Multi-target Screening Research Articles

Doping control analysis of 121 prohibited substances in equine hair by liquid chromatography–tandem mass spectrometry

Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describes a sensitive ultra-high performance liquid chromatography – tandem mass spectrometry (UHPLC–MS/MS) method for the detection of 121 drugs and/or their metabolites in equine hair covering ten classes of prohibited substances with estimated limits of detection between 0.1 and 10 pg/mg. To our knowledge, this is the first report of a screening method in equine hair which can cover such a broad range and well over one hundred prohibited substances in a single analytical run. This method has been validated for its specificity, precision and extraction recovery. Applicability of this method has been demonstrated by: (i) the successful identification of clenbuterol, 2-(1-hydroxyethyl) promazine sulfoxide, acepromazine and tetrahydrozoline in genuine equine mane samples; as well as (ii) the detection of drugs from artificially incurred mane hair samples which have been prepared by soaking blank hair samples in solutions of drug targets.

Multi-functional neuroprotective activity of neohesperidin dihydrochalcone: a novel scaffold for Alzheimer's disease therapeutics identified via drug repurposing screening

Multi-target screening identifies neohesperidin dihydrochalcone for Alzheimer's disease therapeutics, which exhibits strong BACE1 and amyloid aggregation inhibition along with antioxidant activity.

Recent applications of gas chromatography with high-resolution mass spectrometry.

Gas chromatography coupled to high-resolution mass spectrometry is a powerful analytical method that combines excellent separation power of gas chromatography with improved identification based on an accurate mass measurement. These features designate gas chromatography with high-resolution mass spectrometry as the first choice for identification and structure elucidation of unknown volatile and semi-volatile organic compounds. Gas chromatography with high-resolution mass spectrometry quantitative analyses was previously focused on the determination of dioxins and related compounds using magnetic sector type analyzers, a standing requirement of many international standards. The introduction of a quadrupole high-resolution time-of-flight mass analyzer broadened interest in this method and novel applications were developed, especially for multi-target screening purposes. This review is focused on the development and the most interesting applications of gas chromatography coupled to high-resolution mass spectrometry towards analysis of environmental matrices, biological fluids, and food safety since 2010. The main attention is paid to various approaches and applications of gas chromatography coupled to high-resolution mass spectrometry for non-target screening to identify contaminants and to characterize the chemical composition of environmental, food, and biological samples. The most interesting quantitative applications, where a significant contribution of gas chromatography with high-resolution mass spectrometry over the currently used methods is expected, will be discussed as well.

A sediment extraction and cleanup method for wide-scope multitarget screening by liquid chromatography–high-resolution mass spectrometry

Previous studies on organic sediment contaminants focused mainly on a limited number of highly hydrophobic micropollutants accessible to gas chromatography using nonpolar, aprotic extraction solvents. The development of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) permits the spectrum of analysis to be expanded to a wider range of more polar and ionic compounds present in sediments and allows target, suspect, and nontarget screening to be conducted with high sensitivity and selectivity. In this study, we propose a comprehensive multitarget extraction and sample preparation method for characterization of sediment pollution covering a broad range of physicochemical properties that is suitable for LC-HRMS screening analysis. We optimized pressurized liquid extraction, cleanup, and sample dilution for a target list of 310 compounds. Finally, the method was tested on sediment samples from a small river and its tributaries. The results show that the combination of 100 °C for ethyl acetate-acetone (50:50, neutral extract) followed by 80 °C for acetone-formic acid (100:1, acidic extract) and methanol-10 mM sodium tetraborate in water (90:10, basic extract) offered the best extraction recoveries for 287 of 310 compounds. At a spiking level of 1 μg mL-1, we obtained satisfactory cleanup recoveries for the neutral extract-(93 ± 23)%-and for the combined acidic/basic extracts-(42 ± 16)%-after solvent exchange. Among the 69 compounds detected in environmental samples, we successfully quantified several pharmaceuticals and polar pesticides.

