarthritis (OA), the most common of all arthritic conditions, is a social and financial burden to all nations. The most recent research has significantly advanced our understanding of the cause of OA and risk factors associated with it. These findings have provided useful information that has helped in the daily management of patients with OA. Some preventative measures and a number of therapeutic agents and drugs are available, which may help to reduce the progression of OA in certain patients. Moreover, the most recent progress in research has significantly enhanced our knowledge of the factors involved in the development of the disease and of the mechanisms responsible for its progression. This has allowed identification of several new therapeutic targets in a number of pathophysiological pathways. Consequently, the field is opening up to a new era in which drugs and agents that can specifically block important mechanisms responsible for the structural changes that occur in OA can be brought into development and eventually into clinical trials.     

Cellular interplay in pulmonary arterial hypertension: Implications for new therapies

Pulmonary arterial hypertension (PAH) is a complex and multifactorial disease characterized by vascular remodeling, vasoconstriction, inflammation and thrombosis. Although the available therapies have resulted in improvements in morbidity and survival, PAH remains a severe and devastating disease with a poor prognosis and a high mortality, justifying the need of novel therapeutic targets. An increasing number of studies have demonstrated that endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts of the pulmonary vessel wall, as well as platelets and inflammatory cells have a role in PAH pathogenesis. This review aims to integrate the interplay among different types of cells, during PAH development and progression, and the impact of current therapies in cellular modulation. The interplay among endothelial cells, smooth muscle cells and fibroblasts present in pulmonary vessels wall, platelets and inflammatory cells is regulated by several mediators produced by these cells, contributing to the pathophysiologic features of PAH. Current therapies are mainly focused in the pulmonary vascular tone and in the endothelial dysfunction. However, once they have not been effective, novel therapies targeting other PAH features, such as inflammation and platelet dysfunction are emerging. Further understanding of the interplay among different vascular cell types involved in PAH development and progression can contribute to find novel therapeutic targets, decreasing PAH mortality and morbidity in the future.     

Chemokines in cardiovascular remodeling: clinical and therapeutic implications

Chemokines are newly discovered molecules that mediate the migration of leukocytes into inflammed tissues and control the inflammatory reactions in various immune-mediated diseases. Both in animal models and in human specimens, chemokine expression is associated with atherosclerotic lesion development and vascular remodeling and restenosis after angioplasty. Furthermore, recent studies have demonstrated that chemokines play an important role in the pathophysiology of acute coronary syndromes, post-infarction left ventricular remodeling and chronic heart failure. The capacity to control activation and movement of inflammatory cells suggests that chemokines and their receptors might provide novel targets for therapeutic intervention in a number of conditions characterized by chronic inflammation, including cardiovascular diseases. The present review summarizes current knowledge regarding the potential pathogenic role of chemokines in major cardiovascular disorders, as well as the modulation of the chemokine network as a novel, interesting therapeutic modality in this field.     

Defining the molecular nexus of cancer, type 2 diabetes and cardiovascular disease

The metabolic syndrome is characterized by a state of metabolic dysfunction resulting in the development of several chronic diseases that are potentially deadly. These metabolic deregulations are complex and intertwined and it has been observed that many of the mechanisms and pathways responsible for diseases characterizing the metabolic syndrome such as type 2 diabetes and cardiovascular disease are linked with cancer development as well. Identification of molecular pathways common to these diverse diseases may prove to be a critical factor in disease prevention and development of potential targets for therapeutic treatments. This review focuses on several molecular pathways, including AMPK, PPARs and FASN that interconnect cancer development, type 2 diabetes and cardiovascular disease. AMPK, PPARs and FASN are crucial regulators involved in the maintenance of key metabolic processes necessary for proper homeostasis. It is critical to recognize and identify common pathways deregulated in interrelated diseases as it may provide further information and a much more global picture in regards to disease development and prevention. Thus, this review focuses on three key metabolic regulators, AMPK, PPARs and FASN, that may potentially serve as therapeutic targets.     

