Identification of the human testis protein phosphatase 1 interactome

Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins—PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.     

Tuberous sclerosis preclinical studies: timing of treatment, combination of a rapamycin analog (CCI-779) and interferon-gamma, and comparison of rapamycin to CCI-779

Background

Protein-protein interactions (PPIs) are challenging but attractive targets for small chemical drugs. Whole PPIs, called the 'interactome', have been emerged in several organisms, including human, based on the recent development of high-throughput screening (HTS) technologies. Individual PPIs have been targeted by small drug-like chemicals (SDCs), however, interactome data have not been fully utilized for exploring drug targets due to the lack of comprehensive methodology for utilizing these data. Here we propose an integrative in silico approach for discovering candidates for drug-targetable PPIs in interactome data.

Results

Our novel in silico screening system comprises three independent assessment procedures: i) detection of protein domains responsible for PPIs, ii) finding SDC-binding pockets on protein surfaces, and iii) evaluating similarities in the assignment of Gene Ontology (GO) terms between specific partner proteins. We discovered six candidates for drug-targetable PPIs by applying our in silico approach to original human PPI data composed of 770 binary interactions produced by our HTS yeast two-hybrid (HTS-Y2H) assays. Among them, we further examined two candidates, RXRA/NRIP1 and CDK2/CDKN1A, with respect to their biological roles, PPI network around each candidate, and tertiary structures of the interacting domains.

Conclusion

An integrative in silico approach for discovering candidates for drug-targetable PPIs was applied to original human PPIs data. The system excludes false positive interactions and selects reliable PPIs as drug targets. Its effectiveness was demonstrated by the discovery of the six promising candidate target PPIs. Inhibition or stabilization of the two interactions may have potential therapeutic effects against human diseases.     

SARS病毒中S蛋白的hAPN受体接合功能域分析(英文)

目的:获得SARS冠状病毒S蛋白与CD13相互作用的信息,发现其可能的配体-受体作用区域和结合位点,为SARS蛋白功能研究以及设计抗SARS药物和疫苗提供线索。方法:在比较基因组学的基础上,通过运用多序列比对、同源性分析和进化分析等手段预测并确定SARS冠状病毒S蛋白与CD13相互作用的区域和结合位点,并用分子模拟和分子对接分析的方法模建S蛋白与CD13在预测区域的相互作用。结果:获得了SARS冠状病毒S蛋白与CD13相互作用的信息,发现了一个冠状病毒S蛋白与CD13相互作用的功能域,以及位于此功能域中的4个可能的相互作用的位点。分子模拟验证了其中一个可能的相互作用的位点。结论:CD13可能是SARS冠状病毒S蛋白结合的一个靶点,它们之间的相互作用可能是SARS病毒感染的途径之一。同时,本研究也为运用生物信息方法寻找蛋白质作用靶点的线索提供了一种策略。     

Aminoacyl-tRNA synthetases: essential and still promising targets for new anti-infective agents

The emergence of resistance to existing antibiotics demands the development of novel antimicrobial agents directed against novel targets. Historically, bacterial cell wall synthesis, protein, and DNA and RNA synthesis have been major targets of very successful classes of antibiotics such as beta-lactams, glycopeptides, macrolides, aminoglycosides, tetracyclines, rifampicins and quinolones. Recently, efforts have been made to develop novel agents against validated targets in these pathways but also against new, previously unexploited targets. The era of genomics has provided insights into novel targets in microbial pathogens. Among the less exploited--but still promising--targets is the family of 20 aminoacyl-tRNA synthetases (aaRSs), which are essential for protein synthesis. These targets have been validated in nature as aaRS inhibition has been shown as the specific mode of action for many natural antimicrobial agents synthesized by bacteria and fungi. Therefore, aaRSs have the potential to be targeted by novel agents either from synthetic or natural sources to yield specific and selective anti-infectives. Numerous high-throughput screening programs aimed at identifying aaRS inhibitors have been performed over the last 20 years. A large number of promising lead compounds have been identified but only a few agents have moved forward into clinical development. This review provides an update on the present strategies to develop novel aaRS inhibitors as anti-infective drugs.     

