The potential impact of structural genomics on tuberculosis drug discovery

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) in humans, is a devastating infectious organism that kills approximately two million people annually. The current suite of antibiotics used to treat TB faces two main difficulties: (i) the emergence of multidrug-resistant (MDR) strains of M. tuberculosis, and (ii) the persistent state of the bacterium, which is less susceptible to antibiotics and causes very long antibiotic treatment regimes. The complete genome sequences of a laboratory strain (H37Rv) and a clinical strain (CDC1551) of M. tuberculosis and the concurrent identification of all the open reading frames that encode proteins within this organism, present structural biologists with a wide array of protein targets for structure determination. Comparative genomics of the species that make up the M. tuberculosis complex has also added an array of genomic information to our understanding of these organisms. In response to this, structural genomics consortia have been established for targeting proteins from M. tuberculosis. This review looks at the progress of these major initiatives and the potential impact of large scale structure determination efforts on the development of inhibitors to many proteins. Increasing sophistication in structure-based drug design approaches, in combination with increasing numbers of protein structures and inhibitors for TB proteins, will have a significant impact on the downstream development of TB antibiotics.     

Single nucleotide polymorphism discovery and haplotype analysis of Ca2+-dependent K+ channel beta-1 subunit

The large-conductance, Ca-dependent K channel plays a key role in the control of vascular tone. Variation in the gene encoding the beta-1 subunit of the Ca-dependent K channel (KCNMB1) has been reported to be associated with hypertension, however, variants in KCNMB1 have not been systematically characterized to date. In this study, we have performed the most comprehensive evaluation to date of single nucleotide polymorphisms in KCNMB1 using genomic DNA from 60 individuals of European, African and native American ancestry. We identified and characterized single nucleotide polymorphisms in the exons, intron/exon junctions, upstream region and 3' untranslated regions of KCNMB1 using denaturing high-performance liquid chromatography combined with direct DNA sequencing. A total of 25 single nucleotide polymorphisms in KCNMB1 were identified. Seven of the polymorphisms (28%) are novel single nucleotide polymorphisms not reported previously. Allele frequencies range from less than 1.7 to 50% and 19 single nucleotide polymorphisms had a minor allele frequency greater than 5%. A lack of strong linkage disequilibrium among the 25 single nucleotide polymorphisms was observed in all three race/ethnicity groups; therefore the identification of haplotype 'tag' single nucleotide polymorphisms for genetic association studies is not likely to be appropriate for KCNMB1. Multiple species comparative analysis and in-silico functional analysis were performed to identify potential functionally important single nucleotide polymorphisms within the gene. These data highlight that a tag single nucleotide polymorphism approach will not be appropriate for the study of genes such as KCNMB1, although potentially important functionally significant single nucleotide polymorphisms are suggested for future studies investigating the influence of this gene's variability on disease and drug response.     

Polymorphisms in human muscarinic receptor subtype genes

A wide range of polymorphisms have been reported in muscarinic receptor subtype genes, mostly in M? and M? and, to a lesser extent, M? receptors. Most studies linking such genetic variability to phenotype have been performed for brain functions, but a more limited amount of information is also available for cardiac and airway function. Unfortunately, for none of the phenotypes under investigation a robust association with genotype has emerged. Moreover, it remains mostly unclear whether a reported association indicates a causative role of the polymorphism under investigation or merely a role as indicator of other polymorphisms affecting expression and/or function of the receptor. Also, most data on genotype-phenotype associations of muscarinic receptor subtypes are based on cross-sectional samples. Mechanistic studies linking polymorphisms to molecular, cellular, and tissue functions are largely missing. Finally, studies on a possible impact of muscarinic receptor polymorphisms on drug responsiveness are also largely missing. Thus, the field of genomics of muscarinic receptor subtypes is still in an early stage and a considerably greater number of studies will be required to judge the role of muscarinic receptor gene variability in physiology, pathophysiology, and drug treatment.     

New bioinformatics tools for viral genome analyses at Viral Bioinformatics--Canada

Viruses are much smaller than prokaryotes and eukaryotes, and it is now practical to sequence closely related members of virus families, strains, or even different isolates recovered during the course of an outbreak. However, comparative analysis of viral genomes requires the development of novel bioinformatics tools that allow us to align, edit, compare and interact with these genomes at all levels, from whole genome, to gene family, to single nucleotide polymorphisms. Comparative viral genomics can lead to the identification of the core characteristics that define a virus family, as well as the unique properties of viral species or isolates that contribute to variations in pathogenesis. This paper describes a number of tools, mainly developed for Viral Bioinformatics--Canada, that can be used for annotation and comparative genomic analysis of poxviruses. Nonetheless, these tools are also broadly applicable to other virus families.     

