High-performance liquid chromatographic–electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening

Within the pharmaceutical industry, significant resources have been applied to the identification of new drug compound leads through the use of high-throughput screening (HTS). To meet the demand for rapid analytical characterization of biologically active samples identified by HTS, the technique of high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) has been utilized, and the application of this technique specifically for the integration of natural product sample mixtures into modern HTS is reviewed. The high resolution provided by reversed-phase HPLC coupled with the gentle and relatively universal ionization facilitated by the electrospray process has had significant impact upon a variety of procedures associated with the HTS of natural products, including extract sample diversity evaluation, dereplication, structure elucidation, preparative isolation, and affinity-based biological activity evaluation.     

Design,Synthesis and Biological Activity of (20S,21S)‐7‐Cyclohexyl‐21‐fluorocamptothecin Carbamates as Potential Antitumor Agents

(20S,21S)‐7‐Cyclohexyl‐21‐fluorocamptothecin was discovered by a fluorine drug design strategy with potent antitumor activity and increased metabolic stability. In continuous efforts to find novel antitumor agents derived from natural product camptothecin, 20‐carbamates of the active compound (20S,21S)‐7‐cyclohexyl‐21‐fluorocamptothecin have been designed and synthesized. Among them, one compound with the diethylamino group showed greater antiproliferative activity than the other 20‐carbamate derivatives. The following biological activity assays indicated that the above compound is a valuable lead compound with excellent Topo I inhibitory activity and solution stability.     

Comparison of a Molecular and an Immobilized Gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] as Catalyst in the Asymmetric Danishefsky‐Hetero‐Diels‐Alder‐Reaction

On‐column reaction gas chromatography combines the power of separation and rapid analysis of reactants and reaction products with screening of reactions in a single step. Not only conversions but the reaction rates at various temperatures can be obtained from single measurements, making this approach superior to the time‐consuming measurements typically performed in reaction progress analysis. However, this approach has only been used in the investigation of interconversion processes, rearrangement reactions, and only a few examples of higher‐order reactions are known. Here we present the screening of immobilized gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] in the Danishefsky‐hetero‐Diels‐Alder‐reaction by enantioselective on‐column reaction gas chromatography utilizing cryogenic focusing to achieve catalytic conversions in this higher‐order reaction and subsequent separation of the enantiomeric product mixture to determine the enantiomeric ratio. The results obtained by this approach could be transferred to the conventional batch reaction at a larger scale, demonstrating that on‐column reaction chromatography provides reliable results in the screening of enantioselective reactions. Chirality 26:243–248, 2014. © 2014 Wiley Periodicals, Inc.     

Plant natural fragments,an innovative approach for drug discovery

Plant natural products (PNP) (e.g., secondary vegetal metabolites and their derivatives) have been a productive source of active ingredients for the pharmaceutical industry. The High Throughput Screening of Plant Natural Products (PNP-HTS) with extracts or isolated compounds has shown to be time consuming, expensive, and not as successful as expected. Recently building upon the innovative fragment-based drug discovery (FBDD) a disruptive approach was developed based on PNP. The fragment approach involves elaboration and/or isolation of weakly binding small molecules with molecular weights between 150 and 250 Da. This method is fundamentally different from HTS in almost every aspect (i.e., size of the compound library, screening methods, and optimization steps from hit to lead). Due to their nature, vegetal natural fragments have unique three-dimensional (3D) properties, high Fsp³, low aromaticity, and large chemo-diversities which represent potential opportunities for developing novel drugs. Preliminary results using vegetal natural fragments appear to be a promising and emerging field which offers valuable prospects for developing new drugs.

     

Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo‐4

Mutations in the multidrug resistance transporter of Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host–parasite system have never been analysed. The fluorochrome Fluo‐4 is a well‐documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo‐4 fluorescence uptake as a measure of compartmental Fluo‐4 concentration accumulation in the different compartments of the host–parasite system. We performed a ‘reverse Fluo‐4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca²⁺ fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.     

Plant natural products: Back to the future or into extinction?