Network-based drug repositioning: A novel strategy for discovering potential antidepressants and their mode of action

Current target-oriented paradigm for novel antidepressant discovery has been difficult to succeed and the failures always bring huge economic losses. Although abundant ledge of disease related genes and drug action targets has been accumulated, the successful application of the knowledge for new drug discovery is limited. Here, we predicted and validated potential antidepressants and molecular targets from DrugBank recorded drugs using a novel network-based drug repositioning approach. This approach predicted relationships between drug and targets through network-based integration of drug chemical similarity, therapeutic similarity and protein–protein interactions. We predicted genome-wide relations of drugs and targets, and then screened drugs that connect to depression-related targets of known antidepressants. Six drugs were predicted and experimentally validated to have antidepressant-like effects in the tail suspension test (TST) and forced swimming test (FST) in mice. Alverine, which is a gastrointestinal antispasmodic drug, was further validated to display antidepressant-like effects in the learned helplessness and chronic unpredictable stress models of depression. Four targets, including serotonin transporter, norepinephrine transporter, serotonin 1A receptor and serotonin 2A receptor, were included in the predictable system and confirmed as primary sites of action for alverine. The results suggest that alverine may be an effective antidepressant drug and the network-based drug repositioning may be a promising drug discovery paradigm for complex multi-genetic diseases such as depression.     

Evaluation of kinase inhibitor selectivity by chemical proteomics

Small-molecule inhibitors of protein kinases constitute a novel class of drugs for therapeutic intervention in a variety of human diseases. Most of these agents target the relatively conserved ATP-binding site of protein kinases and have only been tested against a rather small subset of all human protein kinases. Therefore, the selectivity of protein kinase inhibitors has remained a widely underestimated, but highly important issue in drug development programs. In this review, we focus on the recent advancement of chemical proteomic methods to evaluate drug selectivity in an unbiased, comprehensive way. Efficient affinity purification procedures using immobilized kinase inhibitors combined with the sensitivity of mass spectrometry detection permit the mapping of drug targets on a proteome-wide scale. Data from this type of assessment can be used to set up tailor-made selectivity panels, which guide compound development in the context of the most relevant off-targets during lead optimization. In cases in which identified alternative targets are of validated clinical relevance, chemical proteomics provides the opportunity to repeatedly exploit a once established kinase inhibitor principle for additional target kinases and can thereby dramatically shorten the time toward highly selective, preclinical candidates. Moreover, the identification of alternative targets for preclinical or clinical drugs can provide new insights into their cellular modes of action, which might help to define those disease settings in which the most beneficial therapeutic effect is likely to occur.     

Dopamine receptor interacting proteins: targeting neuronal calcium sensor-1/D2 dopamine receptor interaction for antipsychotic drug development

D2 dopamine receptors (D2Rs) represent an important class of receptors in the pharmacological development of novel therapeutic drugs for the treatment of schizophrenia. Recent research into D2R signaling suggests that receptor properties are dependent on interaction with a cohort of dopamine receptor interacting proteins (DRIPs) within a macromolecular structure termed the signalplex. One component of this signalplex is neuronal calcium sensor 1 (NCS-1) a protein found to regulate the phosphorylation, trafficking, and signaling profile of the D2R in neurons. It has also been found that NCS-1 can contribute to the pathology of schizophrenia and may play a role in the efficacy of antipsychotic drug medication in the brain. In this review we discuss how the selective targeting of a DRIP, such as NCS-1, can be utilized as a novel strategy of drug design for the creation of new therapeutics for a disease such as schizophrenia. Using a fluorescence polarization assay we explore how the ability to detect changes in D2R/NCS-1 interaction can be exploited as an effective screening tool in the isolation and development of lead compounds for antipsychotic drug development. This line of work explores a novel direction in targeting D2Rs via their signalplex components and supports the notion that receptor interacting proteins represent an emerging new class of molecular targets for pharmacological drug development.     

