Meeting the Challenge: Using Cytological Profiling to Discover

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Meeting the challenge: using cytological profiling to discover chemical probes from traditional Chinese medicines against Parkinson’s disease Chao Wang, Xinzhou Yang, George D Mellick, and Yunjiang Feng ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00245 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 16, 2016

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ACS Chemical Neuroscience

Meeting the challenge: using cytological profiling to discover chemical probes from traditional Chinese medicines against Parkinson’s disease Chao Wang,† Xinzhou Yang,‡ George D. Mellick,†* and Yunjiang Feng†* † ‡

Eskitis Institute for Drug Discovery, Griffith University, Brisbane 4111, College of Pharmacy, South-Central University for Nationalities, Wuhan 430074

ABSTRACT: Parkinson’s disease (PD) is an incurable neurodegenerative disorder with a high prevalence rate worldwide. The fact that there are currently no proven disease-modifying treatments for PD underscores the urgency for a more comprehensive understanding of the underlying disease mechanism. Chemical probes have been proven to be powerful tools for studying biological processes. Traditional Chinese medicine (TCM) contains a huge reservoir of bioactive small molecules as potential chemical probes that may hold the key to unlocking the mystery of PD biology. The TCM-sourced chemical approach to PD biology can be advanced through the use of an emerging cytological profiling (CP) technique that allows unbiased characterization of small molecules and their cellular responses. This comprehensive technique, applied to chemical probe identification from TCM and used for studying the molecular mechanisms underlying PD, may inform future therapeutic target selection and provide a new perspective to PD drug discovery. KEYWORDS: Parkinson’s disease, chemical probe, traditional Chinese medicine, cytological profiling

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in substantia nigra and the presence of intracellular inclusion bodies containing the protein alpha-synuclein (called Lewy bodies). Current medications targeting the dopaminergic system only provide symptomatic relief but are not able to stop, slow or reverse the progression of neurodegeneration.1 There is a huge unmet clinical need for neuroprotective drugs that can actually treat the root cause of the disease. Indeed, many putative neuroprotective agents have been identified in the laboratory, but none has been unequivocally proven to have a diseasemodifying effect in PD patients. Such disheartening failures are largely due to a poor understanding of the molecular mechanism underlying PD etiology and pathogenesis; in other words, it is not known what causes PD and it is unclear precisely what to target.2 The emerging use of “chemical biology” and the use of chemical probes, especially those derived from natural products, to explore the biological processes, offers a “probe to drug” approach to address the above obstacles to the development of effective neuroprotective therapies.3 CHEMICAL PROBES Chemical probes are small-molecule modulators that can be used to interrogate the roles of their molecular targets in physiology and pathology.4 Chemical probes have proven to be powerful tools in biological research, not only because they are complementary to classical genetic approaches, such as conditional knockout and RNA interference, but also because they have unique advantages.5 From an overall perspective, chemical probes take a step beyond genetics by revealing how genes, proteins and other biomolecules function together at the

system level.6 When applied to cells or organisms, probes can rapidly and reversibly switch a protein on or off via a binding interaction. Unlike genetics, which involves introducing mutations, mostly with delayed and lasting effects, probes produce perturbations that are flexibly tunable in a dosedependent manner and often readily transferred across cell types and species that may lack genetic tools. Another advantage of chemical probes is that they can disrupt a particular function of a protein; hence they can distinguish between effects induced by the regulation of different functions of the protein.7 When coupled with other agents or genetic perturbations, chemical probes are capable of dissecting the biological networks at high resolution.8 However, chemical probes must have sufficient aqueous solubility and membrane penetrability, and exhibit potent, selective activity with a well-defined mechanism of action (MOA) to be useful for biological questions.4, 7 High-quality chemical probes have provided proof of concept for the potential druggability of a molecular target or a pathway in several diseases. For example, the Bromo and extra terminal (BET) family, which includes BRD2, BRD3, BRD4 and BRDT proteins, is a novel class of bromodomaincontaining transcriptional regulators that are targets for chemical probes. BRDs have been considered as interesting but not druggable targets by many pharmaceutical companies due to lack of potent and selective inhibitors.9 However, the recent release of the first chemical probes with nanomolar affinity for BET BRDs enabled rapid deconvolution of the role of BRDs in diseases such as cancer and autoimmunity and uncovered some therapeutic opportunities associated with BET inhibition.10 Based on the published data, pharmaceutical companies initiated drug discovery programs targeting the BET family for the treatment of cancer, cardiovascular disease,

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type 2 diabetes and Alzheimer’s disease, and several BET inhibitors have progressed to the clinical stages.11 Given that chemical probes serve both as powerful tools to understand biological systems and as seeds to spur the development of new drugs, significant efforts have been made in the utilization of bioactive small molecules to combat PD, one of the world’s medical puzzles. Table 1 gives a list of small-molecule tools used to probe PD biology that have progressed to clinical trial as disease-modifying or neuroprotective agents.1 Table 1. Small-Molecule Tools Progressed to Clinical Trial as Disease-Modifying or Neuroprotective Agents for the Treatment of PD. Compounds

Mechanism of action

Coenzyme Q10 *

Modulator of mitochondrial function

Creatine *

Modulator of mitochondrial function

Deferiprone

Iron chelator

Inosine

Urate precursor

Isradipine CR

Calcium antagonist

PYM-50028

Oral neurotrophic factor modulator

* These clinical trials have been terminated due to futility, according to ClinicalTrials.gov.

