Illuminating drug discovery with biological pathways

Systems biology promises to impact significantly on the drug discovery process. One of its ultimate goals is to provide an understanding of the complete set of molecular mechanisms describing an organism. Although this goal is a long way off, many useful insights can already come from currently available information and technology. One of the biggest challenges in drug discovery today is the high attrition rate: many promising candidates prove ineffective or toxic owing to a poor understanding of the molecular mechanisms of biological systems they target. A "systems" approach can help identify pathways related to a disease and can suggest secondary effects of drugs that might cause these problems and thus ultimately improve the drug discovery pipeline.     

Quantitative analysis of cell signaling and drug action via mass spectrometry‐based systems level phosphoproteomics

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems‐level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS‐based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine‐directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.     

Lipidomics analysis in drug discovery and development

Despite being a relatively new addition to the Omics' landscape, lipidomics is increasingly being recognized as an important tool for the identification of druggable targets and biochemical markers. In this review we present recent advances of lipid analysis in drug discovery and development. We cover current state of the art technologies which are constantly evolving to meet demands in terms of sensitivity and selectivity. A careful selection of important examples is then provided, illustrating the versatility of lipidomics analysis in the drug discovery and development process. Integration of lipidomics with other omics’, stem-cell technologies, and metabolic flux analysis will open new avenues for deciphering pathophysiological mechanisms and the discovery of novel targets and biomarkers.     

Weak affinity chromatography as a new approach for fragment screening in drug discovery

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–μM in dissociation constant (K_D)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.     

Ensemble-based docking: From hit discovery to metabolism and toxicity predictions

This paper describes and illustrates the use of ensemble-based docking, i.e., using a collection of protein structures in docking calculations for hit discovery, the exploration of biochemical pathways and toxicity prediction of drug candidates. We describe the computational engineering work necessary to enable large ensemble docking campaigns on supercomputers. We show examples where ensemble-based docking has significantly increased the number and the diversity of validated drug candidates. Finally, we illustrate how ensemble-based docking can be extended beyond hit discovery and toward providing a structural basis for the prediction of metabolism and off-target binding relevant to pre-clinical and clinical trials.     

Identification of new inhibitors for low molecular weight protein tyrosine phosphatase isoform B

The National Cancer Institute Diversity Set II (1356 compounds) and Diversity Set III (1597 compounds) were screened via in silico methods as potential inhibitors of low molecular weight protein tyrosine phosphatase (LWM-PTP) isoform B (EC 3.1.3.48). Those candidates that demonstrated comparable or better docking scores than that of pyridoxal 5′-phosphate (PLP), one of the most potent known inhibitors of LMW-PTP with a competitive inhibitor dissociation constant (K_is) of 7.6 μM (pH 5.0), were analyzed via in vitro kinetic assays against LMW-PTP isoform B. While none of the compounds tested in vitro was significantly better that PLP, five compounds showed comparable inhibition. These five compounds are very diverse in structure and represent new therapeutic leads for inhibition of this isozyme.     

Systems biology of lipid metabolism: From yeast to human

Lipid metabolism is highly relevant as it plays a central role in a number of human diseases. Due to the highly interactive structure of lipid metabolism and its regulation, it is necessary to apply a holistic approach, and systems biology is therefore well suited for integrated analysis of lipid metabolism. In this paper it is demonstrated that the yeast Saccharomyces cerevisiae serves as an excellent model organism for studying the regulation of lipid metabolism in eukaryotes as most of the regulatory structures in this part of the metabolism are conserved between yeast and mammals. Hereby yeast systems biology can assist to improve our understanding of how lipid metabolism is regulated.     

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.     

Resistin: A journey from metabolism to cancer

Resistin, a small secretory molecule, has been implicated to play an important role in the development of insulin resistance under obese condition. For the past few decades, it has been linked to various cellular and metabolic functions. It has been associated with diseases like metabolic disorders, cardiovascular diseases and cancers. Numerous clinical studies have indicated an increased serum resistin level in pathological disorders which have been reported to increase mortality rate in comparison to low resistin expressing subjects. Various molecular studies suggest resistin plays a pivotal role in proliferation, metastasis, angiogenesis, inflammation as well as in regulating metabolism in cancer cells. Therefore, understanding the role of resistin and elucidating its’ associated molecular mechanism will give a better insight into the management of these disorders. In this article, we summarize the diverse roles of resistin in pathological disorders based on the available literature, clinicopathological data, and a compiled study from various databases. The article mainly provides comprehensive information of its role as a target in different treatment modalities in pre as well as post-clinical studies.     

Addressing the malaria drug resistance challenge using flow cytometry to discover new antimalarials

A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 μM) overall and ring stage in vitro antimalarial activity.     

