Molecular Approaches to Receptors as Targets for Drug Discovery

Abstract

The cloning of a great number of receptors and channels has revealed that many of these targets for drug discovery can be grouped into superfamilies based on sequence and structural similarities. This review presents an overview of how molecular biological approaches have revealed a plethora of receptor subtypes, led to new definitions of subtypes and isoforms, and played a role in the development of highly selective drugs. Moreover, the diversity of subtypes has molded current views of the structure and function of receptor families. Practical difficulties and limitations inherent in the characterization of the ligand binding and signaling properties of expressed recombinant receptors are discussed. The importance of evaluating drug-receptor interactions that differ with temporally transient and distinct receptor conformational states is emphasized. Structural motifs and signal transduction features are presented for the following major receptor superfamilies: ligand-gated ion channel, voltage-dependent ion channel, G-protein coupled, receptor tyrosine-kinase, receptor protein tyrosine-phosphatase, cytokine and nuclear hormone. In addition, a prototypic receptor is analyzed to illustrate functional properties of a given family. The review concludes with a discussion of future directions in receptor research that will impact drug discovery, with a specific focus on orphan receptors as targets for drug discovery. Methods for classifying orphan receptors based upon homologies with members of existing superfamilies are presented together with molecular approaches to the greater challenge of defining their physiological roles. Besides revealing new orphan receptors, the human genome sequencing project will result in the identification of an abundance of novel receptors that will be molecular targets for the development of highly selective drugs. These findings will spur the discovery and development of an exciting new generation of receptor-subtype specific drugs with enhanced therapeutic specificity.     

Molecular Approaches in the Diagnosis of Dermatophytosis

Dermatophytosis is one of the most common infectious diseases in the world and can be caused by several dermatophyte species. These species are closely related in genetic structure in spite of different phenotypic and ecological features. The morphological similarity, variability, and polymorphism of dermatophytes have meant that species identification for dermatophytes is time consuming and requires a significant degree of knowledge and technological expertise. Molecular biology-based techniques have solved problems concerning the morphology-based identification of dermatophytes and have improved our knowledge on the epidemiology of dermatophytosis. Further development of molecular diagnosis of dermatophytosis requires the investigation of additional molecular markers for diagnostic tools targeting multiple loci as well as the improvement of techniques.     

Structural Propensities of Human Ubiquitination Sites: Accessibility,Centrality and Local Conformation

The existence and function of most proteins in the human proteome are regulated by the ubiquitination process. To date, tens of thousands human ubiquitination sites have been identified from high-throughput proteomic studies. However, the mechanism of ubiquitination site selection remains elusive because of the complicated sequence pattern flanking the ubiquitination sites. In this study, we perform a systematic analysis of 1,330 ubiquitination sites in 505 protein structures and quantify the significantly high accessibility and unexpectedly high centrality of human ubiquitination sites. Further analysis suggests that the higher centrality of ubiquitination sites is associated with the multi-functionality of ubiquitination sites, among which protein-protein interaction sites are common targets of ubiquitination. Moreover, we demonstrate that ubiquitination sites are flanked by residues with non-random local conformation. Finally, we provide quantitative and unambiguous evidence that most of the structural propensities contain specific information about ubiquitination site selection that is not represented by the sequence pattern. Therefore, the hypothesis about the structural level of the ubiquitination site selection mechanism has been substantially approved.     

Monte Carlo Investigations for Relative Efficiency of Stratified Random Sampling Scheme for Estimation of Relative Risk in Case-Control Studies

Monte Carlo Investigations have been widely used in Sample Surveys in Comparing the efficiency of various methods when exact mathematical comparisons are not possible. In this paper the same has been used for comparing the efficiency of Stratified Random Sampling with respect to Simple Random Sampling for estimation of Relative Risk in Case-Control Studies. The data used relate to a Case Control study on peptic ulcer. On the basis of Monte Carlo Investigations on 50 samples of size 10–20 (Cases and Controls), it has been observed that there is considerable gain in efficiency in using Stratified Random Sampling over Simple Random Sampling. The sensitivity of the results with the change in Sample Size has also been investigated.     