Figure 1. Chemical structures of small-molecule tools listed in Table 1.

There is a paucity of small-molecule tools required to understand the molecular mechanism underlying PD. More research is needed to discover chemical probes modulating diverse targets relevant to PD. The use of such probes to dissect the complexity of disease networks may shift the traditional “one target, one drug” paradigm12 in PD therapy towards a more effective “multi-target, multi-drug” model13. In this respect, traditional Chinese medicine is a unique source for the discovery of chemical probes against PD. TRADITIONAL CHINESE MEDICINE Traditional Chinese medicine (TCM) has been a mainstay of the Chinese healthcare system for thousands of years.

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Enormous knowledge has been accumulated through the observation of clinical efficacy and safety of TCM targeting a wide variety of diseases, especially complex chronic disorders. According to TCM theory, manifestations of diseases result from specific dynamic imbalances in the whole-body system. The goal of TCM practice is to help restore the balance. Therefore, TCM embraces a far more experience-based and holistic approach to treat the dysfunction of the living organisms as compared with Western medicine. TCMs can be prescribed singly or as a formula comprising many “herbs” such as plants, animals or minerals, each of which contains a considerable number of chemical substances. Although there are usually hundreds or even thousands of compounds in a TCM recipe, relatively few of them are bioactive. From a chemical point of view, there is a high extent of overlap between TCM components and Western drugs,14 which may suggest that TCM and Western medicine are essentially converged on the molecular base. The difference in this regard is that TCM recipes are multicomponent and multi-target agents acting in the same way as the combination therapy of multi-component drugs.15 Their therapeutic effects are achieved through collectively modulating the disease network by the bioactive ingredients. Quite a few herbs (Table 2) and herbal formulae (Table 3) have been used to treat PD in China. Increasing interest is being devoted in the neuroprotective evaluation of herbal materials on the treatment or prevention of PD.16, 17 Cellular and animal models of PD have significantly contributed to our understanding of PD in humans. This is despite the fact that these models generally fail to recapitulate many of the pathological features of the disease. Three neurotoxins, 6hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) and rotenone, are commonly used agents to mimic various aspects of PD in vitro or in vivo and assess the neuroprotective effects of herbal materials.17 The extracts, fractions, or pure compounds obtained from herbs listed in Table 2 and extracts from herbal formulae in Table 3 have been tested on the neurotoxic models and showed some positive results.16 The major mechanisms for these neuroprotective effects include antioxidant, antiapoptosis, mitochondrial protection, antiinflammation, and autophagy induction. For example, green tea extract from the leaves of Camellia sinensis (L.), O. Kuntze, can attenuate 6-OHDAinduced nuclear factor-κB (NF-κB) activation and cell death in SH-SY5Y cells.18 Polyphenols derived from green tea have also shown to be protective in SH-SY5Y cells and in a rat model of PD, through inhibition of the ROS-nitrogen monoxide pathway.19, 20 Moreover, two catechins, (-)epicatechin gallate and (-)-epigallocatechin-3-gallate, isolated from green tea polyphenols have protective effects against 6OHDA-induced apoptosis in PC12 cells.21 The herbal formula Yeoldahanso-tang has neuroprotective effects on a different PD model via autophagy enhancement.22 Up to now the molecular base of TCM still remains largely unknown. Thousands of years’ accumulation in TCM knowledge opens up an opportunity to access a huge unexplored reservoir of bioactive natural products continually treated as a source of inspiration for biological and drug discovery. The identification of chemical probes against different targets from TCM may lead to a combination therapy for PD.


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Table 2. 24 Genera of Herbal Medicines Used to Treat PD. (Adapted from ref 16)

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Families

Plant genera and species

Araliaceae

Acanthopanax senticosus (Rupr. et Maxim.) Harms.

Zingiberaceae

Alpinia oxyphylla Miq.

Leguminosae

Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao; Astragalus membranaceus (Fisch.) Bge.

Theaceae

Camellia sinensis (L.) O. Kuntze

Leguminosae

Cassia obtusifolia L.; Cassia tora L.