病毒-宿主相互作用的系统生物学与宿主靶向抗病毒策略

以病毒蛋白为靶的抗病毒药物面临易产生耐药、抗病毒谱较窄等诸多问题,宿主分子靶向已经成目前抗病毒药物研究的重要策略,宿主靶标的辨识是宿主靶向药物设计的关键。病毒-宿主相互作用的系统生物学研究将成为抗病毒药物宿主靶标辨识和宿主-病毒联合靶向治疗策略设计提供有力工具。近年来通过蛋白质组学、大规模基因沉默、基因芯片等实验得到了大量的病毒感染相关宿主分子和病毒-宿主分子相互作用关系,为在病毒-宿主分子网络水平揭示病毒生存策略奠定了基础。整合病毒感染基因表达谱和人蛋白相互作用网络可以构建病毒感染激活网络,进而通过网络分析获得关键的宿主因子。正在发展的动态蛋白质组学和动态网络分析技术将为建立更加真实的病毒-宿主分子网络模型,进而辨识有效的宿主靶标提供有力工具。     

Novel antimalarial drug targets: hope for new antimalarial drugs

Malaria is a major global threat, that results in more than 2 million deaths each year. The treatment of malaria is becoming extremely difficult due to the emergence of drug-resistant parasites, the absence of an effective vaccine, and the spread of insecticide-resistant vectors. Thus, malarial therapy needs new chemotherapeutic approaches leading to the search for new drug targets. Here, we discuss different approaches to identifying novel antimalarial drug targets. We have also given due attention to the existing validated targets with a view to develop novel, rationally designed lead molecules. Some of the important parasite proteins are claimed to be the targets; however, further in vitro or in vivo structure-function studies of such proteins are crucial to validate these proteins as suitable targets. The interactome analysis among apicoplast, mitochondrion and genomic DNA will also be useful in identifying vital pathways or proteins regulating critical pathways for parasite growth and survival, and could be attractive targets. Molecules responsible for parasite invasion to host erythrocytes and ion channels of infected erythrocytes, essential for intra-erythrocyte survival and stage progression of parasites are also becoming attractive targets. This review will discuss and highlight the current understanding regarding the potential antimalarial drug targets, which could be utilized to develop novel antimalarials.     

Novel antimalarial drug targets: hope for new antimalarial drugs

Malaria is a major global threat, that results in more than 2 million deaths each year. The treatment of malaria is becoming extremely difficult due to the emergence of drug-resistant parasites, the absence of an effective vaccine, and the spread of insecticide-resistant vectors. Thus, malarial therapy needs new chemotherapeutic approaches leading to the search for new drug targets. Here, we discuss different approaches to identifying novel antimalarial drug targets. We have also given due attention to the existing validated targets with a view to develop novel, rationally designed lead molecules. Some of the important parasite proteins are claimed to be the targets; however, further in vitro or in vivo structure–function studies of such proteins are crucial to validate these proteins as suitable targets. The interactome analysis among apicoplast, mitochondrion and genomic DNA will also be useful in identifying vital pathways or proteins regulating critical pathways for parasite growth and survival, and could be attractive targets. Molecules responsible for parasite invasion to host erythrocytes and ion channels of infected erythrocytes, essential for intra-erythrocyte survival and stage progression of parasites are also becoming attractive targets. This review will discuss and highlight the current understanding regarding the potential antimalarial drug targets, which could be utilized to develop novel antimalarials.     

The hunt for antimitotic agents: an overview of structure-based design strategies

ABSTRACT

Introduction: Structure-based drug discovery offers a rational approach for the design and development of novel anti-mitotic agents which target specific proteins involved in mitosis. This strategy has paved the way for development of a new generation of chemotypes which selectively interfere with the target proteins. The interference of these anti-mitotic targets implicated in diverse stages of mitotic cell cycle progression culminates in cancer cell apoptosis.

Areas covered: This review covers the various mitotic inhibitors developed against validated mitotic checkpoint protein targets using structure-based design and optimization strategies. The protein-ligand interactions and the insights gained from these studies, culminating in the development of more potent and selective inhibitors, have been presented.

Expert opinion: The advent of structure-based drug design coupled with advances in X-ray crystallography has revolutionized the discovery of candidate lead molecules. The structural insights gleaned from the co-complex protein-drug interactions have provided a new dimension in the design of anti-mitotic molecules to develop drugs with a higher selectivity and specificity profile. Targeting non-catalytic domains has provided an alternate approach to address cross-reactivity and broad selectivity among kinase inhibitors. The elucidation of structures of emerging mitotic drug targets has opened avenues for the design of inhibitors that target cancer.     

