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Natural Product Reports

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Current developments in natural products chemistry

Volume 24 | Number 6 | December 2007 | Pages 1197–1432

ISSN 0265-0568

Volume 24 | N

umber 6 | 2007 N

PR Pages 1197–1432

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Chemical BiologyREVIEWJason Micklefield et al. Cellular targets of natural products 0265-0568(2007)24:6;1-R

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REVIEW www.rsc.org/npr | Natural Product Reports

Cellular targets of natural products

Neil Dixon, Lu Shin Wong, Torsten H. Geerlings and Jason Micklefield*

Received (in Cambridge, UK) 12th June 2007First published as an Advance Article on the web 17th October 2007DOI: 10.1039/b616808f

Covering: 2001 to 2006 inclusive

Natural products have evolved, at least in part, to bind to biological macromolecules, particularlyproteins. As a result, natural products are able to interact with many specific targets within the cell.Indeed for many years this has been central in the drug development process. Today, however, naturalproducts are finding increasing use as probes to interrogate biological systems as part of chemicalgenomics and related research. In order to demonstrate the utility of natural products in these efforts,the biological activities of many of the major classes of natural products is discussed, according to thecellular organelle and localisation of their specific molecular targets. Emphasis is given to newlydiscovered compounds and activities that either provide interesting insights into a specific biologicalfunction, or that form the basis for potentially new therapeutic approaches.

1 Introduction2 Nucleus2.1 Genetic integrity2.2 Telomerase inhibition2.3 Gene expression3 Ribosome3.1 Prokaryotic vs. eukaryotic ribosomes3.2 Prokaryotic translation initiation and peptide

translocation3.3 Prokaryotic ribosomal subunit assembly3.4 Eukaryotic initiation factor 4A (eIF4A)3.5 Eukaryotic peptide elongation and translocation3.6 tRNA synthetase inhibitors4 Endoplasmic reticulum4.1 Sarcoplasmic reticulum5 Golgi apparatus5.1 Natural product targets within the Golgi6 Vesicles7 Cytoskeleton7.1 Products targeting actin7.2 Microtubule stabilisation and dynamics7.3 Other products targeting tubilin7.4 Antimitotic peptides and kinesin inhibition8 Mitochondria8.1 Modulators of the intrinsic apoptotic pathway8.2 Modulators of respiration and energy-generating

systems9 Cytoplasm9.1 MAP kinase9.2 The ubiquitin–proteasome pathway9.3 Type I fatty acid synthase9.4 Type II fatty acid synthase10 Cell membrane10.1 Chemokines

School of Chemistry & Manchester Interdisciplinary Biocentre, The Uni-versity of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.E-mail: [email protected]; Tel: +44 (0)161 3064509

10.2 Multidrug-resistant cells10.3 Vacuolar-(H+)-ATPase11 Conclusion12 References

1 Introduction

For over three billion years microorganisms have evolved to pro-duce nucleic acids, proteins and other biological macromoleculesalongside smaller metabolites.1 It is thus hardly surprising thatthere exists a small metabolite, or natural product, ligand for manyof the distinct macromolecular targets that exist within today’scells. Natural products are biosynthesised by protein catalysts, andas such natural product and protein structures must necessarilyhave co-evolved to bind to one another selectively.2 Indeed, it islikely that during evolution, there has existed a natural productligand that could bind to all possible protein folds. In additionto this, many metabolites are known which can regulate theirown biosynthesis or degradation through interaction with thegenes, particularly mRNAs, which encode the enzymes responsiblefor their metabolism.3,4 Thus whilst primary metabolites servedefined roles in the normal growth and development of livingorganisms, it seems likely that the secondary metabolites haveevolved specifically to interact with and modulate the function ofbiological macromolecules.

Most secondary metabolites are produced by bacteria, fungi,plants and marine organisms. Some of these molecules are knownto provide a selective advantage against microbial attack ordefence against infestation and disease,5 whilst others functionas signalling molecules in quorum sensing,6 as pheromones orpigments facilitating reproduction. However, the role of many ofthese molecules and their specific molecular targets within theproducing cell or local environment remains unclear. In fact, it hasbeen suggested that the majority of natural products do not haveany significant biological activity or functional role.7 However,evolution could still favour their retention, as part of the widerchemical diversity. Through the presence of promiscuous enzymes

1288 | Nat. Prod. Rep., 2007, 24, 1288–1310 This journal is © The Royal Society of Chemistry 2007

Neil Dixon received his MChem in 2000 and PhD in 2005 from the University of Leeds, where he investigated interactions between syntheticnatural product mimics and the osteoporosis target enzyme V-ATPase, with Terry Kee. He is currently a postdoctoral research associate withJason Micklefield at the Manchester Interdisciplinary Biocentre, where he is investigating small molecule mediated gene regulation.

Lu Shin Wong received a PhD in Chemistry in 2005 from the University of Southampton working with Mark Bradley on solid phasecombinatorial synthesis. Subsequently he joined the Manchester Interdisciplinary Biocentre as a postdoctoral research associate with JasonMicklefield. His current research interests are focused on the application of chemical biology to surface chemistry and biomolecular-arraytechnologies.

Torsten Hans Geerlings completed his PhD in biochemistry in 2003 at the Vrije Universiteit Amsterdam researching the maturation andprocessing of ribosomal RNA in the yeast Saccharomyces cerevisiae. He then moved to Rijksuniveristeit Groningen as a postdoctoralresearcher focused on enhancing protein secretion in the yeast Hansenula polymorpha. Currently he is working as a postdoctoral researchassociate in the Manchester Interdisciplinary Biocentre with Jason Micklefield and John McCarthy studying the interaction between smallnatural and synthetic ligands and RNA aptamers.

Jason Micklefield graduated from the University of Cambridge in 1993 with a PhD in Organic Chemistry, working with Prof. Sir AlanR. Battersby to complete the first total synthesis of haem d1. He then moved to the University of Washington, USA, as NATO postdoctoralfellow with Prof. Heinz G. Floss investigating various biosynthetic pathways and enzyme mechanisms. In 1995 he began his independentresearch career at Birkbeck College, University of London, before moving to Manchester in 1998, where he is now Reader in ChemicalBiology. His research interests are at the chemistry–biology interface and include the redesign of nucleic acid, small molecule control of geneexpression, nonribosomal peptide biosynthesis, biocatalysis and enzyme mechanism.

Neil Dixon Lu Shin Wong Torsten HansGeerlings

Jason Micklefield

and mutations within biosynthetic pathway, this chemical diversitycan evolve, until a natural product with potent biological activityis selected, which would increase the fitness of the population.7 De-spite this, specific targets have been discovered for many secondarymetabolites within human cells. Indeed, a significant portion of thenew pharmaceuticals released for clinical use are derived from orinspired by natural products, and this has been one of the majorcornerstones of pharmaceutical drug development.8 The fact thatmany natural products are known that can selectively bind toand modulate the function of many targets within the humancell is not surprising. Most macromolecular targets in humanshave functional counterparts, possessing sequence similarity, inlower eukaryotes and prokaryotes.2 As a result, the biologicalactivity of natural products in human cells is a consequence oftheir co-evolution alongside similar targets within the producingor neighbouring organism in the environment.

In addition to pharmaceutical drug development, the enormouspotential of natural products as modulators of biomolecularfunction has become increasingly evident with the advent ofchemical genomics and increasingly powerful high-throughputscreening methodologies. Unlike pharmaceutical research, whichfocuses on only those cellular targets that are deemed thera-

peutically relevant, chemical genomics aims to uncover smallmolecules that can specifically modulate every function of all geneproducts in a given cell.9 In this respect, natural products haveproved invaluable constituents of compound libraries in chemicalgenomics. Moreover, this research has uncovered a plethora ofhitherto unidentified targets to which natural products interactwith exquisite selectivity. As a result of this and the continuingneed for improved pharmaceuticals, there has been an increaseddrive to expand the number and structural diversity of availablenatural product compound libraries. To this end, genome miningand microbial metagenomic approaches along with heterologousexpression systems have been developed to reveal the massive,untapped, natural product resources within the environment thatremain intractable or cryptic.10 Coupled with this, powerful toolsfor combinatorial biosynthetic engineering have been introducedfor the biosynthesis of ‘unnantural’ natural products.10 Theseapproaches not only enable lead optimisation of existing structuraltemplates, but offer the potential for the de novo biosynthesis ofcompletely new structural entities.

In this review we aim to demonstrate the breadth of differentcellular targets with which the main structural classes of naturalproducts interact across the whole cell. To this end, the eukaryotic

This journal is © The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 1288–1310 | 1289

cellular organelles (Fig. 1) are used to subdivide and categorisethe different natural products according to the functions that theymodulate within the cell. Eukaryotic cells are more complex andoffer more compartmentalised macromolecular targets with whichnatural products can interact. Indeed, the interaction of naturalproducts with certain specific eukaryotic cellular targets has beenwidely studied, particularly in the context of pharmaceuticaldevelopment. It is thus not possible to cover the entire historicalbasis by which these cellular targets have been interrogated withnatural products. Instead the review focuses on those more recentlydiscovered natural products that have been identified and shownto exhibit the most potent, or interesting, biological effects againstselected targets. In particular, emphasis will be given to thosenatural product entities that have arisen out of modern high-throughput screening regimes or chemical genomics research, aswell as those that offer potential new directions in pharmaceuticaldevelopment. Whilst the structure of this review is based aroundthe eukaryotic organelle, the interaction of natural productswith similar cellular targets in prokaryotes, for example in thedevelopment antimicrobial agents, will be briefly discussed.