Intrinsically disordered proteins and biomolecular condensates as drug targets

Intrinsically disordered domains represent attractive therapeutic targets because they play key roles in cancer, as well as in neurodegenerative and infectious diseases. They are, however, considered undruggable because they do not form stable binding pockets for small molecules and, therefore, have not been prioritized in drug discovery. Under physiological solution conditions many biomedically relevant intrinsically disordered proteins undergo phase separation processes leading to the formation of mesoscopic highly dynamic assemblies, generally known as biomolecular condensates that define environments that can be quite different from the solutions surrounding them. In what follows, we review key recent findings in this area and show how biomolecular condensation can offer opportunities for modulating the activities of intrinsically disordered targets.     

In vivo investigation of homocysteine metabolism to polyamines by high-resolution accurate mass spectrometry and stable isotope labeling

Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity.     

An optimised version of the secretome protein enrichment with click sugars (SPECS) method leads to enhanced coverage of the secretome

The secretome, the entirety of all soluble proteins either being secreted or proteolytically released by a cell, plays a key role in inter‐cellular communication of multi‐cellular organisms. Pathological alterations contribute to diseases such as hypertension, cancer, autoimmune disorders or neurodegenerative diseases. Hence, studying disease‐related perturbations of the secretome and the secretome itself covers an important aspect of cellular physiology. We recently developed the secretome protein enrichment with click sugars (SPECS) method that enables the analysis of secretomes of in vitro cell cultures even in the presence of FCS with MS. So far, SPECS facilitated the identification of protease substrates of BACE1, SPPL3 and ADAM10. Though, the SPECS method has already enabled deep insights into secretome biology, we aimed to improve the SPECS protocol to obtain even more information from MS‐based secretome analysis and reduce the amount of input material. Here, we optimised the reaction buffer, the pH and replaced Dibenzocyclooctyne (DBCO) PEG12‐biotin with the more water‐soluble variant DBCO‐sulpho‐biotin to finally provide an optimised protocol of the recently published SPECS protocol. Overall, the number of quantified glycoproteins and their average sequence coverage was increased by 1.6‐ and 2.4‐fold, respectively. Thus, the opzimised SPECS protocol allows reducing the input material by half without losing information. These improvements make the SPECS method more sensitive and more universal applicable to cell types with limited availability.     

Apoptosis induction with electric pulses — A new approach to cancer therapy with drug free

Electrical pulses have been widely used in biomedical fields, whose applications depend on the parameters such as durations and electric intensity. Conventional electroporation (0.1-1 kV/cm, 100 μs) has been used in cell fusion, transfection and electrochemotherapy. Recent studies with high-intensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact, resulting in an application of intracellular manipulation. The most recent development is the finding that parameters between those two ranges could be used to induce apoptosis of cancer cells. Proposal of apoptosis induction and tumor inhibition has advantages to pursue the treatment of cancer free of cytotoxic drugs.     

Dissecting the human plasma proteome and inflammatory response biomarkers

A central focus of clinical proteomics is to search for biomarkers in plasma for diagnostic and therapeutic use. We studied a set of plasma proteins accessed from the Healthy Human Individual's Integrated Plasma Proteome (HIP²) database, a larger set of curated human proteins, and a subset of inflammatory proteins, for overlap with sets of known protein biomarkers, drug targets, and secreted proteins. Most inflammatory proteins were found to occur in plasma, and over three times the level of biomarkers were found in inflammatory plasma proteins and their interacting protein neighbors compared to the sets of plasma and curated human proteins. Percentage overlaps with Gene Ontology terms were similar between the curated human set and plasma protein set, yet the set of inflammatory plasma proteins had a distinct ontology‐based profile. Most of the major hub proteins within protein‐protein interaction networks of tissue‐specific sets of inflammatory proteins were found to occur in disease pathways. The present study presents a systematic approach for profiling a plasma subproteome's relationship to both its potential range of clinical application and its overlap with complex disease.     

Epigenetic-based therapy: From single- to multi-target approaches

The treatment of cancer has traditionally been based on the identification of a single molecule and/or enzymatic function (target) responsible for a particular phenotype, and therefore on the ability to stimulate, attenuate or inhibit its activity through the use of selective compounds. However, cancer is no longer considered a disease caused by a single factor, but is now recognized as a multi-factorial disorder. Genetic, epigenetic and metabolic factors all contribute to neoplasia, causing significant changes in molecular networks that govern cell growth, development, death and specialization. Consequently, many antitumor therapies are no longer directed against a single target but the biological system as a whole, in which functions determining the onset and maintenance of a physio-pathological state are modulated. The field of epi-drug discovery is currently in a transitional phase where the search for putative anticancer drugs is shifting from single-target-oriented molecules to network-active compounds and to epi-drugs used in combination with other epi-agents and with traditional chemotherapeutics. This review illustrates the pros and cons of each therapeutic option, providing examples in support of single-target and multi (network)-target epi-drug approaches.     