Functional Genomics- and Network-driven Systems Biology Approaches for Pharmacogenomics and Toxicogenomics

Recent advances in genomic technologies have enabled the identification of thousands of genetic variations that are associated with hundreds of complex human diseases or traits in genome-wide association studies (GWAS). The large number of genetic loci uncovered for each disease or trait along with the difficulty in pinpointing the underlying genes and mechanisms further testify to the complexity of human pathophysiology. To alleviate the challenges of GWAS, systems biology approaches have been utilized to map the molecular mechanisms underlying complex human diseases/traits via the integration of genetic variation, functional genomics (such as genetics of gene expression), pathways, and molecular networks. Similar approaches have been applied to a spectrum of drug metabolizing enzymes to discover novel functional genetic variations that affect the expression or activities of these enzymes as well as to define the regulatory pathways/networks of genes involved in drug metabolism and toxicology in key human tissues. We envision that the increased coverage of functional genetic polymorphisms, the availability of drug metabolism-centered gene networks, and the maturing methodologies previously developed for understanding complex human diseases can be applied to pharmacogenomic and toxicogenomic studies to further our understanding of inter-individual variability in drug efficacy and toxicity and eventually help direct personalized medicine.     

Pharmacogenetics, pharmacogenomics, and individualized medicine

Individual variability in drug efficacy and drug safety is a major challenge in current clinical practice, drug development, and drug regulation. For more than 5 decades, studies of pharmacogenetics have provided ample examples of causal relations between genotypes and drug response to account for phenotypic variations of clinical importance in drug therapy. The convergence of pharmacogenetics and human genomics in recent years has dramatically accelerated the discovery of new genetic variations that potentially underlie variability in drug response, giving birth to pharmacogenomics. In addition to the rapid accumulation of knowledge on genome-disease and genome-drug interactions, there arises the hope of individualized medicine. Here we review recent progress in the understanding of genetic contributions to major individual variability in drug therapy with focus on genetic variations of drug target, drug metabolism, drug transport, disease susceptibility, and drug safety. Challenges to future pharmacogenomics and its translation into individualized medicine, drug development, and regulation are discussed. For example, knowledge on genetic determinants of disease pathogenesis and drug action, especially those of complex disease and drug response, is not always available. Relating the many gene variations from genomic sequencing to clinical phenotypes may not be straightforward. It is often very challenging to conduct large scale, prospective studies to establish causal associations between genetic variations and drug response or to evaluate the utility and cost-effectiveness of genomic medicine. Overcoming the obstacles holds promise for achieving the ultimate goal of effective and safe medication to targeted patients with appropriate genotypes.     

The use of proteomics to study infectious diseases

Technology surrounding genomics, or the study of an organism's genome and its gene use, has advanced rapidly resulting in an abundance of readily available genomic data. Although genomics is extremely valuable, proteins are ultimately responsible for controlling most aspects of cellular function. The field of proteomics, or the study of the full array of proteins produced by an organism, has become the premier arena for the identification and characterization of proteins. Yet the task of characterizing a proteomic profile is more complex, in part because many unique proteins can be produced by the same gene product and because proteins have more diverse chemical structures making sequencing and identification more difficult. Proteomic profiles of a particular organism, tissue or cell are influenced by a variety of environmental stimuli, including those brought on by infectious disease. The intent of this review is to highlight applications of proteomics used in the study of pathogenesis, etiology and pathology of infectious disorders. While many infectious agents have been the target of proteomic studies, this review will focus on those infectious diseases which rank among the highest in worldwide mortalities, such as HIV/AIDS, tuberculosis, malaria, measles, and hepatitis.     

Functional Genomics Approaches in Arthritis

The post-genomic era of functional genomics and target validation will allow us to narrow the bridge between clinically correlative data and causative data for complex diseases, such as arthritis, for which the etiological agent remains elusive. The availability of human and other annotated genome sequences, and parallel developments of new technologies that allow analysis of minute amounts of human and animal cells (peripheral blood cells and infiltrating cells) and tissues (synovium and cartilage) under different pathophysiological conditions, has facilitated high-throughput gene mining approaches that can generate vast amounts of clinically correlative data. Characterizing some of the correlative/causative genes will require reverting to the hypothesis-driven, low throughput method of complementary experimental biology using genomic approaches as a tool. This will include in silico gene expression arrays, genome-wide scans, comparative genomics using various animal models (such as rodents and zebrafish), bioinformatics and a team of well trained translational scientists and physicians. For the first time, the "genomic tools" will allow us to analyze small amounts of surgical samples (such as needle biopsies) and clinical samples in the context of the whole genome. Preliminary genomic analysis in osteoarthritis has already resurrected the debate on the semantic issues in the definition of inflammation. Further analyses will not only facilitate the development of unbiased hypotheses at the molecular level, but also assist us in the identification and characterization of novel targets and disease markers for pharmacological intervention, gene therapy, and diagnosis.     