Fig. 1 Cellular organelles: (1) Eukaryotic cell; (2) Nucleus; (3) Ribosome;(4) Endoplasmic reticulum (ER); (5) Golgi apparatus; (6) Vesicles;(7) Cytoskeleton; (8) Mitochondria; (9) Cytoplasm; (10) Cell membrane.Adapted from a freely available image from http://en.wikipedia.org/wiki/Organelles.

2 Nucleus

The nucleus is the core of the eukaryotic cell and is separated fromthe cytoplasm by the nuclear envelope, a double membrane layer,with integral nuclear pore complexes that allow water-solublemolecules (<60 kDa) to diffuse freely. The natural products thatare known to target this cellular organelle act upon or disrupt thetwo principle functions of the nucleus, which are the maintenanceof genetic integrity and the control of gene expression.

2.1 Genetic integrity

There are many known natural products that directly interfere withgenetic integrity, including DNA alkylators and intercalators. Inaddition, other natural products exist which target elements thatmaintain genetic integrity such as DNA polymerases, helicases,topoisomerases, transcription factors (e.g. p53) and telomerases.Of the many known DNA alkylators, the thiocyanate-containingalkaloid fasicularin 1 from the ascidian (sea squirt) Nephteisfasicularis is particularly interesting. Recent studies have demon-strated that fasicularin undergoes intramolecular displacement ofthe thiocyanate group to form an aziridinium ion intermediate 2that selectively alkylates N7 of guanine within dsDNA, leadingto strand cleavage (Fig. 2).11 This is notable as the first exampleof a natural product that has been shown to alkylate DNA, viaan aziridinium ion intermediate in a mechanistically analogousfashion to mechlorethamine and other well known nitrogenmustards. The properties of the potent antitumour agents thatinclude CC-1065 along with duocarmycins A and SA have beenwidely reviewed previously.12 However, yatakemycin 3, which wasrecently isolated from Streptomyces sp. TP-A0356, is the firstmember of this family of natural products to contain a centralalkylation subunit ‘sandwiched’ between DNA-binding subunits.13

Indeed, yatakemycin was found to preferentially alkylate adenineover guanine (25 : 1) in dsDNA, leading to strand cleavage (Fig. 2),and represents the most potent member of this class of antitumourcompounds to be isolated to date (IC50 3.0 pM).

There are numerous natural products that have been shown toinhibit DNA polymerases in eukaryotic cells. The fungal aromaticpolyketide dehydroaltenusin 4 is particularly interesting in that itselectively inhibits (with an IC50 of 0.5 lM) mammalian DNA

Fig. 2 (A) N7-alkylation of guanine by alkaloid fasicularin 1. (B) Yatakemycin 3, which preferentially alkylates adenine N3.


polymerase a, which is needed to initiate DNA replication.14

Unlike most other DNA polymerase inhibitors, dehydroaltenusin4 has no inhibitory activity towards other replicative DNApolymerases, such as d and e, which continue DNA replication.15

Dehydroaltenusin has proved useful for analyzing the cellularreplication systems due to its chemical stability in vivo, and iscurrently being investigated as a candidate drug for anticancertreatment. On the other hand, sulfoquinovosyl diacylglycerol(SQDG) 5, has been identified as a selective mammalian DNApolymerase e (pol e) inhibitor.16 This glycolipid, which was isolatedfrom the marine red alga Gigartina tenella, displays IC50 <40 nMactivity towards pol e whilst being three orders of magnitude lessactive towards other mammalian DNA polymerases. In additionto its polymerisation activity, DNA polymerase b (pol b) also hasan intrinsic deoxyribose phosphatase (dRP) activity. This aids inits function as part of the primary DNA repair mechanism, asit dephosporylates the 3′-OH ready for ligation to the incomingdeoxyribonucleotide triphosphate.15 Bioassay-directed isolationfrom an unidentified sponge of the Demospongiae family hasyielded three new natural products 6–8, along with two knownditerpenoid natural products 9 and 10 that display significant pol bdRP inhibitory activity (IC50 15–30 lM).17 This second activityconstitutes an additional target for the discovery of potential cyto-toxicity of DNA-damaging anticancer agents. Similarly, bioassay-guided fractionation of the organic extracts from Cladogynusorientalis, Hymenache donacifolia and Heteropsis integerrima gavefurther pol b dRP inhibitors, stigmasterol 11 (IC50 43.6 lM) and b-sitosterol 12 (IC50 43.3 lM), and from Acacia pilispina b-sitosterol-b-D-glucoside 13 (IC50 72.4 lM) was identified.18 Furthermore, itwas shown that none of these sterols showed any inhibitory activitytowards the polymerase activity of DNA pol b. Selective pol b dRPinhibitors may be clinically useful for anticancer therapy whenused in combination with DNA-damaging antitumour agents.

Also worthy of discussion are the related isomalabaricanetriterpenoids including stelliferin riboside 14, isolated from the

sponge Rhabdastrella globostellata, which were recently reportedto be the first natural products with the ability to promotestabilisation of DNA binding to DNA-polymerase a.19 In addition,two novel fungal tetralols, nodulisporol 15 and nodulisporone16, recently isolated from Nodulisporium sp., have been shown toselectively inhibit human DNA polymerase k with IC50 values of168 and 82 lM respectively, whilst showing no inhibition towardsany other DNA polymerase or metabolic DNA enzymes tested.20

Finally, cytotoxic furanosesterterpene 17 was recently isolatedfrom a marine sponge Psammocinia sp.21 and shown to inhibittwo other enzymes involved in DNA replication, topoisomerase Iand polymerase R-primase, with IC50 values both in the lM range.

2.2 Telomerase inhibition

Telomerase is a ribonucleoprotein that functions as a reversetrancriptase maintaining the guanine-rich telomeres at the terminiof eukaryotic chromosomes. Expression of telomerase is essentialduring embryogenesis and in certain specialised cells includingstem cells. In addition, telomerase is also responsible for immortal-isation of cancer cells.22 Indeed, the fact that telomerase activity isfound in about 90% of human tumours, but not in normal cells, hasmade telomerase inhibition an important target for developmentof new anticancer agents.23

Telomeres contain the simple repeat sequences of TTAGGGand are thought to form G-quadraplex structures in vivo. Thenonribosomal peptide derived telomestatin 18, from Streptomycesanulatus 3533-SV4, was one of the first natural products thatwas shown to stabilise G-guadraplex structures and therebyinhibit telomerase activity.24,25 Indeed, modelling studies sug-gest that telomestatin can stabilise the human telomeric repeatd[AG3(T2AG3)3] by binding between G-tetrad motifs 19, throughH-bonding, p–p and electrostatic interactions between the oxazolerings and the guanine bases.25 The cytotoxic agent daidzin20,26 a flavonoid glycoside from soybean, is also suggested tointeract with G-quadruplex. Electrospray ionisation mass spec-troscopy, polyacrylamide gel electrophoresis, circular dichroismspectroscopy and molecular simulation studies predict that daidzinbinds in the diagonal loop region of the G-quadruplex with1 : 1 and 1 : 2 (DNA:ligand) stoichometries.27 Moreover, it issuggested that similar molecular interactions might explain howdietary isoflavones exert their anticancer effects (although theagonist/antagonist properties of the various isoflavones towards


cancer cells is a matter of some contention). Anti-telomerasebioassay-guided fractionation of the extracts of Japanese marinesponge Dictyodendrilla verongiformis also led to the isolationof dictyodendrin A 21 and related structures.28 The authorsincorrectly claimed this to be first marine natural product that wasshown to inhibit telomerase, but a polysaccharide from the marinemicroalga dinoflagellate Gymnodinium sp. has previously beenreported.29 Finally, the forward chemical genetics approach ledto the identification of two unrelated antibiotics, chrolactomycin22 and UCS1025A 23, with strong telomerase inhibitory effects(IC50 of 0.5 lM and 1.3 lM respectively).30 This identification wasachieved by screening a microbial natural products library againsta yeast strain with shortened telomere length, which enabled com-pounds that induced selective growth defects to be identified andthen evaluated using the telomere repeat amplification protocol(TRAP) assay.

2.3 Gene expression

Natural products are known to bind to numerous biomoleculartargets involved in transcription including RNA polymerases, heli-cases, topoisomerases, transcription factors and proteins involvedin nuclear membrane trafficking. In addition, natural productscan inhibit the production of rRNA and the ribosome within thenucleolus sub-organelle.

DNA topoisomerases play a crucial role in numerous DNAmetabolic events; not only replication, recombination, and chro-mosome segregation at mitosis, but also transcription by regulat-ing the coiling grade of DNA, and thus control its accessibility toenzymes that bind to relaxed stretches of DNA. In light of this,DNA topoisomerases are important cellular targets for chemicalintervention in the treatment of cancer. Oleanane 24 and therelated lupane triterpenoids isolated from the bark of Phyllanthusflexuosus were recently identified as human topoisomerase I andII inhibitors.31 The most potent members of this family have IC50

values in the range of 10–39 lM. Moreover, they are thoughtto be inhibitors of catalysis, which makes them an ideal leadstructure for drug development. The lanostane-type triterpeneacid 25 isolated from the sclerotium of the fungus Poria cocoshas been shown to selectively inhibit topoisomerase II with anIC50 value of 5.0 lM.32 Triterpene 25 shows no inhibitory effectagainst topoiosmerase I and exhibits weak or moderate inhibitoryactivity against mammalian DNA polymerases.