蛋白质相互作用研究方法及其应用

过去10年来,蛋白质组学得到迅速发展,蛋白质间的相互作用作为蛋白质组学的重要内容,更是成为国内外竞相研究的重点,研究方法的快速发展为蛋白质间相互作用的研究奠定了坚实基础。着重就经典的噬菌体展示、酵母双杂交以及新近发展起来的串联亲和纯化、荧光共振能量转移技术和表面等离子共振等蛋白质相互作用研究方法的原理及应用作一综述并展望其发展前景。     

APL的发病分子机制和治疗进展

急性前髓细胞性白血病(APL)是急性髓细胞样白血病(AML)的一个亚型,它的分子生物学特征为95%的患者有15号染色体前髓细胞性白血病基因(PML)与17号染色体视黄酸受体(RARα)基因的融合易位表达.维甲酸(ATRA)单药治疗能使APL患者达90%的缓解率,化疗药物与ATRA的联合应用更能降低患者复发率,改善生存;三氧化二砷(ATO)能使复发患者缓解率达到90%.这篇文章主要综述APL的发病分子机制和治疗进展.     

Genetic Approaches for Studying Myiasis-causing Flies: Molecular Markers and Mitochondrial Genomics

“Myiasis-causing flies” is a generic term that includes species from numerous dipteran families, mainly Calliphoridae and Oestridae, of which blowflies, screwworm flies and botflies are among the most important. This group of flies is characterized by the ability of their larvae to develop in animal flesh. When the host is a live vertebrate, such parasitism by dipterous larvae is known as primary myiasis. Myiasis-causing flies can be classified as saprophagous (free-living species), facultative or obligate parasites. Many of these flies are of great medical and veterinary importance in Brazil because of their role as key livestock insect-pests and vectors of pathogens, in addition to being considered important legal evidence in forensic entomology. The characterization of myiasis-causing flies using molecular markers to study mtDNA (by RFLP) and nuclear DNA (by RAPD and microsatellite) has been used to identify the evolutionary mechanisms responsible for specific patterns of genetic variability. These approaches have been successfully used to analyze the population structures of the New World screwworm fly Cochliomyia hominivorax and the botfly Dermatobia hominis. In this review, various aspects of the organization, evolution and potential applications of the mitochondrial genome of myiasis-causing flies in Brazil, and the analysis of nuclear markers in genetic studies of populations, are discussed.     

Molecular Approaches to Understanding Salinity Adaptation of Estuarine Animals

The molecular processes by which estuarine organisms adjustto salinity change are the central focus of this review, withemphasis on identifying the relevant mechanisms in euryhalinecrustaceans using the techniques of molecular biology. Thisreview is not intended to be complete with respect to ecologicaland physiological aspects but rather is an attempt to outlinea molecular approach which other investigators may find usefulas they address their own specific questions. Membrane transportersof sodium ions serve as the major focus, beginning with an examinationof candidate transport systems in gill epithelial cells. Particularemphasis is placed on the recent identification and sequencingof a putative Na⁺/H⁺ antiporter cDNA from gills of the greenshore crab Carcinus maenas     

Relative Prevalence of Grapevine Leafroll-Associated Virus Species in Wine Grape-Growing Regions of California

Some diseases manifest as one characteristic set of symptoms to the host, but can be caused by multiple pathogens. Control treatments based on plant symptoms can make it difficult to effectively manage such diseases, as the biology of the underlying pathogens can vary. Grapevine leafroll disease affects grapes worldwide, and is associated with several viral species in the family Closteroviridae. Whereas some of the viruses associated with this disease are transmitted by insect vectors, others are only graft-transmissible. In three regions of California, we surveyed vineyards containing diseased vines and screened symptomatic plants for all known viral species associated with grapevine leafroll disease. Relative incidence of each virus species differed among the three regions regions, particularly in relation to species with known vectors compared with those only known to be graft-transmitted. In one region, the pathogen population was dominated by species not known to have an insect vector. In contrast, populations in the other surveyed regions were dominated by virus species that are vector-transmissible. Our survey did not detect viruses associated with grapevine leafroll disease at some sites with characteristic disease symptoms. This could be explained either by undescribed genetic diversity among these viruses that prevented detection with available molecular tools at the time the survey was performed, or a misidentification of visual symptoms that may have had other underlying causes. Based on the differences in relative prevalence of each virus species among regions and among vineyards within regions, we expect that region and site-specific management strategies are needed for effective disease control.     