Asthma from a pharmacogenomic point of view

Pharmacogenomics, a fascinating, emerging area of biomedical research is strongly influenced by growing availability of genomic databases, high-throughput genomic technologies, bioinformatic tools and artificial computational modelling approaches. One main area of pharmacogenomics is the discovery of new drugs and drug targets with molecular genetic, genomic or even bioinformatic methods; the other is the study of how genomic differences influence the variability in patients' responses to drugs. From a genetic point of view, asthma is multifactorial, which means that the susceptibility to the disease is determined by interactions between multiple genes, and involves important non-genetic factors such as the environment for their expression. In this review, we summarize collective evidence from linkage and association studies that have consistently reported suggestive linkage or association of asthma or its associated phenotypes to polymorphic markers and single nucleotide polymorphisms in selected chromosomes. Genes that have been found implicated in the disease are potential new drug targets and several pharmacological investigations are underway to utilize these new discoveries. Next, we will focus on the inter-individual variability in anti-asthmatic drug responses and review the recent results in this topic.     

The future of plant drug discovery

To have a future in the pharmaceutical industry, plant drug discovery must compete with combinatorial chemistry and high-throughput pharmacologic screening (HTPS). Plant functional genomics coupled with HTPS may achieve this; thus, functional biology can identify ‘libraries’ of candidate plant species from which individuals can be prioritized by ‘differential HTPS’. The full genomic potential of a species for bioactivity can be accessed by cellular mutagenesis and elicitation, with HTPS identifying clones with novel activity. The comparison of ‘positive’ clonal phenotypes with their almost identical ‘negatives’ facilitates the identification of active compounds and ‘genomic’ products. The logical conclusion is to use combinatorial genomics together with HTPS to direct plant cellular ‘evolution’ continuously towards metabolites with specific pharmacologic activity. Mankind would then become the orchestrator of plant secondary metabolism, rather than its passive beneficiary.     

Mechanisms and individuality in chromium toxicity in humans

With regards to health, chromium (Cr) is an ambiguous chemical element. Although it is considered to be an important micronutrient, it also is connected with several pathologies, including carcinogenicity. The mechanism of action of Cr and its compounds in humans is not yet fully understood. Currently, three possible mechanisms have been proposed for carcinogenesis: Cr(VI)-induced multistage carcinogenesis, genomic instability, and epigenetic modification. Therefore, in addition to the toxicity of this metal and its ions, human susceptibility to Cr-induced pathologies depends on external factors and individual characteristics, such as enzymatic polymorphisms, carriers, endogenous reducing system, adduct formation and stability, and efficiency of DNA repair mechanisms, among other factors. In fact, the variability of individual molecular constitutive factors, such as individual polymorphisms, creates an individualized environment for Cr toxicity. This mini-review contemplates the essential variables in this process.     

Development of novel biomedicine based on genome science.

Towards the post genomic sequencing era, conventional drug discovery is drastically improving genomic technologies and computational advances. The completion of the entire genome sequence of many experimental organisms as well as the human organism allow us to compare several genomic sequences, comparative genomics, to get valuable information for gene discovery and functional genomics. Pharmacogenomic studies and chemical genomic investigations are quickly becoming fundamental techniques for genomic drug discovery. Additionally, progress in microchip and microarray technology has been stimulating genomic drug discovery studies. This paper reviews recent progress in human genome research, basic elements in the new strategy for drug discovery based on genome science, and future perspectives for the bio and pharmaceutical industries.     

Genetic variations and haplotypes of UDP-glucuronosyltransferase 1A locus in a Korean population