Multi-Target Screening and Experimental Validation of Natural Products from Selaginella Plants against Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder which is considered to be the most common cause of dementia. It has a greater impact not only on the learning and memory disturbances but also on social and economy. Currently, there are mainly single-target drugs for AD treatment but the complexity and multiple etiologies of AD make them difficult to obtain desirable therapeutic effects. Therefore, the choice of multi-target drugs will be a potential effective strategy inAD treatment. To find multi-target active ingredients for AD treatment from Selaginella plants, we firstly explored the behaviors effects on AD mice of total extracts (TE) from Selaginella doederleinii on by Morris water maze test and found that TE has a remarkable improvement on learning and memory function for AD mice. And then, multi-target SAR models associated with AD-related proteins were built based on Random Forest (RF) and different descriptors to preliminarily screen potential active ingredients from Selaginella. Considering the prediction outputs and the quantity of existing compounds in our laboratory, 13 compounds were chosen to carry out the in vitro enzyme inhibitory experiments and 4 compounds with BACE1/MAO-B dual inhibitory activity were determined. Finally, the molecular docking was applied to verify the prediction results and enzyme inhibitory experiments. Based on these study and validation processes, we explored a new strategy to improve the efficiency of active ingredients screening based on trace amount of natural product and numbers of targets and found some multi-target compounds with biological activity for the development of novel drugs for AD treatment.

Multi-functional activities of citrus flavonoid narirutin in Alzheimer’s disease therapeutics: An integrated screening approach and in vitro validation

Alzheimer’s disease (AD) is a complex disorder and the disease mechanism is yet to be properly characterized. Over the years, “amyloid cascade” emerges as principal pathogenic event in AD. β-Secretase 1 (BACE1) controls the rate-limiting step in amyloid beta (Aβ) generation and Aβ rapidly aggregates to form neurotoxic amyloid fibrils. Oxidative stress is one of the principal mediators of the observed neurotoxicity of amyloid fibril. The disease pathogenesis involves induction of multiple signaling cascades and the cross-talk therein. Thus inhibiting multiple targets could be an attractive therapeutic strategy. Here, a multi-target virtual screening protocol has been devised and used to screen an in house developed phytochemical library. Narirutin comes out as a multi-potent phytochemical. Steady-state and time-resolved fluorescence along with molecular modelling studies demonstrate its binding to the BACE1 active site that induces a conformational transition of the protein from open to closed form which precludes substrate recognition. Narirutin possesses strong Aβ aggregation inhibitory potential, evident from ANS and Thioflavin-T binding assay and confirmed by AFM study. ABTS·+ scavenging assay shows moderate antioxidant activity for narirutin. The applicability of a multi-target screening strategy in AD therapeutics is thus demonstrated and the identified hit, narirutin, shows strong multi-potent activity.

Getting SMARt in drug discovery: chemoinformatics approaches for mining structure–multiple activity relationships

In light of the high relevance of polypharmacology, multi-target screening is a major trend in drug discovery.

Recent advances of liquid chromatography–(tandem) mass spectrometry in clinical and forensic toxicology — An update

Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is a well-established and widely used technique in clinical and forensic toxicology as well as doping control especially for quantitative analysis. In recent years, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in biological matrices have been developed. Such methods have proven particularly useful for analysis of so-called new psychoactive substances that have appeared on recreational drug markets throughout the world. Moreover, the evolvement of high resolution MS techniques and the development of data-independent detection modes have opened new possibilities for applications of LC–(MS/MS) in systematic toxicological screening analysis in the so called general unknown setting. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2010.

Body burden of pesticides and wastewater-derived pollutants on freshwater invertebrates: Method development and application in the Danube River

While environmental risk assessment is typically based on toxicant concentrations in water and/or sediment, awareness is increasing that internal concentrations or body burdens are the key to understand adverse effects in organisms. In order to link environmental micropollutants as causes of observed effects, there is an increasing demand for methods to analyse these chemicals in organisms. Here, a multi-target screening method based on pulverised liquid extraction (PuLE) and a modified QuEChERS approach with an additional hexane phase was developed. It is capable to extract and quantify organic micropollutants of diverse chemical classes in freshwater invertebrates. The method was tested on gammarids from the Danube River (within the Joint Danube Survey 3) and target compounds were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, a non-target screening using high resolution-tandem mass spectrometry (LC-HRMS/MS) was conducted. A total of 17 pollutants were detected and/or quantified in gammarids at low concentrations. Pesticide concentrations ranged from 0.1 to 6.52 ng g−1 (wet weight), those of wastewater-derived pollutants from 0.1 to 2.83 ng g−1 (wet weight). The presence of wastewater-derived pollutants was prominent at all spots sampled. Using non-target screening, we could successfully identify several chlorinated compounds. These results demonstrate for the first time the presence of pesticides and wastewater-derived pollutants in invertebrates of the Danube River.