The nuclear factor (NF)-jB is normally sequestered in thecytoplasm by a family of protein inhibitors (IjBs). Upon receivinga stimulus, IjBs are phosphorylated and subsequently degraded.This allows NF-jB to translocate to the nucleus, bind to DNAand in turn activate the expression of genes involved in tumourpromotion, angiogenesis, and metastasis.33 Several diterpenoids,including oridonin 26 and related products, from the herb Isodonrubescens34 have been identified as potent inhibitors of NF-jBtranscription activity and the expression of its downstream targets.All of the diterpenoids directly interfere with the DNA-bindingactivity of NF-jB to its response DNA sequence. However,several of the compounds were identified to have secondarytranscriptional effects. It is suggested these diterpenoids exhibita novel mechanism of regulation, having an impact on thetranslocation of NF-jB by interfering with the shuttling of NF-jBbetween the nucleus and the cytoplasm.


3 Ribosome

The translation of genetic material into functional proteinsdepends upon the ribosome, a ribonucleoprotein containingribosomal RNA (rRNA) composed of a small and a large subunit.Protein synthesis in all organisms begins with the small subunit,with the first transfer RNA (tRNA) binding to the messengerRNA (mRNA). Having located the first codon on the mRNA, thelarge subunit binds and the assembled ribosome begins proteinsynthesis.35 The publication in 2000 of high-resolution crystalstructures of prokaryotic large and small subunits has proven to bepivotal in the understanding of ribosome function at a molecularlevel (Fig. 3).36–38

Fig. 3 Structure of the ribosome from the bacterium Thermus thermo-philus (A), showing the 50S subunit (B) and the 30S subunit.From M. M. Yusupov, G. Z. Yusupova, A. Baucom, K. Lieberman,T. N. Earnest, J. H. D. Cate and H. F. Noller, Science, 2001, 292, 883–896.Reprinted with permission from AAAS. www.sciencemag.org.

3.1 Prokaryotic vs. eukaryotic ribosomes

In both the eukaryotic and prokaryotic ribosomes, the functionalregions are highly conserved, and the main difference betweenthe two is the size of the rRNA component, which is largerin eukaryotes. The eukaryotic rRNA also contains many so-called variable regions,35 which differ greatly in size betweenspecies. Furthermore, the assembly of eukaryotic ribosomes isconsiderably more complicated than the assembly of prokaryoticribosomes. Historically, the difference between eukaryotic andprokaryotic ribosomes has been an area of intense interest inrelation to the development of new antibacterials.39–44 Antibioticshave long been known to selectively inhibit various aspects ofprokaryotic ribosome activity, but only with the recent elucidationof high-resolution crystal structures of the ribosome with thesesmall molecules bound have their modes of interaction at themolecular level been revealed.43–49 Indeed, these exquisite insightshave sparked renewed interest in the development of rationallydesigned and highly specific antibiotics targeted at the prokaryoticribosome.

3.2 Prokaryotic translation initiation and peptide translocation

Initiation factors (IFs) are proteins involved in the assembly ofribosomes at the start of translation and represent a potentialtarget for the development of antibacterial drugs with an entirelynew mode of action. For example, the oligosaccharide evernimicin27 inhibits bacterial protein synthesis by blocking the interactionof IF2 with the large ribosomal 50S subunit.50 This binding by

evernimicin blocks the IF2-dependent placement of fMet-tRNAin the 70S initiation complex, thus preventing the participation offMet-tRNA in the formation of the first peptide bond.

Two recent independent studies demonstrated that the amino-glycoside kasugamycin (Ksg) 28, unlike other previously studiedantibiotics of this class, acts by binding at a novel site on theribosome, the mRNA channel of the 30S subunit.47,48 Structuraland biochemical data suggest that Ksg indirectly inhibits P-sitetRNA binding during translational initiation through perturba-tion of the mRNA–tRNA codon–anticodon interaction during30S canonical initiation (Fig. 4).

Fig. 4 (A) Structure of kasugamycin (Ksg) 28. (B) The initiator tRNA(orange) positioned at the P site of the 30S ribosomal subunit with mRNAand Ifs (not shown). (C) Ksg (red) binds in a region overlapping withthe P-site codon AUG and the E-site codon. The mRNA–tRNA codon–anticodon is thus perturbed, leading to release of the initiator. Reprintedby permission from Macmillan Publishers Ltd: Nature Chemical Biology(Nat. Chem. Biol., 2006, 13, 871–878), copyright 2006. http://www.nature.com/nchembio/index.html.

GE82832 is a newly reported antibiotic from the bacteriumStreptosporangium cinnabarinum that is active against both Gram-positive and Gram-negative bacteria but not against eukaryotes.Although its complete structure has yet to be elucidated, thisagent is known to possess a molecular mass of 1286 Da andcontain aromatic and peptidic components. Despite this lackof information on its chemical structure, a detailed study of its


biological mode of action has been conducted, and GE82832 wasfound to inhibit the first translocation event. This event moves thenewly formed dipeptide and allows the subsequent formation ofthe second peptide bond. Further studies revealed that GE82832was localised to the region of the 30S subunit close to ribosomalprotein S13, which is thought to regulate the concerted movementof the ribosomal subunits required for the translocation of thegrowing peptide.51 This site of action is different from previouslyknown translocation inhibitors and represents an entirely noveltarget of antibiotic action.

3.3 Prokaryotic ribosomal subunit assembly

Apart from the inhibition of the protein synthesis cycle, it hasnow been shown that a number of natural products also inhibitthe formation of the prokaryotic ribosomal subunits themselves.52

Notably, the semi-synthetic polyketide derived macrolide ABT-773 29 was the first antibiotic which was shown to have aninhibitory effect on the assembly of the large 50S subunit inStreptococcus pneumoniae.53 Subsequently, erythromycin A andazithromycin where also shown to demonstrate a similar effecton Haemophilus influenzae.54 However, ABT-773 was observed toretard to the same extent the assembly of the both 50S and 30Ssubunits in H. influenzae, suggesting that its mode of action maysimply be the inhibition of the translation process, resulting ina global decrease in the number of ribosomal proteins availablefor assembly.54 The dipeptide TAN-1057A 30 (from bacteriaof the Flexibacter genus), a known inhibitor of the peptidyl-transferase centre, was also shown to inhibit the formation ofthe prokaryotic large 50S ribosomal subunit with an IC50 of 9 lgmL−1.55 In contrast, the well known aminoglycosides neomycin 31

and paromomycin have been shown to inhibit the formation of theprokaryotic small 30S subunit with a lesser effect on the formationof the 50S subunit.56

3.4 Eukaryotic initiation factor 4A (eIF4A)

Although the main body of research is concerned with naturalproducts and analogues that interact with prokaryotic ribosomaltargets, there is a growing body of literature describing naturalproducts that inhibit eukaryotic ribosomal function, and thesemolecules have become useful experimental probes. For example,the marine natural product pateamine57 32, isolated from Mycalesp., has been identified as a selective modulator of eIF4a, which is aprototypical member of the DEAD-box family of eukaryotic ATP-dependent RNA helicases. eIF4a is thought to unwind mRNAand together with other translation factors, prepare mRNAtemplates for ribosome recruitment during translation initiation.The activity of pateamine is particularly notable given that specifictargeting of individual members of this family of helicases hasproved problematic since they share many highly conserved aminoacid motifs.

Similarly, hippuristanol58,59 33, a polyoxygenated steroid iso-lated from the coral Isis hippuris, was identified from a chemicalgenetics screen for translation initiation inhibitors. Further studiesdemonstrated that hippuristanol binds to the C-terminal domainof eIF4A, blocking the subsequent binding of the mRNA, anddistinguishes between eIF4A-dependent and -independent modesof translation initiation in vitro and in vivo. Additionally, it wasshown that poliovirus replication was delayed when infectedcells were exposed to hippuristanol, which offers the intriguingpossibility of using similar compounds as antiviral drugs.

3.5 Eukaryotic peptide elongation and translocation

Polyketide macrolide 13-deoxytedanolide (13-DT) 34, isolatedfrom the marine sponge Mycale adhaerens, was first identified asa potent antitumour agent. However, it was only recently thatits cytotoxicity was shown to result from binding to the 60Seukaryotic ribosomal subunit causing inhibition of polypeptideelongation in yeast. Importantly, 13-DT does not exhibit this effectin E. coli, and is thus the first macrolide to be found which canselectively inhibit eukaryotic protein biosynthesis.60


While the ability to differentiate between prokaryotic and eu-karyotic protein synthesis has been a major theme of antibacterialresearch, the development of potential antifungals based on thediscrimination between fungal, animal and plant biosynthesisof proteins is less well explored. Although fungal, plant andmammalian eukaryotic elongation factor 2 (eEF2) possessedhighly conserved amino acid sequences, it has now been shownthat sodarin 35 and its analogues selectively inhibited fungaleEF2, with no effect on its equivalents in plants or mammals.61

These findings offer the possibility for a rational developmentof novel antifungal agents and new molecular tools to study theinteractions between eEF2 and the large ribosomal subunit, ofwhich little is currently known.