Asteraceae

Chrysanthemum morifolium Ramat.; Chrysanthemum indicum L.

Orobanchaceae

Cistanche deserticola Y. C. Ma; Cistanche tubulosa (Schrenk) Wight; Cistanche salsa

Convolvulaceae

Cuscuta australis R. Br.; Cuscuta chinensis Lam.

Oleaceae

Fraxinus sielboldiana Blume; Fraxinus rhynchophylla Hance; Fraxinus chinensis Roxb.; Fraxinus szaboana Lingelsh.; Fraxinus stylosa Lingelsh.

Orchidaceae

Gastrodia elata Bl.

Ginkgoaceae

Ginkgo biloba L.

Cucurbitaceae

Gynostemma pentaphyllum (Thunb.) Makino

Guttiferae

Hypericum perforatum L.

Umbelliferae

Ligusticum chuanxiong Hort.

Ranunculaceae

Paeonia lactiflora Pall.

Araliaceae

Panax ginseng C. A. Mey.; Panax notoginseng (Burk.) F. H. Chen

Polygalaceae

Polygala tenuifolia Willd.; Polygala sibirica L.

Polygonaceae

Polygonum cuspidatum Sieb. et Zucc.

Leguminosae

Psoralea corylifolia L.

Leguminosae

Pueraria lobata (Willd.) Ohwi; Pueraria thomsonii Benth.

Crassulaceae

Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba; Rhodiola rosea L.

Labiatae

Salvia miltiorrhiza Bge.

Labiatae

Scutellaria baicalensis Georgi

Celastraceae

Tripterygium wilfordii Hook F.

CYTOLOGICAL PROFILING One of the challenges facing researchers working to discover active molecules from TCM preparations is that of purifying important compounds of interest from complex mixtures. The crucial question then becomes: how should we best evaluate the biological activity of the mixtures and the pure compounds? Most chemical screens to date have been performed with large chemical libraries tested against a specific target or a predefined phenotype. Both strategies measure very limited regions of biological space and, by their nature cannot reveal potent effects that are not being specifically looked for in the screen.23, 24 Thus, molecules that have interesting but unexpected activity will be discarded if they fail to show the desired effects.24 Conversely, molecules identified in a targeted screen, with modest activity against a specific target – but promiscuous activity at other “off-target” sites, may not be suitable as chemical probes to answer biological questions.4 Instead, emerging broad screens for biological function, known as multiparametric phenotypic profiling, have the advantage of picking up the best “lock” for each “key”

produced by chemical variation.23 In particular, the last decade has seen the development of an unbiased image-based screening technique called “cytological profiling” (CP).23, 25-27 This technique employs high content analysis (HCA), a combination of automated fluorescence microscopy and image processing, to profile small molecules and their cellular effects based on numerous quantifiable phenotypic features. In a typical CP experiment, cells are cultured in 384-well plates, treated with compounds over a sufficient period of time for phenotypic changes to occur, then fixed and stained with various fluorescent probes. After a wash, the plates are scanned in each fluorescent channel by automated microscope. Images are stored and subsequently submitted to automated image analysis for cell state quantification. The use of CP to interrogate a wide spectrum of biological effects relies on two principles. First, divergent fluorescent probes should be employed to visualize a broad range of important cellular components (e.g., organelles, cytoskeleton).25 Antibodies targeting key signaling pathways can also be included to provide information on specific pathway



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Table 3. Herbal Medicines and Their Contents in 11 Formulae Used to Treat PD. (Adapted from ref 16)

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Formulae

Chinese name

Herbal medicines and their contents

半夏厚朴汤

6 g Pinellia ternate Breitenbach, 3 g Poria cocos (Schw.) Wolf, 3 g Magnolia obovata Thunberg, 2 g Perilla frutescens Britton ar. Acuta Lubo, 1 g Zingiber officcinale Roscoc

补肾养肝熄风汤

15 g Rehmanniae Radix Praeparata, 15 g Rehmannia glutinosa Libosch., 15 g Uncariae Ramulus Cum Uncis, 15 g Paeonia lactiflora Pall., 9 g Polygoni Multiflori Radix Praeparata

Chuanxiong Chatiao Pulvis

川芎茶调散

12 g Ligusticum chuanxiong Hort., 12 g Schizonepeta tenuifolia Briq., 6 g Angelicae Dahuricae Radix, 6 g Notopterygii Rhizoma et Radix, 6 g Glycyrrhizae Radix et Rhizoma, 3 g Asari Radix et Rhizoma, 4.5 g Saposhnikovia divaricata (Turcz.) Schischk., 12 g Mentha haplocalyx Briq., 4.5 g Green Tea

Huanglian Jiedu Decoction

黄连解毒汤

9 g Coptis chinensis Franch, 6 g Scutellaria baicalensis Georgi, 6 g Phellodendron amurense Rupr, 9 g Gardenia jasminoides Ellis