Multi-target screening mines hesperidin as a multi-potent inhibitor: Implication in Alzheimer's disease therapeutics

Alzheimer's disease (AD) is the most frequent form of neurodegenerative disorder in elderly people. Involvement of several pathogenic events and their interconnections make this disease a complex disorder. Therefore, designing compounds that can inhibit multiple toxic pathways is the most attractive therapeutic strategy in complex disorders like AD. Here, we have designed a multi-tier screening protocol combining ensemble docking to mine BACE1 inhibitor, as well as 2-D QSAR models for anti-amyloidogenic and antioxidant activities. An in house developed phytochemical library of 200 phytochemicals has been screened through this multi-target procedure which mine hesperidin, a flavanone glycoside commonly found in citrus food items, as a multi-potent phytochemical in AD therapeutics. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of hesperidin to the active site of BACE1 induces a conformational transition of the protein from open to closed form. Hesperidin docks close to the catalytic aspartate residues and orients itself in a way that blocks the cavity opening thereby precluding substrate binding. Hesperidin is a high affinity BACE1 inhibitor and only 500 nM of the compound shows complete inhibition of the enzyme activity. Furthermore, ANS and Thioflavin-T binding assay show that hesperidin completely inhibits the amyloid fibril formation which is further supported by atomic force microscopy. Hesperidin exhibits moderate ABTS+ radical scavenging assay but strong hydroxyl radical scavenging ability, as evident from DNA nicking assay. Present study demonstrates the applicability of a novel multi-target screening procedure to mine multi-potent agents from natural origin for AD therapeutics.

"Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

A ranking method for the concurrent learning of compounds with various activity profiles.

BackgroundIn this study, we present a SVM-based ranking algorithm for the concurrent learning of compounds with different activity profiles and their varying prioritization. To this end, a specific labeling of each compound was elaborated in order to infer virtual screening models against multiple targets. We compared the method with several state-of-the-art SVM classification techniques that are capable of inferring multi-target screening models on three chemical data sets (cytochrome P450s, dehydrogenases, and a trypsin-like protease data set) containing three different biological targets each.ResultsThe experiments show that ranking-based algorithms show an increased performance for single- and multi-target virtual screening. Moreover, compounds that do not completely fulfill the desired activity profile are still ranked higher than decoys or compounds with an entirely undesired profile, compared to other multi-target SVM methods.ConclusionsSVM-based ranking methods constitute a valuable approach for virtual screening in multi-target drug design. The utilization of such methods is most helpful when dealing with compounds with various activity profiles and the finding of many ligands with an already perfectly matching activity profile is not to be expected.Electronic supplementary materialThe online version of this article (doi:10.1186/s13321-014-0050-6) contains supplementary material, which is available to authorized users.

Ultracentrifugation-based multi-target affinity selection mass spectrometry

A rate-zonal density gradient ultracentrifugation-based affinity selection mass spectrometry approach has been developed for simultaneous multi-target screening.

Highly Multiplexed Targeted Sequencing of Recurrent Fusion Genes in Acute Leukemia