3.6 tRNA synthetase inhibitors

Another means by which protein synthesis on the ribosome canbe indirectly inhibited is by a reduction in the levels of availableaminoacyl-tRNA feedstocks; in this respect inhibitors of tRNAsynthetase have received considerable attention. Of the naturalproducts that inhibit tRNA synthetase, microcin C (McC) 36 frombacteria of the Enterobacteriaceae family is notable. McC acts bya ‘Trojan horse’ two-step mechanism whereby the hexapeptidechain of the inactive form 36 facilitates uptake by susceptiblecells, followed by release of the N-acylphosphoramidate aspartyl-adenylate 37, which is believed to be the active inhibitorycompound.62

4 Endoplasmic reticulum

The endoplasmic reticulum (ER) consists of an interconnectednetwork of tubules, vesicles and sacs, and serves a number of func-tions including protein synthesis, calcium sequestration, steroidproduction, glycogen storage and production, and insertion ofmembrane proteins. Furthermore, the ER is also the organelle inwhich the synthesis of most lipids used in organelle membranesis finished before these metabolites are distributed to their correct

location in the cell. Natural products are known which interactwith various targets in the ER. For example, cephalostatin-1 38,a bis-steroidal marine natural product, has been shown to rapidlyinitiate an endoplasmic reticulum stress response characterised byphosphorylation of the a-subunit of eukaryotic initiation factor-2. Due to the fact that cephalostatin-1 is able to induce cell deathindependently of the classical mitochondrial apoptosis pathway,it may be advantageous for the treatment of tumours which arechemo-resistant due to defects in the mitochondrial pathway.63

The polyketide versipelostatin 39 produced by Streptomycesversipellis 4083-SVS6, was recently shown to down-regulate grp78gene expression.64 GRP78 is a molecular chaperone in the ERwhich associates with proteins as they traverse the ER. GRP78is expressed during stress such as glucose starvation, inhibitionof protein glycosylation, perturbation of ER function/proteinmovement by tunicamycin and brefeldin A respectively, andsuppression of the ER-calcium-ATPase pump by thapsigargin 45(see Section 4.1). In addition to showing promise as an cancerchemotherapeutic agent, versipelostatin may be a useful tool tostudy molecular chaperones in mammalian cells.

4.1 Sarcoplasmic reticulum

The sarcoplasmic reticulum is a specialised form of ER, foundin muscle tissue, which can store the Ca2+ needed for mus-cle action. Bastadins 40–42 from the sponge Ianthella basta(Pallas),65 were shown to bind to the ryanodine receptor in theRyR1–FKBP12 complex within the sarcoplasmic reticulum. Forexample, 40 and 41 exhibited antagonistic IC50 values of 13and 29 lM, respectively, toward the RyR1–FKBP12 complex.By contrast, 42 was shown to be an agonist for the receptor(EC50 <14 lM). This is the first report of antagonism of theSR channel by a bastadin analogue and suggests a bimodalmechanism of action.66,67 Xestospongins (e.g. 43 and 44), are bis-1-oxaquinolizidines produced by the Pacific sponge Xestospongiaexigua,68 which block inositol-1,4,5-trisphosphate (IP-3) receptor-mediated Ca2+ release from cerebellar ER membranes.69 IP-3 andryanodine receptors are known to induce spatial and temporalrelease of Ca2+ signals in most animal cells and often coexistwithin the endoplasmic/sarcoplasmic reticulum membrane. In


addition to inhibiting the IP-3 receptor, xestospongins were alsofound to lead to sensitisation of the ryanodine receptor-1 channel,enhancing Ca2+-induced Ca2+ release. It has been suggested thatthe xestospongins can be used to help elucidate the important roleof IP-3 and ryanodine receptors in cell signalling.70

It has previously been demonstrated that the sesquiterpene-lactone thapsigargin 45 is a potent inhibitor of the endo/sarcoplasmic calcium ATPase (SERCA).71 Second-generationSERCA inhibitors based on the 2-deoxy analogue 46 (producedby Laser trilobum), and semi-synthetic analogues (e.g. 47 and 48)with modified O8 acyl groups have recently been prepared anddemonstrated to be almost equipotent to thapsigarin as SERCAinhibitors.72 In addition, 12-Boc-aminododecanoyl derivative 48was also able to induce apoptosis.

5 Golgi apparatus

The Golgi apparatus is responsible for packaging proteins, lipidsand other macromolecules biosynthesised in the eukaryotic cell.Historically, natural products have been essential tools for theelucidation of the structure and function of the Golgi apparatus.73

Nocodazole 49, brefeldin A 50 and ilimaquinone 51 and havebeen used to study the role of microtubules in the maintenanceof the Golgi,74 cause fusion of the Golgi with ER75 and to inducemicrotubule-independent Golgi fragmentation,76 respectively.

5.1 Natural product targets within the Golgi

The marine sponge metabolite ilimaquinone 51, originally isolatedfrom Hippiospongia metachromia,77 has a wide range of biologicalactivities,78 including vesiculation of the Golgi apparatus andinterference with intracellular protein trafficking. Preincubationwith S-adenosylmethionine confers resistance to ilimaquinone-induced fragmentation of the Golgi apparatus, supporting thehypothesised interaction with methylation enzymes, specificallyAdoHcy hydrolase, as the cause of Golgi vesiculation. However,the mode of action is still the subject of debate.6 The Golgi-disrupting effects of brefeldin A (BFA) 50, a macrolide antibioticthat was first isolated from Penicillium decumbens, and a numberof its C4 and C7 ester analogues, have recently been visualisedby confocal microscopy.79 The studies demonstrated that the C7position would be an ideal handle for the synthesis of BFAbioconjugates and affinity columns.

The effect of norrisolide 52, isolated from the nudibranchChromodoris norrisi, on the Golgi membranes has been studiedand demonstrated to be the only known compound to inducean irreversible vesiculation of these membranes.80 More recently,the same researchers have prepared norrisolide analogues toenable the visualisation of vesiculation, an important processthat occurs during cellular mitosis.81 In addition, an iodinatednorrisolide analogue was shown to cause complete and irreversiblevesiculation of the Golgi apparatus, providing a potential tool for


the identification of the specific norrisolide biological target withinthe organelle.82

6 Vesicles

The link between the ER, the Golgi stacks and the cell membraneis maintained by a host of single-membrane-bound vesicles, whichtransport cargo between them. Two specialised vesicles exist:the vacuole (which is clearly visible in certain cells using lightmicroscopy) and the lysosomes. The vacuole plays an importantrole in detoxification, pH regulation and osmotic regulation, whilethe lysosomes have a wide range of roles, including fatty acidsynthesis and detoxification of oxygen radicals. Both the vacuoleand the lysosomes can be formed de novo from the ER. Themachinery used to bud off the membranes to form the lysosomesand vacuole is also used for endo- and exocytosis, which playsan important role in cellular uptake and secretion. As a result,altering this machinery impacts on a large range of otherwiseunrelated cellular processes and disease states.

A number of natural products have been observed to causecell death in macrophages with characteristic formation of largevacuoles within cells that are not related to necrotic nor apoptoticcell death pathways. This mechanism of cell death is of particularinterest since it is thought that macrophages in peripheral tissuesregulate the pathological processes in conditions such as tumourdevelopment, inflammation, and atherosclerosis. However, theprecise mechanism of this mode of cell death is currently unknown.Two recently discovered natural products with a related triterpenebisdesmoside structure that display this effect are enterolosaponinA 53 from Securidaca inappendiculata83 roots and structurallyrelated contortisilioside B from Enterolobium contortisiliquum.84

Both were found to be cytotoxic towards BAC1.2F5 mousemacrophages with LD50 values of 3 and 3.4 lM respectively.

Botulinum toxins are the most lethal human toxins known, andingestion causes neuroparalysis by inhibition of the release of theneurotransmitter acetylcholine from the vesicles at the terminiof motor neurones. Once internalised into nerve cells, the toxinacts by cleaving members of the soluble NSF attachment receptor(SNARE) proteins, which are responsible for vesicular exocytosisand release of the acetylcholine. While incidences of botulismcontinue to be reported due to inadequate food preparation or

wound infection, this neurotoxin has also received significantattention more recently as a potential biological warfare agent.As a result, there is considerable interest in the development of arapidly acting and safe antidote. Toosendanin 54 is a triterpenoidfrom the bark of Melia toosendan that has been known since the1980’s to inhibit botulism both in vivo and in vitro. Its effectswere previously attributed to the inhibition of various potassiumchannels, but a new study has reported that treatment withtoosendanin results in resistance of SNAP-25 (a SNARE protein)in the synaptic vesicles to proteolytic cleavage by botulinumtoxin A.85

In the biliary system, multidrug resistance-associated protein2 (Mrp2) is employed by hepatocytes (liver cells) as an effluxpump for a variety of organic anions (e.g. bilirubin, conjugatedglucuronides and sulfated bile acids) that largely comprise thebile acid-independent fraction of bile flow. Impairment, down-regulation or altered localisation of Mrp2 has been associatedwith some cases of cholestasis (impairment of bile output) in liverdisease, and thus regulation of Mrp2 represents an important ther-apeutic target. A recent study using the natural product genipin55, from Gardenia jasminoides, suggests that it may enhance thebile acid-independent secretory capacity of hepatocytes, mainlyby stimulation of exocytosis and insertion of Mrp2 into the cellmembrane, although the molecular mechanism by which it inducesthe redistribution of Mrp2 was not identified.86 It is also unknownif these changes of Mrp2 are related to genipin’s antidiabetic effectsin cellular mitochondria (see Section 8.2).