Natural product substances have historically served as the most significant source of new leads for pharmaceutical development. However, with the advent of robotics, bioinformatics, high throughput screening (HTS), molecular biology-biotechnology, combinatorial chemistry, in silico (molecular modeling) and other methodologies, the pharmaceutical industry has largely moved away from plant derived natural products as a source for leads and prospective drug candidates. Can, or will, natural products ever recapture the preeminent position they once held as a foundation for drug discovery and development? The challenges associated with development of natural products as pharmaceuticals are illustrated by the Taxol^® story. Several misconceptions, which constrain utilization of plant natural products, for discovery and development of pharmaceuticals, are addressed to return natural products to the forefront.     

Penicimutanin C,a New Alkaloidal Compound,Isolated from a Neomycin‐Resistant Mutant 3‐f‐31of Penicillium purpurogenum G59

Penicimutanin C, a new alkaloidal compound, was isolated from the neomycin‐resistant mutant strain 3‐f‐31 of the marine‐derived fungus, Penicillium purpurogenum G59, together with four known compounds. The structure of penicimutanin C, including the absolute configuration, was determined by spectroscopic and chemical methods. The absolute configuration of penicimutanin A was also re‐confirmed by Marfey's and chiral HPLC analyses of the hydrolyzed products. Penicimutanins C and A inhibited the proliferation of five human cancer cell lines to some extent. Penicimutanin C is the third dimer of diketopiperazine and penicimutanolone, which are only produced by mutants of P. purpurogenum G59 isolated to date, and it showed cytotoxic activity against human cancer cell lines. The neomycin‐resistant screening strategy has been previously successfully used to discover new compounds by activating silent metabolites in fungi, and the present results provide an additional example of the effectiveness of this method.     

Fragment screening: an introduction

There are clearly many different philosophies associated with adapting fragment screening into mainstream Drug Discovery Lead Generation strategies. Scientists at Astex, for instance, focus entirely on strategies involving use of X-ray crystallography and NMR. However, AstraZeneca uses a number of different fragment screening strategies. One approach is to screen a 2000 compound fragment set (with close to "lead-like" complexity) at 100 microM in parallel with every HTS such that the data are obtained on the entire screening collection at 10 microM plus the extra samples at 100 microM; this provides valuable compound potency data in a concentration range that is usually unexplored. The fragments are then screen-specific "privileged structures" that can be searched for in the rest of the HTS output and other databases as well as having synthesis follow-up. A typical workflow for a fragment screen within AstraZeneca is shown below (Figure 24) and highlights the desirability (particularly when screening >100 microM) for NMR and X-ray information to validate weak hits and give information on how to optimise them. In this chapter, we have provided an introduction to the theoretical and practical issues associated with the use of fragment methods and lead-likeness. Fragment-based approaches are still in an early stage of development and are just one of many interrelated techniques that are now used to identify novel lead compounds for drug development. Fragment based screening has some advantages, but like every other drug hunting strategy will not be universally applicable. There are in particular some practical challenges associated with fragment screening that relate to the generally lower level of potency that such compounds initially possess. Considerable synthetic effort has to be applied for post-fragment screening to build the sort of potency that would be expected to be found from a traditional HTS. However, if there are no low-hanging fruit in a screening collection to be found by HTS then the use of fragment screening can help find novelty that may lead to a target not being discarded as intractable. As such, the approach offers some significant advantages by providing less complex molecules, which may have better potential for novel drug optimisation and by enabling new chemical space to be more effectively explored. Many literature examples that cover examples of fragment screening approaches are still at the "proof of concept" stage and although delivering inhibitors or ligands, may still prove to be unsuitable when further ADMET and toxicity profiling is done. The next few years should see a maturing of the area, and as our understanding of how the concepts can be best applied, there are likely to be many more examples of attractive, small molecule hits, leads and candidate drugs derived from the approaches described.     

Design,Synthesis, and Cholinesterase Inhibition Assay of Coumarin‐3‐carboxamide‐N‐morpholine Hybrids as New Anti‐Alzheimer Agents