In acute leukemia, recurrent chromosomal translocations which result in the fusion of two genes are frequent. Some of these markers have a well established prognostic and therapeutic impact and are routinely screened at diagnosis by cytogenetics and RT-PCR. Yet, due to the limitations of these methods, only a few among the dozens of known rearrangements are systematically tested. Many abnormalities which could provide important clinical information thus remain ignored, mainly due to the impossibility of performing a cost effective multi-targeted screening. To overcome this obstacle, we have developed a simple and parsimonious assay which allows a reliable detection of dozens of fusion genes in only a few hours.Our assay was designed to screen simultaneously more than 50 translocations involving 70 genes, recurrent in acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid (CML) leukemia. cDNA samples obtained from leukemic cells are first incubated with a mix of oligonucleotides probes which are complementary to the ends of the exons, at the abnormal junctions on the fusion mRNAs (Figure). For most genes, different probes were designed to detect different transcripts resulting from alternative genomic breakpoints (for example on exons 1, 13, 14 and 19 for BCR, and on exons 2 and 3 for ABL). The mix we created thus regroups more than 150 probes and targets more than 400 different fusion transcripts. All left probes have a common tail (T1) at their 5′ ends, all right probes a common tail (T2) at their 3′ ends. Additional probes were also included to detect the most frequent mutations of NPM1 (A, B, D). If one translocation is present in the sample, two probes hybridize side to side on the fusion cDNA. A DNA ligase is next used to create a covalent link between these probes, allowing their amplification by PCR with the T1 and T2 primers. If a PCR product is amplified, the two partners are identified by sequencing analysis.To test this method, we applied it to a retrospective series of 430 patients, (252 AML and 178 childhood ALL). In B-ALL (147 cases), all 33 ETV6-RUNX1, 6 BCR-ABL and 5 TCF3-PBX1 as well as 6 MLL rearrangements (3 AF4; 1 ENL, 1 AF9 and 1 AFF4) identified at diagnosis by conventional methods were detected, as well as 5 previously unnoticed P2RY8-CRLF2 junctions. We only failed to detect one MLL-AF4 fusion detected by cytogenetics but not by RT-PCR, probably due to the poor quality of the RNA. Further analysis of 10 MLL-AF4 positive adult ALL patients allowed the correct identification of this fusion in all cases. In T-ALL (31 cases), we detected 6 known (4 SIL-TAL, 2 CALM-AF10) and 5 previously undetected fusions (2 NUP214-ABL, 1 MLL-ENL, 1 ETV6-ABL, and a new PLZF-ABL junction). In AML, we detected 86 fusions: 23 PML-RARA, 2 PLZF-RARA, 18 CBFB-MYH11, 12 RUNX1-RUNX1T1, 4 NUP98-NSD1, 2 BCR-ABL, 1 DEK-NUP214, 1 CALM-AF10, 1 MOZ-CBP, 22 MLL rearrangements (13 PTD, 3 AF9, 2 AF6, 1 AF10, 1 ENL, 1 AF1Q and 1 MAPRE) and 44 NPM1 mutations. Two PML-RARA cases, one with a BCR2 breakpoint (not included in our design), one weakly positive by RT-PCR but negative by cytogenetics, two t(11;17) validated by FISH but negative by RT-PCR, and one NPM1J mutation (not included in our assay) were not detected. Importantly, 20 translocations in this series, including 14 MLL fusions and a cytogenetically cryptic t(8;21) had not been identified at diagnosis. Furthermore, all these new abnormalities (in AML and ALL) could be confirmed by conventional RT-PCR and sequencing analysis, demonstrating the specificity of the method. In the whole cohort of 430 patients, the three methods thus detected 157 fusions. 85 (54.1%) and 112 (71.3%) had been detected at diagnosis by cytogenetics and RT-PCR respectively, and 152 (96.8%) by our assay. Two were revealed only by cytogenetics, one only by RT-PCR and 30 only by our method.In conclusion, we have developed a simple multiplexed assay which can reveal a very large number of recurrent gene fusions in leukemia. Its short turn-around time (we repeatedly tested up to 40 patients in parallel and the results can be obtained in less than one day) and low cost (only one PCR module, one pyrosequencer and basic molecular biology reagents are needed) make it particularly suitable for a daily practice. Its capacity to detect many abnormalities which are almost never tested in daily practice could provide many important diagnosis and prognosis information, and enable the stratification of patients in prospective clinical trials. [Display omitted] DisclosuresRuminy:Centre Henri Becquerel: Patents & Royalties. Marchand:Centre Henri Becquerel: Patents & Royalties.

Multi-target screening of biological samples using LC-MS/MS: focus on chromatographic innovations.

Multi-target screening of biological fluids is a key tool in clinical and forensic toxicology. A complete toxicological analysis encompasses the sample preparation, the chromatographic separation and the detection. The present review briefly covers the new trends in sample preparation and detection and mainly focuses on the chromatographic stage, since a lot of technical improvements have been proposed over the last years. Among them, columns packed with sub-2μm fully porous particles and sub-3μm core-shell particles allow for significant improvements of resolution and higher throughput. Even if reversed-phase LC remains the most widely used chromatographic mode for toxicological screening, hydrophilic interaction chromatography and supercritical fluid chromatography appear as promising alternatives for attaining orthogonal selectivity, retention of polar compounds, and enhanced MS sensitivity.