Binding mode of conformations and structure-based pharmacophore development for farnesyltransferase inhibitors

Farnesyltransferase (FTase) is one of the prenyltransferase family enzymes that catalyse the transfer of 15-membered isoprenoid (farnesyl) moiety to the cysteine of CAAX motif-containing proteins including Rho and Ras family of G proteins. Inhibitors of FTase act as drugs for cancer, malaria, progeria and other diseases. In the present investigation, we have developed two structure-based pharmacophore models from protein–ligand complex (3E33 and 3E37) obtained from the protein data bank. Molecular dynamics (MD) simulations were performed on the complexes, and different conformers of the same complex were generated. These conformers were undergone protein–ligand interaction fingerprint (PLIF) analysis, and the fingerprint bits have been used for structure-based pharmacophore model development. The PLIF results showed that Lys164, Tyr166, TrpB106 and TyrB361 are the major interacting residues in both the complexes. The RMSD and RMSF analyses on the MD-simulated systems showed that the absence of FPP in the complex 3E37 has significant effect in the conformational changes of the ligands. During this conformational change, some interactions between the protein and the ligands are lost, but regained after some simulations (after 2 ns). The structure-based pharmacophore models showed that the hydrophobic and acceptor contours are predominantly present in the models. The pharmacophore models were validated using reference compounds, which significantly identified as HITs with smaller RMSD values. The developed structure-based pharmacophore models are significant, and the methodology used in this study is novel from the existing methods (the original X-ray crystallographic coordination of the ligands is used for the model building). In our study, along with the original coordination of the ligand, different conformers of the same complex (protein–ligand) are used. It concluded that the developed methodology is significant for the virtual screening of novel molecules on different targets.     

Heat shock protein inhibitors for the treatment of fungal infections

Invasive fungal infections are a leading cause of mortality, especially in immunocompromised patients. Therapy is made difficult by the limited number of antifungal agents currently available which mostly target ergosterol in fungal cell membranes. The paucity of targets allows the development of cross resistance to all drugs with a common target. This highlights the need to develop new therapeutic strategies for fungal disease including agents with novel mechanisms of action. Heat shock protein 90 stabilizes calcineurin which regulates response to stress, allowing for calcineurin dependent stress responses required to survive exposure to antifungal drugs. Heat shock protein 90 inhibition abrogates calcineurin dependent stress responses, changing fungistatic drugs to fungicidal. Targeting a highly conserved protein that has a vital role in many cellular signaling pathways, reduces the potential for emergence of resistance to heat shock proteins inhibitors. This article will review recent patents in novel heat shock protein inhibitor therapy, such as efungumab, which diminish the emergence of antifungal drug resistance and enable greater efficacy of existing antifungals.     

Triterpene derivatives as inhibitors of protein involved in the inflammatory process: molecules interfering with phospholipase A2, cycloxygenase, and lipoxygenase

Over the past years, there was an explosion in the knowledge of the protein target and molecular mechanism associated with various disease types and in the new research of drugs of natural origin. The key idea is to evaluate bioactive natural products interacting with protein domains of different genetic origin but structurally preserved to develop libraries of compounds biologically validated and selected from an evolutionistic point of view. Compared with synthetic compounds, natural products have a major number of unused scaffolds and not comparable to the libraries of synthetic compounds, and could represent a promising starting points for the discovery of new bioactive compounds. Many natural products are reported to interact with proteins involved in serious diseases, such as inflammation and cancer. Recently various chemical classes of plant secondary metabolites have emerged as potential therapeutic compounds in several inflammatory diseases. Owing to the findings that triterpenoids, a common class of plant secondary metabolites, have anti-inflammatory and anti-cancer effects on humans, the interest in their potential application in human health and disease is increasing. The present review describes anti-inflammatory triterpenes derivatives from plant and fungi reported during the last two decades in order to provide an account of this field of investigation, sorting compounds according to their targets, phospholipase A(2) (PLA(2)), cycloxygenase (COX), and lipoxygenase (LOX). The attempt is also being made to enumerate the possible leads for further synthetic and drug discovery program development.     