7 Cytoskeleton

The cytoskeleton is a broad family of cytoplasmic protein fibresthat includes actin, intermediate filaments and microtubules, all ofwhich provide structure to eukaryotic and some prokaryotic cells.Actin is a globular protein which forms a filamentous network justbelow the plasma membrane and is important for maintainingcell shape. In addition, actin filaments function in endo- andpossibly exocytosis as intracellular transport cables in some lowereukaryotes.87,88 In the case of tubulin proteins, several forms havebeen characterised in cells including a- and b-tubulin, whichunidirectionally polymerise into microtubules. These microtubulesare important in the transport of organelles through the cyto-plasm, and the highly dynamic nature of microtubules is utilisedby certain cells to alter the shape of the plasma membrane to allowcellular movement.

7.1 Products targeting actin

Although the importance of actin in the morphology of invasivecancer cells is recognised, no actin-binding agents have yet beenidentified as being of clinical importance.89 Nevertheless, these


agents remain extremely useful experimental probes.90,91 However,there are a large number of associated proteins which control themotility of actin and modulate microfilament growth, shrinkageas well as severing, and are thus potentially interesting targets forsmall-molecule modulation in the future.89,91

Several naturally derived compounds have been found to actas microfilament-disrupting agents. These block or destabiliseactin filaments by binding two distinct regions of the actinmonomer: the ATP-binding cleft, or the barbed end of thefilaments.89 Latrunculin A 56, derived from the Red Sea spongeNegombata magnifica, is often cited as a prototypical example ofthe former, and destabilises the ATP binding cleft by restrictingconformational changes that allow the formation of interactionsbetween the actin monomers. The polyketide derived swinholideA 57, reidispongiolide A 58 and sphinxolides B and C (59 and60), from the marine sponges Theonella swinhoei, Reidispongiacoerulea and Neosiphonia superstes respectively, are all examplesof the second group which inhibit further addition of actinmonomers to the growing end of the microfilaments. Smallmolecules targeting the actin cytoskeleton have also been usedin the development of potential anti-HIV agents. For example, aphage display assay was used to identify analogues (e.g. 61) ofthe batzelladine family of alkaloids from marine sponges, whichwere able to disrupt interactions between the HIV protein Nef andthe cellular proteins p53, p56lck and actin.92 These interactions areessential for HIV propagation and are thus important targets fortherapeutic intervention, but have not been explored with anyexisting antiretroviral agents.

7.2 Microtubule stabilisation and dynamics

The clinical success of the taxanes and Vinca alkaloids inanticancer chemotherapy has highlighted the importance ofmicrotubules in the cell division process. As a result, this areacontinues to be the focus of much research effort.91,93–99 Three

broad categories have been described for natural products thatinteract with tubulin: (i) binders at the so-called ‘vinca domain’of b-tubulin, (ii) those that bind at the colchicine-binding site,and (iii) promoters and stabilisers of microtubule formation (cf.paclitaxel). It has been suggested that alteration of microtubuledynamics by these interactions, rather than the simple stabilisationor depolymerisation of microtubules, appears to be the underlyingbasis of the activity of all these agents.8

Efforts to improve the understanding of interactions betweensmall-molecule natural products and tubulin have been aided byrecent crystallographic structures. Notably, the structures of thetaxane paclitaxel 62, and the polyketide epothilone A 63 frommyxobacterium Sorangium cellulosum bound to tubulin, have bothrecently been elucidated100,101 (Fig. 5). These crystal structuresshow that although both natural products share essentially thesame binding pocket, their supramolecular interactions with thesurrounding peptide residues are completely different. Thesestructures not only show the bioactive conformations of thesecompounds, but address the conflicting experimental results fromprevious efforts to find the common pharmacophores betweenthem. Indeed, there was previously much debate whether therewere any common binding interactions between these two com-pounds and the microtubules.102,103

Another polyketide, laulimalide 64 from Pacific marine sponges,is known to stabilise microtubules. However, laulimalide binds ata separate site to paclitaxel and the epothilones, and is activeagainst paclitaxel-resistant cell lines which possess mutations inthe paclitaxel-binding region.104 In addition, laulimalide and pa-clitaxel were able to act synergistically.105 Similarly, it was recentlydiscovered that the anticoagulant dicoumarol 65, biosynthesisedfrom coumarin in various Penicillium strains, strongly stabilisedthe growing and shortening dynamics at the ends of the micro-tubules in vitro. While paclitaxel binds strongly to the polymericmicrotubule form of tubulin, dicoumarol binds to tubulin dimersin a 1 : 1 stoichiometry with moderate affinity, suggesting different


Fig. 5 (A) Crystal structure of paclitaxel 62 bound to b-tubilin, alongsidethe structure of paclitaxel 62. (B) Epothilone A 63, bound to b-tubilin.Adapted from D. W. Heinz, W.-D. Schubert and G. Hofle, ‘MuchAnticipated – The Bioactive Conformation of Epothilone and Its Bindingto Tubulin’, Angew. Chem., Int. Ed., 2005, 44, 1298–1301. CopyrightWiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

binding sites.106 These findings suggest the use of dicoumaroland other simple coumarins as adjuncts to enhance the efficacyof current anticancer treatments. Cyclostreptin (FR182877) 66(Fig. 6), from Streptomyces sp. 9885, is another notable compoundwhich was found to stabilise polymerised microtubules but notpromote the polymerisation of tubulin.107 Kinetic studies indicatedthat cyclostreptin bound irreversibly and is not displaced byother microtubule stabilisers, suggesting a covalent attachment. Inaddition, LC-MS analysis indicated binding in 1 : 1 stoichiometryto the microtubules at either of two sites (Thr220 and Asn228). It issuggested that bond formation involves the attack of nucleophilicamino acid residues in a Michael addition to position 17 ofcyclostreptin, driven by the release of the strain present in theC2–C17 bridgehead alkene. Whilst cyclostreptin is less activethan paclitaxel, the covalent attachment may offer an avenue toovercome drug resistance, since cancer cells cannot easily preventdrug binding simply by increasing drug efflux or by reducing thebinding site affinity for the compound.

Fig. 6 The mechanism of covalent attachment of cyclostreptin(FR182877) 66 to microtubules.

7.3 Other products targeting tubilin

Pironetin 67, from Streptomyces prunicolor PA-48153, was pre-viously known to inhibit the assembly of microtubules andinhibit the binding of vinblastine to tubulin. A new study,108

employing biotin-labelled pironetin, revealed that the compoundwas covalently bound to tubulin. Subsequent systematic alaninescanning implicated Lys352 at the C-terminal of a-tubulin, whichfaces the b-tubulin of the next heterodimer, as the binding target.This suggested a novel mode of action whereby pironetin binds totubulin via a Michael addition to the unsaturated lactone, whichperturbs the vinca domain of the next b-subunit, thus preventingmicrotubule assembly as well as vinblastine binding.

The diterpenoid pseudolarix acid B 68 from Pseudolarixkaempferi, an indigenous plant of eastern China, has recentlyalso been shown to inhibit tubulin polymerisation.109 Remarkably,a series of experiments including measurements of the intrinsictryptophan fluorescence and CD spectra of tubulin, as wellas DNTB-labelling of cysteine residues and direct competitionexperiments with vinblastine and colchicine, all showed that 68 didnot bind to either of the sites associated with these compounds,and suggested that pseudolarix acid B may target a novel bindingsite on tubulin. In addition, pseudolarix acid B was also noted tohave an effect on the actin cytoskeleton.

7.4 Antimitotic peptides and kinesin inhibition

A number of unusual peptides containing nonproteogenic aminoacids from marine sources are known to disrupt mitosis ineukaryotes by preventing the formation of functional spindlefibres. In most cases, these small peptides bind at the vincadomain, inhibit tubulin-dependent GTP hydrolysis, and interferewith nucleotide turnover at the exchangeable GTP site on b-tubulin. As a result, they inhibit the assembly of tubulin into themicrotubules and, in the cases where it has been studied, stronglysuppress microtubule dynamics at low concentrations.110

Finally, adociasulfate-2 (AS-2) 69 from the marine sponges ofthe genus Haliclona was shown to be the first natural productinhibitor of the kinesin family of ATP-dependent motor proteins.


Kinesins transport various cellular components along the micro-tubules, take part in cell division and intracellular transport, andhave been implicated in neurological and developmental diseasesas well as cancer. AS-2 has an unorthodox mode of actionand is thought to form rod-shaped aggregates that mimic thestructure of microtubules, allowing binding and inhibition of thekinesins.111

8 Mitochondria

Mitochondria are double-membraned organelles whose mainfunction is oxidative respiration in which pyruvate is oxidisedto CO2. The outer mitochondrial membrane is permeable tometabolites while the inner, highly folded, mitochondrial mem-brane contains the proteins needed for respiration and ATPproduction.112,113 The organelles themselves are not static butdivide, fuse, alter their internal structure, and are transported tovarious locations within the cell in response to various stimuli.114

Mitochondria are different from all other organelles (exceptchloroplasts) in that they have their own DNA which codes fora number of mitochondria-specific proteins. In addition to energygeneration, they also play a crucial role in the wider regulation ofmetabolism, cell-cycle control, development, antiviral responsesand apoptosis.114–116

In respect to apoptosis, the mitochondria plays a central rolein the internal so-called ‘intrinsic’ apoptotic pathway, and therelationship between dysfunctional apoptosis and oncogenesishas made this organelle the target of intense research.116–119 Thispathway is characterised by mitochondrial outer membrane per-meabilisation (MOMP) and triggering of a biochemical cascadeof various proteins, including cytochrome c, which finally resultsin cell death.117,120–121 Apart from cancer, the relationships betweenmitochondrial proteins and various pathologies including obesityand diabetes as well as developmental and neurological disorders,are also of interest.114,115,122

8.1 Modulators of the intrinsic apoptotic pathway

There has been tremendous interest in targeting the proteinsinvolved in the intrinsic apoptotic pathway in the hope ofdeveloping new cancer chemotherapeutic agents.117,123 Althoughnewly discovered natural products are often routinely screenedfor their cytotoxicity and potential as chemotherapeutic agents, inmany cases the molecular targets of these small molecules have notbeen clearly established. Indeed, efforts to do so are complicatedby the very large number of signal transducing molecules andfeedback pathways that act on the mitochondria, and the fact thatthe actual targets may be located up- or downstream from themitochondria.