A rapid profiling of hypolipidemic agents in dietary supplements by direct injection tandem mass spectrometry

A new, fast-screening method for identification of various pharmacologically active ingredients in cholesterol-lowering dietary supplements using direct injection mass spectrometry was developed. The optimized method was proven to be useful for multitarget screening: 17 compounds of interest were identified, including monacolin K and its acid form, cynarin, green tea catechines, genistein, daidzein, guggulsterones, allicin and l-arginine. The method was successfully applied for the fingerprint analysis of dietary supplements containing only one or several cholesterol-lowering agents including lovastatin registered as a drug. According to the obtained results, it is apparent that cholesterol-lowering dietary supplements are poorly standardized, as two samples did not contain active ingredients (monacolin K, monacolin K acid form and cynarin). Moreover, the application of the proposed direct injection method to quality control of cholesterol-lowering dietary supplements may be proposed for the routine analysis of a large number of samples in the laboratories of regulatory agencies.

In silico investigation of medicinal spectrum of imidazo-azines from the perspective of multitarget screening against malaria, tuberculosis and Chagas disease

A chemical database of 30 representative imidazo-azines was built and screened against important tropical disease targets by computational docking. After three rounds of screening, an interaction profile was generated and analyzed. On the basis of binding energy and ligand efficiency, it was concluded that in general, imidazo-azine scaffold has a potential of being selective and simultaneous inhibitor against the five receptors Pf-dihydrofolate reductase, Pf-enoyl acyl carrier protein reductase, Pf-protein kinase 7, Mt-pantothenate synthetase and Mt-thymidine monophosphate kinase. Interestingly, two compounds 2-(4-chlorophenyl)-N-cyclohexyl-6-methylH-imidazo[1,2-a]pyridine-3-amine (MCL011) and N-cyclohexyl-2-(4-methoxyphenyl)-6-methylH-imidazo[1,2-a]pyridine-3-amine (MCL017) showed highest binding energy against four targets namely Pf-dihydrofolate reductase, Pf-enoyl acyl carrier protein reductase, Pf-protein kinase 7 and Mt-pantothenate synthetase. Eventually, in order to improve the decision making and success rate in actual efficacy evaluations other criteria such as lead-likeness were envisaged.

Pooling samples for “top-down” molecular exposomics research: the methodology

BackgroundExposomics is the cutting-edge concept of screening the environmental risk factors for disease. In the novel “top-down” approach, we estimate the molecular exposome by measuring all body fluid analytes in a case-controlled study. However, to detect diverse pollutants, a sufficient sample size and multiple analytical methods are required. This may lead to dramatically increased costs and research workload.MethodsTo help reduce complexity, we suggest a sample pooling strategy along with a scheme for combining both general unknown or multi-targeted screening with targeted analysis. The sample pooling method was tested using computer simulations.ResultsBy comprehensively analysis of pooled samples, it is possible to identify environmental risk factors. Factors are initially screened in the pooled case and control population samples, then in the randomized grouped and pooled case and control subpopulation samples. In the sample grouping, five or more pools were suggested for groups having 30 individuals per pool.ConclusionsThis study suggests that sample pooling is a useful strategy for exposomics research, which provides a hypothesis-free method for pollutant risk screening.

Parallel consumption of pregabalin in opioid maintenance treatment detected by multi-target screening in oral fluid

Parallel consumption of pregabalin in opioid maintenance treatment detected by multi-target screening in oral fluid

Multi-targeted screening of botanicals in food supplements by liquid chromatography with tandem mass spectrometry

Safety, quality and composition assessments of food supplements based on botanical ingredients are of major concern, as they have usually not been through a rigorous testing process as required for the approval of therapeutic phytopreparations. Therefore, an efficient multi-targeted method was developed to screen selected botanicals of interest in herbal food supplements. Liquid chromatography coupled with a hybrid triple quadrupole linear ion trap was used for this purpose. Botanicals were characterised by means of appropriate biomarkers, which were unambiguously identified by mass spectrometry using an information dependent acquisition experiment which combined a multiple reaction monitoring survey with dependent enhanced product ion scans. During this procedure, product ion scans of targeted analytes were generated at three collision energies and compared with an in-house library of MS/MS spectra acquired from reference standards of all biomarkers. This generic method enables detection, identification and quantification of 98 biomarkers intended to characterise 79 selected plants.