Kami-shoyo-san

加味逍遥散

3 g Bupleurum falcatum, 3 g Paeonia lactiflora Pall., 3 g Atractylodes lancea, 3 g Angelica acutiloba, 3 g Poria cocos (Schw.) Wolf, 2 g Gardenia jasminoides Ellis, 2 g Paeonia suffruticosa Andr., 1.5 g Glycyrrhiza uralensis Fisch., 1 g Zingiber officcinale Roscoc, 1 g Menthae arvensis

Liuwei Dihuang Pill

六味地黄丸

24 g Rehmanniae Radix Praeparata, 12 g Corni Fructus Praeparata, 9 g Paeonia suffruticosa Andr., 12 g Dioscorea opposita Thunb., 9 g Poria cocos (Schw.) Wolf, 9 g Alisma orientalis (Sam.) Juzep.

San Huang Xie Xin Tang

三黄泻心汤

5 g Coptis chinesis Franch, 5 g Scutellaria baicalensis Georgi, 10 g Rheum officinale Baill.

Tianma Gouteng Yin

天麻钩藤饮

9 g Gastrodia elata Bl., 12 g Uncariae Ramulus Cum Uncis, 18 g Haliotidis Concha, 9 g Gardenia jasminoides Ellis, 12 g Cyathula officinalis Kuan, 9 g Eucommia ulmoides Oliv., 9 g Taxillus chinensis (DC.), 9 g Polygoni Multiflori Caulis, 9 g Fulingshen, 9 g Leonurus japonicas Houtt.

热多寒少汤

17% Pueraria lobata (Willd.) Ohwi, 11% Angelica tenuissima Nakai, 6% Scutellaria baicalensis Georgi, 3% Platycodon grandiflorum (Jacq), 6% Angelica dahurica, 6% Cimicifuga heracleifolia Kom, 6% Raphanus sativa L., 11% Polygala tenuifolia (Willd.), 17% Acorus gramineus Soland., 17% Dimocarpus longan Lour

Zhen Wu Tang

真武汤

30 g Paeonia lactiflora Pall., 10 g Atractylodes macrocephala Koidz, 10 g Typhonium giganteum Engl., 10 g Poria cocos (Schw.) Wolf, 10 g Zingiber officcinale Roscoc

Zhichan Soup

止颤汤

15 g Astragalus Mongholicus, 12 g Salvia miltiorrhiza Bge., 10 g Gastrodia elata Bl., 18 g Uncaria rhynchophylla (Miq.) Miq. ex Havil, 15 g Paeonia lactiflora Pall., 9 g Cimicifugae Rhizoma, 10 g Anemarrhena asphodeloides Bge.

Banxia Houpo Tang Bushen Yanggan Xifeng Decoction

Yeoldahanso-tang

activity. Second, with appropriate probes, fluorescence microscopy and sophisticated image processing systems have the potential to quantify essentially any physiological alterations induced at the single-cell level. All kinds of quantitative cellular descriptors (e.g., morphology, staining intensity, spatial attribute) are useful in capturing these subtle changes. In some cases, the descriptors might have obvious biological meaning (e.g., DNA staining intensity, which measures DNA content per nucleus), and in other cases they might not (e.g., DNA texture, nuclear ellipticity). However, even descriptors with unclear biological meanings have proven to contribute to the characterization of cellular perturbations.25 The ability of CP to generate unbiased multidimensional phenotypic profiles enables a detailed comparison of similarity or dissimilarity in biological space between different samples. Perlman et al.26 showed that compounds having different

potencies, but sharing similar MOA, tend to have similar cytological profiles over a range of concentrations; these were readily distinguished from other compounds with different MOA (Figure 2). Based on such inspiration, many researchers have succeeded in MOA prediction, and new MOA identification, for bioactive molecules at the primary screening stage by comparing their phenotypic profiles with those of reference compounds that have known MOAs.23, 25, 28-37 A classic example of using CP to identify molecules with new MOA comes from a study reported by Tanaka et al.23 They profiled a collection of seven known protein kinase inhibitors and 100 related analogs on 38 cellular features across five cell lines (four cancer cell lines and endothelial cells). Principle component analysis (PCA) applied to the high-dimensional features uncovered h ydroxy-PP, a p y r a z o l o p y r i m i d i n e t h a t


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ACS Chemical Neuroscience perturbations. There is more work to be done to fully reach HCA’s potential in neuroscience.