Evaluation of Local Media Surveillance for Improved Disease Recognition and Monitoring in Global Hotspot Regions

Digital disease detection tools are technologically sophisticated, but dependent on digital information, which for many areas suffering from high disease burdens is simply not an option. In areas where news is often reported in local media with no digital counterpart, integration of local news information with digital surveillance systems, such as HealthMap (Boston Children’s Hospital), is critical. Little research has been published in regards to the specific contribution of local health-related articles to digital surveillance systems. In response, the USAID PREDICT project implemented a local media surveillance (LMS) pilot study in partner countries to monitor disease events reported in print media. This research assessed the potential of LMS to enhance digital surveillance reach in five low- and middle-income countries. Over 16 weeks, select surveillance system attributes of LMS, such as simplicity, flexibility, acceptability, timeliness, and stability were evaluated to identify strengths and weaknesses in the surveillance method. Findings revealed that LMS filled gaps in digital surveillance network coverage by contributing valuable localized information on disease events to the global HealthMap database. A total of 87 health events were reported through the LMS pilot in the 16-week monitoring period, including 71 unique reports not found by the HealthMap digital detection tool. Furthermore, HealthMap identified an additional 236 health events outside of LMS. It was also observed that belief in the importance of the project and proper source selection from the participants was crucial to the success of this method. The timely identification of disease outbreaks near points of emergence and the recognition of risk factors associated with disease occurrence continue to be important components of any comprehensive surveillance system for monitoring disease activity across populations. The LMS method, with its minimal resource commitment, could be one tool used to address the information gaps seen in global ‘hot spot’ regions.     

Bootstrap Confidence Regions for Canonical Variates: Application to Studies of Evolutionary Differentiation

Theory recently developed to construct confidence regions based on the parametric bootstrap is applied to add inferential information to graphical displays of sample centroids in canonical variate analysis. Problems of morphometric differentiation among subspecies and species are addressed using numerical resampling procedures.     

Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

Background

The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives

The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results

Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A_2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A_2A is the adenosine receptor present in platelets; it is known that inosine is not an A_2A ligand. Docking of adenosine and inosine inside A_2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH₂ of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A_2A receptor.

Conclusion

Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.     

A Regression-Based Method for Estimating Risks and Relative Risks in Case-Base Studies

Both the absolute risk and the relative risk (RR) have a crucial role to play in epidemiology. RR is often approximated by odds ratio (OR) under the rare-disease assumption in conventional case-control study; however, such a study design does not provide an estimate for absolute risk. The case-base study is an alternative approach which readily produces RR estimation without resorting to the rare-disease assumption. However, previous researchers only considered one single dichotomous exposure and did not elaborate how absolute risks can be estimated in a case-base study. In this paper, the authors propose a logistic model for the case-base study. The model is flexible enough to admit multiple exposures in any measurement scale—binary, categorical or continuous. It can be easily fitted using common statistical packages. With one additional step of simple calculations of the model parameters, one readily obtains relative and absolute risk estimates as well as their confidence intervals. Monte-Carlo simulations show that the proposed method can produce unbiased estimates and adequate-coverage confidence intervals, for ORs, RRs and absolute risks. The case-base study with all its desirable properties and its methods of analysis fully developed in this paper may become a mainstay in epidemiology.     

On Estimation of Relative Risk in Case Control Studies

An estimator of relative risk in a case control study has been proposed in terms of observed cell frequencies and the probability of disease. The bias of the usual estimator i.e odds ratio as compared to the new estimator has been workedout. The expression of Mean Square Error of proposed estimator has been derived in situations where probability of disease is exactly known and when it is estimated through an independent survey. It has been observed that there is a serious error using odds ratio as an estimate of relative risk when probability of disease is not negligible. In such situations the proposed estimator can be used with advantage.