Glucuronidation catalyzed by UDP-glucuronosyltransferases (UGTs) is one of the most important mechanisms for host defense against xenobiotics and endobiotics. Although genetic polymorphisms of several UGT1A isoforms have been reported separately, the haplotypes in all functional exons have not been identified, and little information is available regarding single nucleotide polymorphisms in Koreans. We analyzed genetic polymorphisms in all functional exons of the UGT1A locus by direct sequencing of genomic DNA from 50 healthy Korean subjects, and their haplotypes were inferred from genotype data using an expectation-maximization algorithm. We identified 67 polymorphisms, including three novel single nucleotide polymorphisms, 233C>T in UGT1A1 (T78M), 292C>T in 1A4 (Q98Stop), and 701T>C in 1A7 (I234T). Two amino acid substitutions, 1A4 Q98Stop and 1A7 I234T, were each associated with a decrease of enzymatic activity, whereas UGT1A1 T78M had no significant influence on catalytic function. The frequencies of the known variants in Koreans differed significantly from those reported in other ethnic groups. Haplotype analysis was performed within the polymorphisms in each UGT1A isoform as well as across the isoforms. Based on strong linkage disequilibrium within UGT1A7, between 1A5 and 1A4, and within 1A3, the complex was divided into three blocks, Block 7, Block 5/4, and Block 3. The haplotypes for each block were subsequently determined, revealing a profile that differed from those of other ethnic groups. These results suggest that genetic polymorphisms of the UGT1A locus differ between Koreans and other ethnic populations. Such differences should be considered in pharmacogenetic studies.     

Use of comparative genomics approaches to characterize interspecies differences in response to environmental chemicals: Challenges,opportunities, and research needs

A critical challenge for environmental chemical risk assessment is the characterization and reduction of uncertainties introduced when extrapolating inferences from one species to another. The purpose of this article is to explore the challenges, opportunities, and research needs surrounding the issue of how genomics data and computational and systems level approaches can be applied to inform differences in response to environmental chemical exposure across species. We propose that the data, tools, and evolutionary framework of comparative genomics be adapted to inform interspecies differences in chemical mechanisms of action. We compare and contrast existing approaches, from disciplines as varied as evolutionary biology, systems biology, mathematics, and computer science, that can be used, modified, and combined in new ways to discover and characterize interspecies differences in chemical mechanism of action which, in turn, can be explored for application to risk assessment. We consider how genetic, protein, pathway, and network information can be interrogated from an evolutionary biology perspective to effectively characterize variations in biological processes of toxicological relevance among organisms. We conclude that comparative genomics approaches show promise for characterizing interspecies differences in mechanisms of action, and further, for improving our understanding of the uncertainties inherent in extrapolating inferences across species in both ecological and human health risk assessment. To achieve long-term relevance and consistent use in environmental chemical risk assessment, improved bioinformatics tools, computational methods robust to data gaps, and quantitative approaches for conducting extrapolations across species are critically needed. Specific areas ripe for research to address these needs are recommended.     

CD14 and toll-like receptors: potential contribution of genetic factors and mechanisms to inflammation and allergy

Innate and adaptive immune responses evolve as protective mechanisms against infectious microorganisms in humans. CD14 and toll-like receptors (TLRs) are examples of pattern recognition receptors that detect antigenic molecules on the surface of gram-positive (peptidoglycans, lipoteichoic acid) and gram-negative (lipopolysaccharide) bacteria. In vitro studies suggest that lipopolysaccharide is a potent inducer of interleukin-12 production that is mediated by both CD14 and TLR4. The associated increase in interferon-gamma steers our immune system away form the allergy-driven type-2 helper T cell phenotype. Epidemiological studies that shed light on the possible protective influences of natural microbial exposure on asthma and atopy development will be discussed. Recent insights into the complex mechanisms of human innate immunity suggest that genetic variability in genes encoding its components may alter the susceptibility to develop atopic disorders and other complex human diseases. The findings of these genetic association studies will be presented. Although highly conserved across a wide range of species, innate immunity genes demonstrate considerable inter-ethnic variability predominantly in the form of single nucleotide polymorphisms. The frequencies of these polymorphisms in CD14 and TLR genes in different ethnic groups will be discussed. Genetic variation in these genes may also play a role in the development of other human diseases that have an inflammatory component. Lastly, the prospect of using immunomodulatory agents targeting on the innate immunity to treat or even prevent asthma and other allergic diseases will be discussed.     

Pharmacogenomics of third-generation aromatase inhibitors

INTRODUCTION: Breast cancer is a common, life-threatening disease among women. Contemporary hormonal therapy with third-generation aromatase inhibitors for estrogen-receptor-positive breast cancers in postmenopausal women is still facing the challenge of interpatient variability in therapeutic response and intensity of adverse effects. AREAS COVERED: This review highlights up-to-date literature regarding genomic findings in the literature pertaining to anastrozole, exemestane and letrozole metabolism, as well as the drug target aromatase. Genetic polymorphisms in phase I and II aromatase inhibitor metabolizing enzymes that contribute to altered responses among different patient genotypes are discussed. Similarly, aromatase CYP19A1 functional genetic polymorphisms are presented in correlation to altered aromatase activity, disease prognosis and severity of aromatase inhibitor adverse effects. EXPERT OPINION: The field of pharmacogenomics has shown remarkable progress over the last few years, notably in cancer. However, large comprehensive genotyping studies, evaluated under clinical settings, are still needed to unravel the potential impact of aromatase inhibitor pharmacogenomics on breast cancer treatment, monitoring and predicting adverse effects.     