Figure 2. Comparison of dose-response cytological profiles between compounds acting through regulation of DNA topoisomerase, protein degradation, HDAC (histone deacetylase), microtubule or actin activity. Compounds with similar MOA showed similar profiles. The x-axis represents increasing dose from 65 pM to 35 µM for all compounds except epothilone B, which is shown from 0.65 pM to 0.35 µM. The y-axis encodes cellular descriptors. Positive deviations from DMSO-treated wells are displayed in red; negative deviations are displayed in green. Figure adapted from ref 26.

exhibited a cell-specific phenotypic signature distinct from all known protein kinase inhibitors including the closely related analog PP2 (Figure 3). Such results indicated that hydroxylPP was likely to induce the differential effects through a new MOA other than kinase inhibition. Using affinity purification, X-ray crystallography and biochemical assay, this new MOA was confirmed to be inhibition of a nonkinase target, carbonyl reductase 1. Much of the work previously done using the CP technique was focused on analyzing pure compounds. It is only recently that CP has been used to screen a large library of natural product extracts for prioritizing isolation of compounds with specific or unique profiles.30, 31, 37 In one case,30 CP screening of an extract library derived from microbial sources revealed a small subset of extracts clustered closely with known antimitotic agents from the reference compound training set (Figure 4A). Subsequent purification of one of the extracts, RLUS1665D, led to the isolation of diketopiperazine XR334 (Figure 4C) that recapitulated the observed antimitotic phenotype of the original extract. The activity of XR334 was confirmed by parallel CP for dilution series of both natural and synthetic products, in which they were clustered together with other microtubule poisons including nocodazole and vinblastine (Figure 4B). Tremendous progress has been made in the application of CP for the discovery of bioactive small molecules against cancer23, 25, 29-35 and bacterial infection,28, 36-40 yet the same strategy in the context of neurological diseases remains largely untapped. Neuroscientists have attempted to score predefined cellular phenotypes (e.g., neurite outgrowth, neurogenesis, cell viability, inclusion formation, apoptosis, cell migration, signal transduction) using HCA as a replacement for manual observation.41 A recent study42 demonstrated, for the first time, the success of HCA in quantifying alpha-synuclein aggregates on a human SH-SY5Y neuroblastoma cell model of PD. The advantages of these automated techniques compared to manual methods are speed and objectivity; however they still share the same limitations of measuring limited regions of biological space, ignoring a large quantity of phenotypic information that could finely differentiate MOAs between different chemical

Figure 3. (A) PCA plot of the phenotypic features. Colored spheres represent a single compound at one concentration (ranging from 6 nM to 40 µM by 3-fold increases); lines connecting the spheres indicate a single compound’s effects over a range of concentrations. Spheres are colored as follows: positive control paclitaxel (green), know protein inhibitors (blue) and their analogs (red). The PCA provides aggregate variables termed “components” made up of a small number of independent variables, which account for the majority of the variability in the dataset. (Adapted from ref 23) (B) Chemical structures of PP2 and hydroxyl-PP.

Research groups in our institute took up the challenge and established a CP platform to profile phenotypic effects of natural products on a robust PD model, human olfactory neurosphere-derived (hONS) cells.43-46 In this assay, cytological parameters were assessed by staining PD patientderived hONS cells with fluorescent probes targeting various cellular pathways and organelles implicated in PD. These included mitochondria, early endosomes, lysosomes, cytoskeleton, apoptosis and autophagy. In total, 38 phenotypic features were generated and followed by fold change analyses. The CP platform was successfully applied to the biological signature examination of 482 structurally diverse natural products in four concentrations (1, 3, 10, and 30 µM).45 Phenotypic analysis showed that all compounds exhibited organelle effects that differed from DMSO control at one dose level at least. Subsequent hierarchical clustering of the



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reservoir of bioactive small molecules that have contributed to clinical efficacy over thousands of years. Such molecules working together in similar or different modes of action may hold the key to unlocking the mystery of PD biology. The TCM-sourced chemical approach to PD biology can be advanced through the use of a CP technique that allows unbiased characterization of natural products and their cellular responses. Chemical probes identified from neuroprotective TCM will lead to a better understanding of molecular mechanism of PD and potential drug targets. AUTHOR INFORMATION Corresponding Author * Email: [email protected] (YF), [email protected] (GM). Mailing address: School of Natural Sciences, Griffith University, 170 Kessels Road, Nathan, Brisbane, Qld 4111, Australia

Author Contributions CW wrote the manuscript under the guidance of YF, GM and XY. YF, GM and XY reviewed and edited the manuscript. Funding Figure 4. (A) Cytological profile of original natural product extracts and microtubule poisons. (Adapted from 30) (B) CP analysis of isolated XR334 (XR334_NP) and synthesized XR 334 (XR334_SYN). (Adapted from ref 30) (C) Chemical structure of XR334.