Numerous plant-derived natural products have been shownto act on the mitochondrial pathway of apoptosis, includingchelerythrine 70. This natural benzophenanthridine alkaloid is aknown protein kinase C (PKC) inhibitor from Chelidonium majus,but was also identified as a new inhibitor of BclXL-Bak duringa high-throughput screening of 107 423 natural product extracts.Chelerythrine binds to the BH3 domain of BclXL with an IC50 of1.5 lM,124 resulting in the displacement of the proapoptotic Baxprotein, leading to cell death via the mitochondrial pathway invarious mammalian cell lines. The structurally related sanguinar-

ine (83, see Section 9.1), derived from the root of Sanguinariacanadensis and other Papaveraceae species, was also observed tohave similar effects.125

Extracts of the kava plant (Piper methysticum) have been usedtraditionally to treat a variety of ailments in the Pacific Islands andare proposed to have tumour-suppressing activity. In screening theindividual compounds from this plant, flavokawain A, B and C(71, 72 and 73 respectively) were found to display apoptotic activityagainst several bladder cancer cell lines. Subsequently, flavokawainA was shown to trigger the intrinsic apoptotic pathway, with theobserved loss of mitochondrial membrane potential, release ofcytochrome c into the cytosol and a decrease in BclXL, whichin turn led to an increase in active Bak levels and caspaseactivation. It has been suggested that flavokawain A may havea dual mode of action, as it was also found to down-regulate theexpression of X-linked inhibitor of apoptosis protein (XIAP) andsurvivin, both major factors in apoptosis resistance in bladdertumours.126 Another natural product that was found to reducethe expression of survivin is ponicidin 74, a diterpenoid from theplant Rabdosia rubescens which is closely related to oridonin (26,see Section 2.3). This compound, in addition to inhibiting NF-jB,34 has been shown to have apoptotic activity in lung cancerA549 and GLC-82 cell lines (IC50 15 and 4.1 lM respectively)but appeared to induce cell death through intrinsic pathways viadown-regulation of anti-apoptotic protein Bcl-2, up-regulation ofpro-apoptotic protein Bax and activation of caspase 9, as well asthe extrinsic pathway involving caspase 8, in addition to survivindown-regulation.127

Two natural products possessing vanillyl moieties, capsaicin 75(the pungent constituent of hot peppers of the genus Capsicum)and resiniferatoxin 76 (a diterpenoid from the latex of AfricanEuphorbia sp.), have long been known to act on vanilloid receptorslocated mainly in primary sensory neurons that are associated withnociception (perception of pain) and neurogenic inflammation.However, these compounds are also antagonists of coenzyme Q,a coenzyme necessary for the mitochondrial electron transportchain, and induce apoptosis in a number of cell lines. It has beensuggested that the onset of apoptosis is due to a rapid increase inthe generation of hydroperoxide radical oxidative species (ROS),a decrease in oxygen consumption and MOMP.128


Hydroxyisovalerylshikonin 77, a compound isolated from thetraditional oriental medicinal herb Lithospermum radix, wasshown using a cDNA array to suppress expression of thegene encoding TRAP1 (tumor necrosis factor receptor-associatedprotein 1). In addition, 77 was found to induce apoptosis invarious lines of human tumour cells independently of this agent’sknown kinase inhibitory effects.129 Characterisation of the newtarget TRAP1 revealed it to be localised in the mitochondria,and it had an anti-apoptotic effect when expressed at high levels.Administration of the ROS scavenger N-acetylcysteine efficientlyprevented apoptosis induced by b-hydroxyisovalerylshikonin, in-dicating that ROS generation was involved in the regulationof TRAP1.

Another natural product which was found to effect a previouslyuntargeted protein in the apoptotic pathway is justicidin A 78,an extract from the plant Justicia procumbens.130 A study ofits effects in colorectal cancer cell lines HT29 and HCT116found that it activated caspase-9 but not caspase-8, with aconcomitant increase of the proapoptotic Bax, suggesting thatjusticidin A initiated the intrinsic apoptotic pathway. Moresignificantly, it was found to decrease the expression of Ku70,a subunit of the Ku complex, which plays a crucial role in DNAdouble-strand break repair. In a separate study, justicidin A wasalso shown to have selective cytotoxic activity against hepatomaHepG2 and Hep3B cells. A battery of biochemical experimentsthen revealed that unlike the earlier study with the colorectalcells, both the intrinsic and extrinsic pathways were induced inhepatic cancer cells, as the proteins in the mitochondrial pathway,including caspase-8, were found to be activated.131 Additionally,

both studies demonstrated that oral administration of justicidinA suppressed growth of HT29 and Hep3B cells transplantedinto immunodeficient NOD-SCID mice, suggesting it may be apractical chemotherapeutic agent for colorectal cancer therapy.

8.2 Modulators of respiration and energy-generating systems

Apart from targeting the mitochondrial apoptotic pathway, natu-ral products also target a number of important proteins involvedin cellular respiratory pathways and respiratory adaptationsto hypoxia (low O2 conditions), such as increased anaerobicrespiration by glycolysis. These too have potential applicationsin cancer therapy since the interiors of many tumours are hypoxicdue to low blood perfusion. Furthermore, some cancers maintaina high level of anaerobic metabolism even in the presence of O2, aphenomenon known as the Warburg effect.116

A notable example targeting hypoxia adaptation is the recentlydiscovered laurenditerpenol 79, isolated from marine red algaLaurencia intricata. This diterpene was found to be a selectiveand potent inhibitor (IC50 0.4 lM) of hypoxia-inducible factor-1 (HIF-1), a transcription factor which promotes tumour celladaptation and survival under hypoxic conditions.132 Experimentshave demonstrated that laurenditerpenol inhibits the mitochon-drial electron transport pathway (IC50 0.8 lM) and suggests itis an inhibitor of mitochondrial respiratory complex I. Thus,laurenditerpenol may block hypoxia-induced HIF-1 activity byincreasing cellular O2 availability under low oxygen concentrationsthrough the inhibition of mitochondrial respiration. That in turnwould promote the oxygen-dependent degradation of the HIF-1a subunit. There are currently no drugs that specifically targethypoxic tumour cells, and therefore such specific HIF-1 inhibitorsrepresent an important new class of potential tumour-selectivetherapeutic agents. The realisation of the importance of HIF-1 as a potential pharmacological target has engendered large-scale screenings of natural products with the aim of discoveringnovel inhibitors. In one such example, 5400 natural productsfrom plants, marine organisms, and microbes were examinedfor HIF-1 inhibitory activity using a cell-based reporter assay.Three potent and specific natural products were identified;manassantin A 80 and the structurally related manassantin B1and 4-O-methylsaucerneol.133 Similarly to laurenditerpenol, thesecompounds inhibit HIF-1 by blocking hypoxia-induced nuclearHIF-1a protein accumulation without affecting HIF-1a mRNAlevels.


One mitochondrial target of major interest is the F0F1-ATPsynthase, which is inhibited by cytotoxic polyketides such asapoptolidin 81, ossamycin, cytovaricin and oligomycin. Theseagents were shown to be extremely selective for certain cancercell lines although the basis of this specificity was unclear.However, a large scale gene expression profile screening demon-strated that, at least in leukaemia cells, the expression of twoenzymes involved in anaerobic respiration, pyruvate kinase andaspartate aminotransferase 2, were positively correlated withsurvival. These findings were confirmed by experiments whereinhibition of the anaerobic respiratory pathways (with oxamateand 2-deoxyglucose) increased the sensitivity of the cells towardsapoptolidin. Thus, these polyketide macrolides appear to beselectively toxic against cells that did not exhibit the Warburgeffect and maintained primarily aerobic respiration.134

One mitochondrial target which is not associated with cancerchemotherapy is uncoupling protein 2 (UCP2), a mitochondrialcarrier protein that mediates proton leak across the inner mi-tochondrial membrane and decreases the yield of ATP fromglucose metabolism. In pancreatic b-cells, UCP2 is known tonegatively regulate glucose-stimulated insulin secretion and thusrepresents a potential target in the treatment of diabetes. The fruitsof Gardenia jasminoides have been used in traditional Chinesemedicine to treat the symptoms of type 2 diabetes. Isolationand testing of the pharmacologically active compounds from thefruits revealed a UCP2-inhibitor, genepin 55 (see Section 6).135

This molecule appears to function as a cell-permeable mimic ofpurine nucleotides, the natural regulators of UCPs, resulting inan increase in mitochondrial membrane potential and ATP levels,closure of KATP channels, as well as increased insulin secretion.