A new series of coumarin‐3‐carboxamide‐N‐morpholine hybrids 5a – 5l was designed and synthesized as cholinesterases inhibitors. The synthetic approach for title compounds was started from the reaction between 2‐hydroxybenzaldehyde derivatives and Meldrum's acid to afford corresponding coumarin‐3‐carboxylic acids. Then, amidation of the latter compounds with 2‐morpholinoethylamine or N‐(3‐aminopropyl)morpholine led to the formation of the compounds 5a – 5l . The in vitro inhibition screen against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) revealed that most of the synthesized compounds had potent AChE inhibitory while their BuChE inhibitions are moderate to weak. Among them, propylmorpholine derivative 5g (N‐[3‐(morpholin‐4‐yl)propyl]‐2‐oxo‐2H‐chromene‐3‐carboxamide) bearing an unsubstituted coumarin moiety and ethylmorpholine derivative 5d (6‐bromo‐N‐[2‐(morpholin‐4‐yl)ethyl]‐2‐oxo‐2H‐chromene‐3‐carboxamide) bearing a 6‐bromocoumarin moiety showed the most activity against AChE and BuChE, respectively. The inhibitory activity of compound 5g against AChE was 1.78 times more than that of rivastigmine and anti‐BuChE activity of compound 5d is approximately same as rivastigmine. Kinetic and docking studies confirmed the dual binding site ability of compound 5g to inhibit AChE.     

Synthesis and Antifungal Activity of 2‐Hydroxy‐4,5‐methylenedioxyaryl Ketones as Analogues of Kakuol

In a study aiming to determine the structural elements essential to the antifungal activity of kakuol, we synthesized a series of 2‐hydroxy‐4,5‐methylenedioxyaryl ketones, and we assayed their in vitro antifungal activity. The most sensitive target organisms to the action of these class of compounds were Phytophthora infestans, Phytium ultimum, Cercospora beticola, Cladosporium cucumerinum, and Rhizoctonia solani. Most of the analogs showed a remarkable in vitro activity, and some of them appeared significantly more effective than the natural product. The biological activity was mainly affected by introducing structural modification on the carbonyl moiety of the natural‐product molecule. In particular, compound 5a , bearing a C?C bond conjugated to the C?O group, was found active with a MIC value of 10 μg ml^?1 against Cladosporium cucumerinum. The results suggest that 2‐hydroxy‐4,5‐methylenedioxyaryl ketones can be considered promising candidates in the development of new antifungal compounds.     

A simple,robust enzymatic‐based high‐throughput screening method for antimicrobial peptides discovery against Escherichia coli

The indiscriminate usage of antibiotics has created a major problem in the form of antibiotic resistance. Even though new antimicrobial drug discovery programs have been in place from the last two decades, still we are unsuccessful in identifying novel molecules that have a potential to become new therapeutic agents for the treatment of microbial infections. A major problem in most screening studies is the requirement of high‐throughput techniques. Given this, we present here an enzyme‐based robust method for screening antimicrobial agent's active against Escherichia coli. This method is based upon the ability of the intracellular innate enzyme to cleave o‐nitrophenyl β‐d ‐galactopyranoside (non‐chromogenic) to o‐nitrophenolate (ONP) (chromogenic) upon the membrane damage or disruption. In comparison with the other currently available methods, we believe that our method provides an opportunity for real‐time monitoring of the antimicrobial agents action by measuring the ONP generation in a user‐friendly manner. Even though this method can be applied to other strain, our experience shows that one has to be careful especially when the pigments or metabolites present in the bacteria have the same wavelength absorbance. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Introduction of New Tools for Chemical Biology Research on Microbial Metabolites

Recently, high-throughput screening (HTS) has become the mainstream technique for drug discovery. Compounds that are synthesized by combinatorial chemistry might be more suitable than natural products to apply to HTS, because the purification procedure is a drawback of using natural products. Nevertheless, natural products remain an extremely important source of drugs. To overcome the demerits of natural products, we are constructing the RIKEN Natural Products Depository (NPDepo) that is focused primarily on microbial metabolites. In this review, I describe (i) engineering pathways for biosynthetic gene clusters of microbial metabolites, (ii) construction of fraction libraries of microbial metabolites, and (iii) the development of a new screening system using a chemical array and a protein library produced by GLORIA.     