phenotypic profiles identified 13 distinct groups (within-group correlations >0.7) that may represent very different MOAs from each other. These results confirmed the ability of CP to capture the subtle changes in hONS cell phenotype and increase the chance of finding biologically diverse small molecule modulators. For those molecules standing out with their strong activities at low concentrations, a further comparison of their CP profiles to those of a reference set of well-annotated compounds is needed to predict their MOA. In addition, the CP platform has been expanded to profile phenotypic effects of natural products on hONS cells from both patients and healthy individuals. A novel alkaloid, iotrochotazine A, isolated from the marine sponge Iotrochota sp., was found to induce disease-specific alteration in PD hONS cells: A decrease in lysosomal staining and an increase in the number of EEA1-associated early endosomes at 1 µM were detected; however, when assayed on hONS cells from two healthy individuals, no significant effects were shown on lysosomal and endosomal markers up to a concentration of 10 µM.43 It would be interesting to know what kind of deficit in the PD model causes such differential effect. Since mutations in the vesicular trafficking protein VPS35 have been recently associated with late-onset PD,47-49 this natural product may aid our understanding of vesicular trafficking pathways in PD pathology. Once the corresponding deficit is identified, screenings for compounds with the ability to reverse the deficit can be performed as a starting point for PD drug discovery. CONCLUSION The fact that there are no treatments to prevent or slow the neurodegenerative process in PD underscores the urgency for a more comprehensive understanding of the underlying disease mechanism. Small molecule approaches to biology not only provide probes to explore the biological processes, but potentially offer novel drug candidates. TCMs contain a huge

CW is supported by scholarships from Griffith University and Chinese Scholarship Council.

REFERENCES (1) Meissner, W. G., Frasier, M., Gasser, T., Goetz, C. G., Lozano, A., Piccini, P., Obeso, J. A., Rascol, O., Schapira, A., and Voon, V. (2011) Priorities in Parkinson’s disease research. Nat. Rev. Drug Discov. 10, 377-393. (2) Stocchi, F., and Olanow, C. W. (2013) Obstacles to the development of a neuroprotective therapy for Parkinson’s disease. Mov. Disord. 28, 3-7. (3) Stockwell, B. R. (2002) Chemical genetic screening approaches to neurobiology. Neuron. 36, 559-562. (4) Arrowsmith, C. H., Audia, J. E., Austin, C., Baell, J., Bennett, J., Blagg, J., Bountra, C., Brennan, P. E., Brown, P. J., and Bunnage, M. E. (2015) The promise and peril of chemical probes. Nat. Chem. Biol. 11, 536-541. (5) Kawasumi, M., and Nghiem, P. (2007) Chemical genetics: elucidating biological systems with small-molecule compounds. J. Invest. Dermatol. 127, 1577-1584. (6) Mruk, K., and Chen, J. K. (2015) Thinking big with small molecules. J. Cell Biol. 209, 7-9. (7) Workman, P., and Collins, I. (2010) Probing the probes: fitness factors for small molecule tools. Chem. Biol. 17, 561-577. (8) Andrusiak, K., Piotrowski, J. S., and Boone, C. (2012) Chemical-genomic profiling: systematic analysis of the cellular targets of bioactive molecules. Bioorg. Med. Chem. 20, 19521960. (9) Kotz, J. (2010) Bet-ting on bromodomains. SciBX. 3. (10) Romero, F. A., Taylor, A. M., Crawford, T. D., Tsui, V., Côté, A., and Magnuson, S. (2016) Disrupting acetyl-lysine recognition: progress in the development of bromodomain inhibitors. J. Med. Chem. 59, 1271-1298. (11) Tough, D. F., Lewis, H. D., Rioja, I., Lindon, M. J., and Prinjha, R. K. (2014) Epigenetic pathway targets for the treatment of disease: accelerating progress in the development of pharmacological tools: IUPHAR Review 11. Br. J. Pharmacol. 171, 4981-5010. (12) Medina-Franco, J. L., Giulianotti, M. A., Welmaker, G. S., and Houghten, R. A. (2013) Shifting from the single to the multitarget paradigm in drug discovery. Drug Discov. Today. 18, 495-501. (13) Hopkins, A. L. (2008) Network pharmacology: the next paradigm in drug discovery. Nat. Chem. Biol. 4, 682-690.