Finally, the mitochondria of protozoal parasites represent apromising target in the search for new anti-protozoals. Forexample, intracellular parasites such as the malaria protozoanPlasmodium falciparum have a range of respiratory pathwaysdistinct from that of their hosts, and recent studies have identifiedlicochalcone A 82 as a potential antiparasitic lead. This moleculeinhibits the respiratory complex II and the bc1 complex, essentialin Plasmodium respiration, with IC50 values of 1.3 and 0.1 lMrespectively, by acting as a competitive antagonist of ubiquinonebinding.136

9 Cytoplasm

The number of natural products that exert a biological effectin the cytoplasm, outside of the various organelles, is huge.Therefore, only those secondary metabolites whose targets haveclearly defined cytoplasmic localisation and are of particularinterest will be discussed here.

9.1 MAP kinase

One of the most important signalling pathways in eukaryotic cellsis the mitogen-activated protein (MAP) kinase cascade pathway,137

which consists of a mitogen-activated protein kinase (MAPK), aMAP kinase kinase (MAPKK), and a MAP kinase kinase kinase(MAPKKK). The MAP kinases’ function is to transfer mostlyextracellular signals to the nucleus. In general, a sensor proteinin the plasma membrane stimulates the MAPKKK, which thenphosphorylates its target MAPKK, which in turn phosphorylatesthe MAPK. The activated MAPK then usually translocates tothe nucleus, where it phosphorylates proteins involved in generegulation, thus modulating gene expression.137 Natural productsthat specifically block or stimulate these pathways can result inaltered expression profiles and significant physiological responses.For example, the benzo[c]phenanthridine alkaloid sanguinarine 83from the poppy Chelidonium majus has been identified as the firstselective mitogen-activated protein kinase phosphatase 1 (MKP-1) inhibitor, using a high-content screening (HCS) assay.138 Theassay developed relies on a powerful method to identify truephenotypic effects of sanguinarine and other small molecules.The fact that MKP-1 is known to be overexpressed in manyhuman tumours makes compounds like 83 potentially usefulprobes in cancer research. 1-Methoxy-canthin-6-one 84, isolatedfrom the plant Ailanthus altissima, has recently been demonstratedto induce apoptosis via activation of MAPK subfamily Jun N-terminal kinase.139 The plant extract was found to have synergisticeffects with the human recombinant tumour necrosis factor(TRAIL), upon the induction of apoptosis in various leukaemiaand carcinoma cell lines wherein suboptimal dosages of bothligands enable apoptosis induction.

9.2 The ubiquitin–proteasome pathway

It can be detrimental for the cell to retain proteins once theirfunction is complete. As a consequence, proteins are normally


produced only transiently in response to certain stimuli and areultimately degraded by a number of mechanisms, including theubiquitin pathway. In this pathway, a protein undergoes polyu-biquitination, which usually involves the covalent attachment offour ubiquitin polypeptides. This serves to target the proteinto the proteasome, which is a large multienzyme complex thatdegrades proteins by proteolysis.140 Natural products and othersmall molecules that are known to interfere with this process canlead to either accumulation or depletion of various proteins inthe cytoplasm depending on whether they block or stimulate thispathway. For example, the cytotoxic agent salinosporamide A 85,from the actinomycete bacteria Salinospora CNB-392, and relatedanalogues have been used as tools to study proteasome-mediatedprotein degradation due to their unique ability to selectivelyinhibit the 20S subunit of the proteasome.141–143 Indeed, 85 wasshown to inhibit the proteasomal chymotrypsin-like proteolyticactivity with an IC50 value of 1.3 nM.142 In addition, seven newcinnabaramides, structurally related to 86, have been isolated froma terrestrial strain of Streptomyces and shown to inhibit the human20S proteasome, with cinnabaramide 86 being the most potent(IC50 = 0.6 nM).143

The medicinal plant Withania somnifera has been used intraditional Indian medicine for treatment of various angiogenicprocesses such as arthritis and menstrual disorders. Extracts ofW. somnifera have been found to contain the active constituentwithaferin A 87, which inhibits cell proliferation in a humanumbilical vein endothelial cell assay via inhibition of NF-jB. Itis suggested that the inhibition is a result of interference with theubiquitin-mediated proteasome pathway.144 Interestingly, recentstudies indicate that the primary molecular target of 87, whichinduces the apoptosis of prostrate cancer cells, is the b5 subunit ofthe proteasome.145 Similarly, celastrol A 88, extracted from the rootbark of Tripterygium wilfordii, has been used for many years as anatural remedy in traditional Chinese medicine for inflammatoryconditions and is also known to induce apoptosis of leukaemiacells. Only recently, however, has the molecular target beenidentified as the 20S proteasome.146 Celastrol A 88 was tested forits ability to inhibit proteasomal activity due to the similarity ofits di-a,b-unsaturated ketone substitution to the antiproteasomaldietary flavanoid quercetin 89.147

Finally, a high-throughput bioluminescence assay has recentlybeen developed that allows screening of plant extracts andchemical compounds for their ability to inhibit the ubiquitin–proteasome pathway by detecting the expression of ubiquitin–luciferase reporter protein. Notably, the assay has led to theidentification of a new pathway inhibitor, physalin B 90 from thetropical herb Physalis angulata, displaying an EC50 value in the lMrange.148

9.3 Type I fatty acid synthase

Stages of fatty acid metabolism occur within several eukaryoticorganelles such as the Golgi, mitochondria and the ER. However,fatty acid synthase (FAS), which is responsible for fatty acidbiosynthesis in eukaryotes, is located within the cytosol and willthus be discussed here. In mammals the FAS is a type I mul-tienzyme, possessing all catalytic activities required, as domains,which are covalently linked together on a single polypeptidechain. The fungal type I FAS on the other hand is comprisedof two polypeptide chains (a and b). Remarkable recent X-raycrystallographic structures show that the mammalian FAS isorganised as a homodimer,149 whilst the fungal FAS is organisedas a heterododecamer (a6b6) (Fig. 7).150,151

It has been shown that human cancer cells express higher levelsof FAS than normal mammalian cells, which has led to thesuggestion that the human FAS would be a potential target forthe development of new antitumour agents.152 Notably, a series ofsynthetic a-methylene-c-butyrolactone analogues of the naturalproduct methylenolactocin 91, from Penicillium sp.,153 were testedas inhibitors of human FAS. The most potent compound, C75 92,was shown to possess antitumour activity in human breast cancercells through the specific interaction with the FAS.152

The difference in architecture between the fungal and mam-malian FAS offers the opportunity for the development ofantifungal agents that selectively target the fungal FAS. In thisrespect, the azaphilone natural products, including CT2108A 93from Penicillium solitum (Westling) strain CT2108, have emergedas new antifungal leads. For example, CT2108A 93 inhibitsCandida FAS with an IC50 of 160 lg mL−1, whilst exhibiting


Fig. 7 (A) Dimeric structure of mammalian FAS (4.5 A). The positionsof the domains are represented by coloured spheres, the diameter ofwhich is approximately the distance that the ACP must approach inorder to deliver the Ppant-tethered substrates to the active sites of thedomains, which are represented by white or blue spheres. Ketoreductase(KR, yellow); enoyl reductase (ER, dark green); dehydratase (DH,light green); malonyl/acetyl-CoA transacylase (MAT, red); b-keto acylsynthase (KS). From T. Maier, S. Jenni and N. Ban, Science, 2006, 311,1258–1262. Reprinted with permission from AAAS. www.sciencemag.org.(B) Heterododecameric (a6b6) fungal FAS complex (5 A). The upper andlower hemispheres of the barrel each contain 3 sets of active sites. Domainsas before, except that malonyl/palmitoyl-CoA transacylase (MPT, red) andacetyl transferase (AT, pink) both function the same as the MAT in themammalian FAS. From S. Jenni, M. Leibundgut, T. Maier and N. Ban,Science, 2006, 311, 1263–1267. Reprinted with permission from AAAS.www.sciencemag.org.

only very low inhibition of the mammalian FAS (IC50 >1000 lgmL−1).154

9.4 Type II fatty acid synthase

Whilst the reactions catalysed by the prokaryotic FAS duringthe assembling of fatty acids are similar to those performedin eukaryotes, the architecture of the synthase is considerablydifferent. For example, in bacteria all reactions are carried out byindividual, monofunctional, enzymes in a dissociated or type IIsystem. Once again, these organisational and structural differenceshave spurred efforts to develop new antimicrobial agents thatinteract specifically with the type II FAS. For example, the greentea extract epigallocatechin gallate (EGCG) 94 has been previouslyshown to exhibit antibacterial as well as antiviral and antitumouractivity.155 More recent studies have shown that the antimicrobial

activity is due to the inhibition of the b-ketoacyl-ACP reductase(fabG) and the enoyl-ACP reductase (fabI) activities within thebacterial type II FAS elongation cycle, with IC50 values between 5and 15 lM.156 The antibacterial activity of 94, however, is not solelydue to the inhibition of fatty acid synthesis, as overexpression ofthe fabG and fabI targets did not confer resistance.

Bischloroanthrabenzoxocinone (BABX) 95 was also identifiedas an inhibitor of the bacterial type II FAS pathway, using a novelhigh-throughput assay that relies on long-chain acyl-CoA insteadof acetyl-CoA substrates. This assay allows specific inhibitors to beidentified for the elongation cycle, as opposed to initiation steps.157

BABX showed IC50 values of 11.4 and 35.3 lg mL−1 against theS. aureus and E. coli FAS, respectively. It has been suggested thatBABX is an inhibitor of the condensation enzyme in the elongationcycle, as BABX did not inhibit fabD but fully inhibited acyl-ACPelongation.