分枝杆菌菌种鉴定DNA微阵列的初步研究与应用

目的制备快速鉴定分枝杆菌菌种的DNA微阵列。方法根据19种分枝杆菌标准株的16SRNA序列设计寡核苷酸探针,制备DNA微阵列,通过反向杂交技术鉴定19种分枝杆菌标准株、9种非分枝杆菌标准株和31株分枝杆菌临床分离株带荧光标记的PCR产物。结果该DNA微阵列与9种非分枝杆菌标准株不杂交,具有分枝杆菌特异性;可将19种分枝杆菌标准株鉴定到群或种,具有较高的分枝杆菌种特异性。应用PCR-SSCP分枝杆菌初步菌种鉴定方法对31株分枝杆菌临床分离株进行初步的鉴定,9株为结核分枝杆菌复合群和22株为非结核分枝杆菌。应用DNA微阵列进一步进行菌种鉴定,9株与探针a杂交阳性,为结核分枝杆菌复合群;2株与探针c杂交阳性,为胞内分枝杆菌;8株与探针d杂交阳性,为堪萨斯分枝杆菌或瘰疬分枝杆菌或猿猴分枝杆菌或胃分枝杆菌;1株与探针k杂交阳性,为戈登分枝杆菌;2株与探针p杂交阳性,为龟分枝杆菌脓肿亚种;2株与探针r杂交阳性,为偶然分枝杆菌;1株与探针s杂交阳性,为草分枝杆菌;2株与探针a和p杂交阳性,为结核分枝杆菌和龟分枝杆菌脓肿亚种复合感染株;1株与探针a和r杂交阳性,为结核分枝杆菌和偶然分枝杆菌复合感染株;3株与该芯片杂交阴性。2株临床分离株经基因测序也证实DNA微阵列鉴定的特异性。结论该DNA微阵列有可能简便、快速、准确地     

Modeling Clinical Phenotype Variability: Consideration of Genomic Variations,Computational Methods,and Quantitative Proteomics

Advances in biomedical and computer technologies have presented the modeling community the opportunity for mechanistically modeling and simulating the variability in a disease phenotype or in a drug response. The capability to quantify response variability can inform a drug development program. Quantitative systems pharmacology scientists have published various computational approaches for creating virtual patient populations (VPops) to model and simulate drug response variability. Genomic variations can impact disease characteristics and drug exposure and response. Quantitative proteomics technologies are increasingly used to facilitate drug discovery and development and inform patient care. Incorporating variations in genomics and quantitative proteomics may potentially inform creation of VPops to model and simulate virtual patient trials, and may help account for, in a predictive manner, phenotypic variations observed clinically.     

Biotechnology in the development of new vaccines and diagnostic reagents against tuberculosis

Tuberculosis (TB) is a disease of global concern. About one third of the world population is infected with Mycobacterium tuberculosis. Every year, approximately 8 million people get the disease and 2 million die of TB. The currently available vaccine against TB is the attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), which has failed to provide consistent protection in different parts of the world. The commonly used diagnostic reagent for TB is the purified protein derivative (PPD) of M. tuberculosis, which is nonspecific because of the presence of antigens crossreactive with BCG and environmental mycobacteria. Thus there is a need to identify M. tuberculosis antigens as candidates for new protective vaccines and specific diagnostic reagents against TB. By using the techniques of recombinant DNA, synthetic peptides, antigen-specific antibodies and T cells etc., several major antigens of M. tuberculosis have been identified, e.g. heat shock protein (hsp)60, hsp70, Ag85, ESAT-6 and CFP10 etc. These antigens have shown promise as new candidate vaccines and/or diagnostic reagents against TB. In addition, recent comparisons of the genome sequence of M. tuberculosis with BCG and other mycobacteria have unraveled M. tuberculosis specific regions and genes. Expression and immunological evaluation of these regions and genes can potentially identify most of the antigens of M. tuberculosis important for developing new vaccines and specific diagnostic reagents against TB. Moreover, advances in identification of proper adjuvant and delivery systems can potentially overcome the problem of poor immunogenicity/short-lived immunity associated with protein and peptide based vaccines. In conclusion, the advances in biotechnology are contributing significantly in the process of developing new protective vaccines and diagnostic reagents against TB.