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(14) Kong, D. X., Li, X. J., Tang, G. Y., and Zhang, H. Y. (2008) How many traditional Chinese medicine components have been recognized by modern western medicine? A chemoinformatic analysis and implications for finding multicomponent drugs. ChemMedChem. 3, 233-236. (15) Herrick, T. M., and Million, R. P. (2007) Tapping the potential of fixed-dose combinations. Nat. Rev. Drug Discov. 6, 513-514. (16) Li, X. Z., Zhang, S. N., Liu, S. M., and Lu, F. (2013) Recent advances in herbal medicines treating Parkinson’s disease. Fitoterapia. 84, 273-285. (17) Song, J. X., Sze, S. C. W., Ng, T. B., Lee, C. K. F., Leung, G. P., Shaw, P. C., Tong, Y., and Zhang, Y. B. (2012) AntiParkinsonian drug discovery from herbal medicines: What have we got from neurotoxic models? J. Ethnopharmacol. 139, 698711. (18) Levites, Y., Youdim, M. B., Maor, G., and Mandel, S. (2002) Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-κB) activation and cell death by tea extracts in neuronal cultures. Biochem. Pharmacol. 63, 21-29. (19) Guo, S., Bezard, E., and Zhao, B. (2005) Protective effect of green tea polyphenols on the SH-SY5Y cells against 6-OHDA induced apoptosis through ROS-NO pathway. Free Radic. Biol. Med. 39, 682-695. (20) Guo, S., Yan, J., Yang, T., Yang, X., Bezard, E., and Zhao, B. (2007) Protective effects of green tea polyphenols in the 6-OHDA rat model of Parkinson’s disease through inhibition of ROS-NO pathway. Biol. Psychiatry. 62, 1353-1362. (21) Nie, G., Jin, C., Cao, Y., Shen, S., and Zhao, B. (2002) Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells. Arch. Biochem. Biophys. 397, 84-90. (22) Bae, N., Ahn, T., Chung, S., Oh, M. S., Ko, H., Oh, H., Park, G., and Yang, H. O. (2011) The neuroprotective effect of modified Yeoldahanso-tang via autophagy enhancement in models of Parkinson’s disease. J. Ethnopharmacol. 134, 313-322. (23) Tanaka, M., Bateman, R., Rauh, D., Vaisberg, E., Ramachandani, S., Zhang, C., Hansen, K. C., Burlingame, A. L., Trautman, J. K., and Shokat, K. M. (2005) An unbiased cell morphology-based screen for new, biologically active small molecules. PLoS Biol. 3, e128. (24) Lorang, J., and King, R. W. (2005) Cytological profiling: providing more haystacks for chemists’ needles. Genome Biol. 6, 228. (25) Mitchison, T. J. (2005) Small-molecule screening and profiling by using automated microscopy. Chembiochem. 6, 3339. (26) Perlman, Z. E., Slack, M. D., Feng, Y., Mitchison, T. J., Wu, L. F., and Altschuler, S. J. (2004) Multidimensional drug profiling by automated microscopy. Science. 306, 1194-1198. (27) Abraham, Y., Zhang, X., and Parker, C. N. (2014) Multiparametric analysis of screening data growing beyond the single dimension to infinity and beyond. J. Biomol. Screen. 19, 628-639. (28) David, R. (2013) Techniques and applications: finding out how antimicrobials work. Nat. Rev. Microbiol. 11, 741-741. (29) Low, J., Chakravartty, A., Blosser, W., Dowless, M., Chalfant, C., Bragger, P., and Stancato, L. (2009) Phenotypic fingerprinting of small molecule cell cycle kinase inhibitors for drug discovery. Curr. Chem. Genomics. 3, 13-21. (30) Ochoa, J. L., Bray, W. M., Lokey, R. S., and Linington, R. G. (2015) Phenotype-guided natural products discovery using cytological profiling. J. Nat. Prod. 78, 2242-2248. (31) Schulze, C. J., Bray, W. M., Woerhmann, M. H., Stuart, J., Lokey, R. S., and Linington, R. G. (2013) “Function-first” lead discovery: mode of action profiling of natural product libraries using image-based screening. Chem. Biol. 20, 285-295.