生物制药的现状和未来（二）：发展趋势与希望

随着基因组和蛋白质组研究的深入,越来越多的与人类疾病发展相关的靶标被确定,使得我们能够研发更精确的药物来防治这些疾病。这意味着生物制药将有更多机会获得突破性进展,最终将使更多更好的生物技术药物被批准上市。综述了生物制药发展的几个趋势,主要有:(1)哺乳动物细胞表达的产品将在相当长的时间内占统治地位;(2)治疗性抗体将会是生物制药领域第二次创新高潮;(3)越来越多分子量大、结构复杂的功能蛋白将被开发成生物技术药物,尤其是用于治疗遗传性疾病的药物;(4)对已批准上市的生物技术药物的化学修饰尤其是PEG化以改善药物性能;(5)通过某些药物的定点突变获得第二代新生物技术药物,如胰岛素、EPO和t-PA的突变体;(6)组织工程、细胞治疗和基因治疗充满了机遇和挑战。     

In vivo drug target discovery: identifying the best targets from the genome

A vast number of genes of unknown function threaten to clog drug discovery pipelines. To develop therapeutic products from novel genomic targets, it will be necessary to correlate biology with gene sequence information. Industrialized mouse reverse genetics is being used to determine gene function in the context of mammalian physiology and to identify the best targets for drug development.     

瞬时感受器电位通道与疾病

哺乳动物瞬时感受器电位(transient receptor potential,TRP)通道超家族由TRPC、TRPM、TRPV、TRPA、TRPP和TRPML六个亚家族组成。这些亚家族的29个离子通道几乎表达于所有的组织和细胞。大多对单价和二价阳离子都有通透性。TRP通道与多种生物学功能有关,包括高血压、温度觉、血管炎症、刺激感、肿瘤增生、细胞内离子稳态及神经细胞信号转导。对这些通道的生理功能及其与人类疾病的关系的研究有助于开发具有潜在治疗价值的TRP通道调节剂。     

High-throughput screening of biocatalytic activity: applications in drug discovery

Enzymes catalyze a diverse set of reactions that propel life's processes and hence serve as valuable therapeutic targets. High-throughput screening methods have become essential for sifting through large chemical libraries in search of drug candidates, and several sensitive and reliable analytical techniques have been specifically adapted to high-throughput measurements of biocatalytic activity. High-throughput biocatalytic assay platforms thus enable rapid screening against enzymatic targets, and have vast potential to impact various stages of the drug discovery process, including lead identification and optimization, and ADME/Tox assessment. These advances are paving the way for the adoption of high-throughput biocatalytic assays as an indispensable tool for the pharmaceutical industry.     

Target-based drug discovery,genetic diseases,and biologics

The last fifteen years have witnessed a major strategic shift in drug discovery away from an empiric approach based on incremental improvements of proven therapies, to a more theoretical, target-based approach. This arose as a consequence of three technical advances: (1) generation and interpretation of genome sequences, which facilitated identification and characterization of potential drug targets; (2) efficient production of candidate ligands for these putative targets through combinatorial chemistry or generation of monoclonal antibodies; and (3) high-throughput screening for rapid evaluation of interactions of these putative ligands with the selected targets. The basic idea underlying all three of these technologies is in keeping with Marshall Nirenberg’s dictum that science progresses best when there are simple assays capable of generating large data sets rapidly. Furthermore, practical implementation of target-based drug discovery was enabled directly by technologies that either were originated or nurtured by Marshall, his post-docs and fellows. Chief among these was the genetic code. Also important was adoption of clonal cell lines for pharmacological investigations, as well as the use of hybridomas to generate molecular probes that allowed physical purchase on signaling elements that had previously been only hypothetical constructs. Always the pure scientist, Marshall’s contributions nevertheless enabled fruitful applications in the pharmaceutical industry, several of them by his trainees. Both the successes and the shortcomings of target-based drug discovery are worthy of consideration, as are its implications for the choices of therapeutic goals and modalities by the pharmaceutical industry.