PDZ domains at excitatory synapses: potential molecular targets for persistent pain treatment

Persistent pain, a common clinical condition, could be caused by inflammation, tissue injury secondary to trauma or surgery, and nerve injuries. It is often inadequately controlled by current treatments, such as opioids and nonsteroidal anti-inflammatory drugs. The PDZ (Postsynaptic density 95, Discs large, and Zonula occludens-1) domains are ubiquitous protein interaction modules often found among multi-protein signaling complexes at neuronal synapses. Recent preclinical research shows that targeted disruption of PDZ domain-mediated protein interaction among N-methyl-Daspartate (NMDA) receptor signaling complexes significantly attenuates the development and maintenance of persistent pain without affecting nociceptive responsiveness to acute pain. PDZ domains at excitatory synapses may be new molecular targets for prevention and treatment of persistent pain. Here, we illustrate expression and distribution of the PDZ domain-containing proteins associated with NMDA receptors in the pain-related regions of the central nervous system, review the evidence for their roles in persistent pain states, and discuss potential mechanisms by which these PDZ domain-containing proteins are involved in persistent pain.     

整合化学和定量蛋白质组学方法用于鉴定药物靶点（英文）

Most drugs exert pharmacological effects through interaction with their target proteins.Therefore,drug target identification is a crucial step towards the understanding of the mechanism of drug action.It is also imperative to study the pharmacodynamics of a known drug,with an aim to discover the potentially unrevealed actions and thus refine its future clinical applications.Currently,drug-target identification is either through in vitro affinity chromatography-based approaches or in vivo activity-based protein profiling(ABPP)approaches.However,these approaches generally face difficulties discriminating specific drug targets from non-specific ones.To address this issue,we have come up with a strategy by coupling iTRAQTM(isobaric tags for relative and absolute quantitation)quantitative proteomics approach with clickable ABPP,to specifically and compre hensively identify drug targets in live cells.Using this approach,we identified the protein targets of andrographolide,a natural product with known anti-inflammation and anti-cancer effects,in live cancer cells.The identified target list not only confirmed the known functions of the drug but also revealed its potential novel application as a tumor metastasis inhibitor.We have also used this strategy,combining with a cleavable probe to identify the protein targets of aspirin and its binding sites.Our results revealed the roles of aspirin ininhibition of protein synthesis and induction of autophagy,which have been functionally validated.Our strategy is widely applicable to the identification of protein targets of covalent drugs.     

Survival and apoptotic signals in the action of microtubule-targeting antitumor drugs

Microtubule-targeting agents include some of the most successful drugs in cancer chemotherapy, such as taxanes. However, the development of drug resistance has prompted the search for novel therapeutics. The hallmark of microtubule-interfering drugs is mitotic arrest that eventually leads to apoptosis. The interaction between apoptosis and the cell cycle is essential to preserve homeostasis and genomic integrity, and offers new targets for cancer treatment. Elucidation of the molecular mechanism that couples microtubule damage to the onset of apoptosis is expected to reveal novel sites of therapeutic intervention. Microtubule damage induces both survival and pro-apoptotic signals, and inhibition of survival signaling could improve the efficacy of microtubule-targeting agents.     

Microbial drug efflux proteins of the major facilitator superfamily

Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.     

New therapeutic approaches targeted at the late stages of the HIV-1 replication cycle

Owing to the serious clinical consequences associated with acquisition of resistance to current antiretroviral drugs, discovery of new drug targets and development of novel anti-HIV-1 therapeutic agents have become a high research priority. The late stages of HIV-1 replication involve the processes of assembly, budding and maturation, and comprise several new potential therapeutic targets which have not (yet) been targeted by any of the antiretroviral drugs approved at present. The structural protein Gag plays a central role in these stages through its different regions and mature Gag proteins working in concert. In this article, we highlight a number of steps in the late stages of HIV-1 replication that represent promising targets for drug discovery. Recent progress in development of related inhibitors targeting at CA, zinc fingers of NC, p6-Tsg101 interaction, lipid rafts of plasma membrane, proteolytic cleavage sites in Gag and gp160 processing is also reviewed.