Isoalantolactone restores the sensitivity of gram‐negative Enterobacteriaceae carrying MCR‐1 to carbapenems

Polymyxin B has been re‐applied to the clinic as the final choice for the treatment of multidrug‐resistant gram‐negative pathogenic infections, but the use of polymyxin B has been re‐assessed because of the emergence and spread of the plasmid‐mediated mcr‐1 gene. The purpose of this study was to search for an MCR inhibitor synergistically acting with polymyxin to treat the infection caused by this pathogen. In this study, we used the broth microdilution checkerboard method to evaluate the synergistic effect of isoalantolactone (IAL) and polymyxin B on mcr‐1‐positive Enterobacteriaceae. Growth curve analysis, time‐killing assays and a combined disc test were used to further verify the efficacy of the combined drug. Colonization of the thigh muscle in mice, survival experiments and lung tissue section observations was used to determine the effect of synergy in vivo after Klebsiella pneumoniae and Escherichia coli infection. We screened a natural compound, IAL, which can enhance the sensitivity of polymyxin B to mcr‐1‐positive Enterobacteriaceae. The results showed that the combined use of polymyxin B and IAL has a synergistic effect on mcr‐1‐positive Enterobacteriaceae, such as K pneumoniae and E coli, not only in vitro but also in vivo. Our results indicate that IAL is a natural compound with broad application prospects that can prolong the service life of polymyxin B and make outstanding contributions to the treatment of gram‐negative Enterobacteriaceae infections resistant to polymyxin B.     

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 × 10⁶ compound library, the identification of novel chemical “hits” for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10⁶ compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.     

Synthesis,Anticonvulsant Activity and Metabolism of 4‐chlor‐3‐methylphenoxyethylamine Derivatives of Trans‐2‐aminocyclohexan‐1‐ol

In this study, we report the synthesis, spectral characterization, antiepileptic activity and biotransformation of three new, chiral, N‐aminoalkyl derivatives of trans – 2 aminocyclohexan‐1‐ol: 1 (R enantiomer), 2 (S enantiomer) and 3 (racemate). Antiepileptic activity of the titled compounds was studied using MES and scMet. Moreover, in this study, the biotransformation of 1 , 2 and 3 in microbial model (Cunninghamella), liver microsomal assay as well as in silico studies (MetaSite) was evaluated. Studies have indicated that 1 , 2 and 3 have good antiepileptic activity in vivo, comparable to valproate. Biotransformation assays showed that the most probable metabolite (indicated in every tested assays) was M1 . The microbial model as well as in silico study showed no difference in biotransformation between tested enantiomers. However, in a rat liver microsomal study compound 1 and 2 (R and S enantiomer) had different main metabolite – M2 for 1 and M1 for 2 . MS/MS fragmentation allowed us to predict the structures of obtained metabolites, which were in agreement with 1°alcohol ( M1 ) and carboxylic acid ( M2 ). Our research has shown that microbial model, microsomal assay, and computational methods can be included as useful and reliable tools in early ADME‐Tox assays in the process of developing new drug candidates. Chirality 27:163–169, 2015. © 2014 Wiley Periodicals, Inc.     

High-throughput screening: update on practices and success

High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.     

Jasminum polyanthum Franch. as a natural source of (−)‐methyl jasmonate: an alternative to the use of the synthetic standard

Introduction – Methyl jasmonate (MJ) contains two chiral centres at C‐3 and C‐7 in its chemical structure, which implies that it can exist in four possible stereoisomeric forms, namely (+)‐MJ, (?)‐MJ, (+)‐epiMJ and (?)‐epiMJ. The absolute configuration of the two side chains of MJ affects the biological activity associated with this compound. Objective – To isolate pure (?)‐MJ from a natural source, Jasminum polyanthum Franch., with the intention of increasing the knowledge about its biological properties, including its effect on the biosynthesis of plant metabolites. Methodology – The method used was based on steam distillation extraction (SDE) as an extraction technique followed by high‐performance liquid chromatography (HPLC) as a purification procedure. The HPLC flow‐rate as well as the number of fractions accumulated were optimised to achieve the concentration and purity required. Results – The employment of 0.3 mL/min as HPLC flow‐rate and the accumulation of three HPLC fractions allowed the required enantiomeric purity (95%) and concentration (0.36 mg/L in each HPLC fraction) to efficiently obtain (?)‐methyl jasmonate from Jasminum polyanthum Franch. to be achieved. Conclusion – The approach proposed may enable the properties and effect of pure (?)‐MJ on plant responses to be studied. The use of a natural source to obtain (?)‐MJ is presented as an alternative to the enantioselective synthesis and enantiomeric resolution from the standard racaemic mixture. Copyright © 2009 John Wiley & Sons, Ltd.     

High-throughput screening assay for the identification of compounds regulating self-renewal and differentiation in human embryonic stem cells

High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high-content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high-content assays should accelerate progress in basic and translational hESC biology.