From circadian rhythms to cancer chronotherapeutics

Mammalian circadian rhythms result from a complex organization involving molecular clocks within nearly all “normal” cells and a dedicated neuroanatomical system, which coordinates the so-called “peripheral oscillators.” The core of the central clock system is constituted by the suprachiasmatic nuclei that are located on the floor of the hypothalamus. Our understanding of the mechanisms of circadian rhythm generation and coordination processes has grown rapidly over the past few years. In parallel, we have learnt how to use the predictable changes in cellular metabolism or proliferation along the 24h time scale in order to improve treatment outcome for a variety of diseases, including cancer. The chronotherapeutics of malignant diseases has emerged as a result of a consistent development ranging from experimental, clinical, and technological prerequisites to multicenter clinical trials of chronomodulated delivery schedules. Indeed large dosing-time dependencies characterize the tolerability of anticancer agents in mice or rats, a better efficacy usually results from treatment administration near the least toxic circadian time in rodent tumor models. Programmable in time multichannel pumps have allowed to test the chronotherapy concepts in cancer patients and to implement chronomodulated delivery schedules in current practice. Clinical phase I and II trials have established the feasibility, the safety, and the activity of the chronotherapy schedules, so that this treatment method has undergone further evaluation in international multicenter phase III trials. Overall, more than 2000 patients with metastatic disease have been registered in chronotherapy trials. Improved tolerability and/or better antitumor activity have been demonstrated in randomized multicenter studies involving large patient cohorts. The relation between circadian rhythmicity and quality of life and even survival has also been a puzzling finding over the recent years. An essential step toward further developments of circadian-timed therapy has been the recent constitution of a Chronotherapy cooperative group within the European Organization for Research and Treatment of Cancer. This group now involves over 40 institutions in 12 countries. It is conducting currently six trials and preparing four new studies. The 19 contributions in this special issue reflect the current status and perspectives of the several components of cancer chronotherapeutics.     

Mass spectrometry-based analysis of glycoproteins and its clinical applications in cancer biomarker discovery

Glycosylation is one of the most important posttranslational modifications of proteins and plays essential roles in various biological processes. Aberration in the glycan moieties of glycoproteins is associated with many diseases. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. Mass spectrometry (MS) has become a powerful tool for glycoprotein analysis. Especially, tandem mass spectrometry can provide highly informative fragments for structural identification of glycoproteins. This review provides an overview of the development of MS technologies and their applications in identification of abnormal glycoproteins and glycans in human serum to screen cancer biomarkers in recent years.     

抗结核活性化合物HY-152E的作用靶点分析

抗结核活性化合物HY-152E是本实验室前期获得的具有良好抗结核活性并拥有授权专利（ZL201210088290.0）的小分子化合物（最低抑菌浓度≤0.09 μg/mL）。为深入探索HY-152E的抗结核机制,本研究利用药物亲和反应靶标稳定性（drug affinity responsive target stability,DARTS）技术并结合蛋白质谱技术,分析可能与HY-152E相互作用的结核分枝杆菌潜在靶标蛋白。将结核分枝杆菌H37Ra的菌体蛋白裂解液与HY-152E共同孵育互作,用不同浓度的链霉菌蛋白酶消化30、45、60 min后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）分离并比较与HY-152E互作前后菌体蛋白耐受蛋白酶消化的差异条带,分别在相对分子质量70 000和45 000~55 000处观察到差异蛋白条带。利用蛋白质谱技术分析差异条带的蛋白信息,共获得86个蛋白信息。结合结核分枝杆菌数据库及蛋白功能信息,最终筛选到9个蛋白可能是HY-152E的抗结核作用潜在靶标。这些潜在靶点的确定,为后续研究HY-152E的抗结核分子机制奠定了基础。     

Chemical genetics and drug screening in Drosophila cancer models

Drug candidates often fail in preclinical and clinical testing because of reasons of efficacy and/or safety.It would be time- and cost-efficient to have screening models that reduce the rate of such false positive candidates that appear promising at first but fail later.In this regard,it would be particularly useful to have a rapid and inexpensive whole animal model that can pre-select hits from high-throughput screens but before testing in costly rodent assays.Drosophila melanogaster has emerged as a potential whole animal model for drug screening.Of particular interest have been drugs that must act in the context of multi-cellularity such as those for neurological disorders and cancer.A recent review provides a comprehensive summary of drug screening in Drosophila,but with an emphasis on neurodegenerative disorders.Here,we review Drosophila screens in the literature aimed at cancer therapeutics.