(32) Sutherland, J. J., Low, J., Blosser, W., Dowless, M., Engler, T. A., and Stancato, L. F. (2011) A robust high-content imaging approach for probing the mechanism of action and phenotypic outcomes of cell-cycle modulators. Mol. Cancer Ther. 10, 242-254. (33) Towne, D. L., Nicholl, E. E., Comess, K. M., Galasinski, S. C., Hajduk, P. J., and Abraham, V. C. (2012) Development of a high-content screening assay panel to accelerate mechanism of action studies for oncology research. J. Biomol. Screen. 17, 10051017. (34) Woehrmann, M. H., Bray, W. M., Durbin, J. K., Nisam, S. C., Michael, A. K., Glassey, E., Stuart, J. M., and Lokey, R. S. (2013) Large-scale cytological profiling for functional analysis of bioactive compounds. Mol. Biosyst. 9, 2604-2617. (35) Young, D. W., Bender, A., Hoyt, J., McWhinnie, E., Chirn, G. W., Tao, C. Y., Tallarico, J. A., Labow, M., Jenkins, J. L., and Mitchison, T. J. (2008) Integrating high-content screening and ligand-target prediction to identify mechanism of action. Nat. Chem. Biol. 4, 59-68. (36) Nonejuie, P., Burkart, M., Pogliano, K., and Pogliano, J. (2013) Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules. Proc. Natl. Acad. Sci. U. S. A. 110, 16169-16174. (37) Nonejuie, P., Trial, R. M., Newton, G. L., Lamsa, A., Perera, V. R., Aguilar, J., Liu, W. T., Dorrestein, P. C., Pogliano, J., and Pogliano, K. (2016) Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis. J. Antibiot. (Tokyo) 69, 353-361. (38) Effenberger, K. A., James, R. C., Urabe, V. K., Dickey, B. J., Linington, R. G., and Jurica, M. S. (2015) The natural product N-palmitoyl-l-leucine selectively inhibits late assembly of human spliceosomes. J. Biol. Chem. 290, 27524-27531. (39) McLeod, S. M., Fleming, P. R., MacCormack, K., McLaughlin, R. E., Whiteaker, J. D., Narita, S., Mori, M., Tokuda, H., and Miller, A. A. (2015) Small-molecule inhibitors of Gram-negative lipoprotein trafficking discovered by phenotypic screening. J. Bacteriol. 197, 1075-1082. (40) Quach, D., Sakoulas, G., Nizet, V., Pogliano, J., and Pogliano, K. (2016) Bacterial cytological profiling (BCP) as a rapid and accurate antimicrobial susceptibility testing method for staphylococcus aureus. EBioMedicine. 4, 95-103. (41) Dragunow, M. (2008) High-content analysis in neuroscience. Nat. Rev. Neurosci. 9, 779-788. (42) Macchi, F., Deleersnijder, A., Van den Haute, C., Munck, S., Pottel, H., Michiels, A., Debyser, Z., Gerard, M., and Baekelandt, V. (2016) High-content analysis of α-synuclein aggregation and cell death in a cellular model of Parkinson’s disease. J. Neurosci. Methods. 261, 117-127. (43) Grkovic, T., Pouwer, R. H., Vial, M. L., Gambini, L., Noël, A., Hooper, J. N., Wood, S. A., Mellick, G. D., and Quinn, R. J. (2014) NMR fingerprints of the drug-like natural-product space identify iotrochotazine A: a chemical probe to study Parkinson’s disease. Angew. Chem. Int. Ed. Engl. 53, 6070-6074. (44) Dashti, Y., Vial, M. L., Wood, S. A., Mellick, G. D., Roullier, C., and Quinn, R. J. (2015) Kororamide B, a brominated alkaloid from the bryozoan Amathia tortuosa and its effects on Parkinson’s disease cells. Tetrahedron. 71, 7879-7884. (45) Vial, M. L., Zencak, D., Grkovic, T., Gorse, A. D., Mackay-Sim, A., Mellick, G. D., Wood, S. A., and Quinn, R. J. (2016) A grand challenge. 2. Phenotypic profiling of a natural product library on Parkinson’s patient-derived cells. J. Nat. Prod. 79, 1982-1989. (46) Wang, D., Feng, Y., Murtaza, M., Wood, S., Mellick, G., Hooper, J. N., and Quinn, R. J. (2016) A grand challenge: unbiased phenotypic function of metabolites from Jaspis splendens against Parkinson’s disease. J. Nat. Prod. 79, 353-361.



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(47) Vilariño-Güell, C., Wider, C., Ross, O. A., Dachsel, J. C., Kachergus, J. M., Lincoln, S. J., Soto-Ortolaza, A. I., Cobb, S. A., Wilhoite, G. J., and Bacon, J. A. (2011) VPS35 mutations in Parkinson disease. Am. J. Hum. Genet. 89, 162-167. (48) Zimprich, A., Benet-Pagès, A., Struhal, W., Graf, E., Eck, S. H., Offman, M. N., Haubenberger, D., Spielberger, S., Schulte, E. C., and Lichtner, P. (2011) A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease. Am. J. Hum. Genet. 89, 168-175. (49) Follett, J., Norwood, S. J., Hamilton, N. A., Mohan, M., Kovtun, O., Tay, S., Zhe, Y., Wood, S. A., Mellick, G. D., and Silburn, P. A. (2014) The Vps35 D620N mutation linked to Parkinson's disease disrupts the cargo sorting function of retromer. Traffic 15, 230-244.


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Meeting the challenge: using cytological profiling to discover chemical probes from traditional Chinese medicines against Parkinson’s disease Chao Wang,† Xinzhou Yang,‡ George D. Mellick,†* and Yunjiang Feng†* †

Eskitis Institute for Drug Discovery, Griffith University, Brisbane 4111, College of Pharmacy, South-Central University for Nationalities, Wuhan 430074

‡


Meeting the Challenge: Using Cytological Profiling to Discover

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