A high-throughput screen using a xylose-inducible plasmidto express fabF antisense RNA in Staphylococcus aureus ledto the identification of phomallenic acid A, B and C (96, 97and 98) as inhibitors of FabF.158 Induction of fabF antisenseRNA reduces levels of fabF and of co-transcribed fabH mRNA,which increases the sensitivity towards inhibitors of these geneproducts. Using this assay, 250 000 natural product extractsfrom actinomycetes and fungi were screened, resulting in theidentification of 96–98 from Phoma sp. as novel FabF inhibitors.These agents were approximately 20-fold superior to that ofthiolactomycin and cerulenin (previously known type II FASinhibitors) against S. aureus and displayed a good spectrum ofactivity against the clinically important pathogens methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, andHaemophilus influenzae.158,159 The same group of researchers,in a similar screening experiment, also identified platensimycin99, from the soil bacterium Streptomyces platensis, as a potentantibacterial against a variety of antibiotic-resistant organismswith little effect on mammalian or fungal cells. Administration ofplatensimycin to S. aureus-infected mice resulted in a 104–105-foldreduction in viable bacteria with no apparent adverse effects tothe mice, indicating this antibiotic is a promising candidate forfurther clinical studies.160 Further studies involving a combination


of biochemical, radiolabelling and crystallographic experimentsestablished that platensimycin was bound to FabF at the malonylsubsite. Notably, the antibiotic formed interactions with a regionof FabF that contains the catalytic site, which is highly conservedbetween species. Furthermore, many intermolecular interactionsbetween the antibiotic and enzyme were mediated by hydrogenbonds to the backbone chain of the protein, interactions whichwould be relatively insensitive to mutations of the amino acidsequence. Together, these results suggest that the development ofresistance through mutation of FabF would be unlikely.

Finally, a number of natural products have also been identifiedas possessing anti-protozoal activity. For example, several naturalproducts were isolated from the ethanolic root extract of Scro-phularia lepidota,161 an endemic plant of the Turkish flora andshown to inhibit FabI, a key regulatory and rate-limiting enzymeof the type II FAS pathway, in Plasmodium falciparum.162 Themost potent product, scrolepidoside 100, was also active againstLeishmania donovani, with an IC50 6.1 lg mL−1. Several of thecompounds isolated also possessed some trypanocidal activityagainst Trypanosoma brucei rhodesiense (IC50 values 29.3–73.0 lgmL−1), which make them interesting leads for the treatment ofAfrican sleeping sickness.

10 Cell membrane

The cell membrane can be considered one of the most crucialparts of any cell. Its primary function is to keep the cell machineryprotected from the external environment. However, embedded inthe cell membrane are a host of other proteins involved in com-munication between the interior of the cell and the surroundingmedium. These are involved in signalling, import of nutrients,export from the cell, cell division, pH balancing and osmoticbalancing. In fact, for any natural product to have any influence ona cell it first needs to either pass this barrier or in some way interactwith it. All cell walls are composed of a phospholipid bilayer,often mixed with sphingolipids and sterols. Membranes are made

selectively permeable by a large range of transporter proteins,which either actively or passively allow certain types of moleculesto pass. In addition to a cell membrane, many organisms alsopossess a cell wall, which serves to both provide structure, as wellas extra protection from the external environment. For example,bacterial cell walls contain either a thick layer of peptidoglycan,or a thin layer of peptidoglycan with a second outer membraneof phospholipids and liposaccharides, whilst plant cell walls aremostly composed of cellulose. In addition, several single-celledfungi also have a cell wall, mostly consisting of chitin.

10.1 Chemokines

Chemokines are small extracellular proteins involved in chemo-taxis, the process by which cells migrate towards nutrients oraway from toxins. Upon secretion they bind to cell membranereceptors and cause cells to move in the direction of an in-creasing gradient of stimulant. They play an important part inthe immune response system by attracting white blood cells.163

Several triterpine glycosides, including 101, have been isolatedfrom the sea cucumber Telenata ananas sp. off the Andaman andNicobar islands and shown to selectively to inhibit the chemokinereceptor CCR5. This receptor is known to be a co-factor for HIV-1 attachment and viral entry into host cells, which has madethese glycosides (e.g. 101) interesting pharmacological leads.164

Similarly, several new cyclopropyl-containing natural products(e.g. 102) were isolated from the Peruvian plant Lippia alva(Verbenaceae) and shown by high-throughput screening to beantagonists for the CCR5 receptor with IC50 values in the 5.0–7.0 lg mL−1 range.165

The Chinese herbal medicine ‘zicao’ from the root of Lithos-permum erythrorhizon is known to contain the active componentshikonin 103 with various biological activities, including inhibitionof HIV-1 binding, via inhibition of chemokine function. It hasbeen suggested that the inhibitory properties are due to theinterference with expression of the CCR5 receptor in additionto interference with receptor signalling.166 Also, in an assay toassess the competition between CCR5 and the viral envelopeglycoprotein gp120, the 120 kDa outer shell glycoprotein of the


HIV virus, several natural product antagonists were isolated fromdifferent sources. The most potent of these, anibamine 104, a novelpyridinium alkaloid from Aniba sp., displays an IC50 of 1 lM.167

The association between a subset of the chemokine receptorCCR2 and the chemoattractant protein MCP-1 is known tomediate various inflammatory processes such as rheumatoidarthritis, multiple sclerosis and atherosclerosis. Using a bioassay-guided fractionation of microbial fermentation and plant sources(decaying leaf litter), several new fungal metabolites were dis-covered and shown to be antagonists of this interaction. Theseincluded emestrin 105, chaetomin 106 and cytochalasin A 107,which all display micromolar range inhibition of MCP-1 bindingto the CCR2 receptor target.168

10.2 Multidrug-resistant cells

Multidrug resistance is often not due to drug-specific resistance ofan organism, but rather the ability of the organism to neutralisegeneric harmful substances at an increased level. Often thisresistance is achieved by an increased number of efflux pumpsin the membrane. These pumps are transmembrane proteins thatare able to use chemical energy (e.g. ATP, Na+ or H+ gradients)to remove a set of substrates from the cytoplasm. These pumpsoften recognise certain drug-like physicochemical properties andthus are able to confer resistance to a great variety of drugs.The ABC (ATP Binding Cassette) superfamily is one of the mostwidespread families of these transporters. From screening a libraryof 85 000 compounds, various drug efflux pump inhibitors havebeen identified. These include EA-371a 108 produced by a newactinomycete strain closely related to Streptomyces vellosus, whichinhibits the MexAB pump of Pseudomonas aeruginosa, responsible

for the intrinsic antibiotic resistance of this strain. In addition,milbemycin a9 109 from the Streptomyces strain MF-EA-371-NS1was shown to inhibit the fluconazole resistance efflux pump CDRof Candida albicans.169

A bioassay-guided fractionation of Erythroxylum pervillei basedon the restoration of sensitivity to the cytotoxic vinblastine hasalso led to the isolation of the novel tropane alkaloid pervilleineA 110. Pervilleine A was shown to have no effect upon either themRNA transcription or expression of the vinblastine efflux pump,P-glycoprotein (ABC superfamily). Treatment of MDR cells with110 (40 lM) led to a nearly 30-fold increase in the intracellularaccumulation of [3H]vinblastine, and in addition 110 was shownto be a competitive inhibitor of vinblastine binding to MDR cellmembranes.170 Using the yeast Saccharomyces cerevisiae trans-formed with various plasmids containing fluconazole resistancegenes from C. albicans, the sterol 111 from Dysidea arenaria wasshown to be the first marine natural product to reverse fluconazoleresistance mediated by a MDR efflux pump. Indeed, treatmentwith 111 increases the fluconazole sensitivity of the engineeredyeast strains by up to 35 times.171

Dihydro-b-agarofuran sesquiterpenes from Celastraceae (ofgeneral structure 112) have been previously shown to reverse


MDR in human cancer cells and Leishmania.172–174 More re-cently, the mechanism of action of these sesquiterpenes has beenelucidated, using mouse cells transfected with human MDR-1resistance genes.175 In fact, 28 structural variants of 112 were shownto inhibit [3H]colchicine and tetramethylrosamine P glycoprotein-mediated efflux at submicromolar concentrations. The sameresearchers have subsequently isolated 6 structurally similarsesquiterpenes from Zinowiewia costaricensis, which are alsopotent inhibitors of P-glycoprotein-mediated efflux, using anassay to detect for the reversal of daunorubicin cytotoxicity uponvarious human cell lines.176

10.3 Vacuolar-(H+)-ATPase

Although, as the name suggests, the vacuolar-(H+)-ATPases (V-ATPases) are found in the vacuoles, they have also been found onthe plasma membrane of various cells, and it is from this cellularlocation that they are of most interest as therapeutic targets.Disease states implicated by aberrant V-ATPase function includeabnormal secretion of digestive enzymes, diabetes, cardiovascularand clotting disorders, neurological disorders such as Alzheimer’sdisease, disorders involving abnormal receptor-mediated uptake

processes, glaucoma, defective urinary acidification, osteoporosisand cancer.177–179

Using the National Cancer Institute 60-cell antitumour screen,a novel class of benzolactone enamide ligands was identified ashaving a ‘V-ATPase like’ activity profile similar to that of theV-ATPase inhibitor concanamycin A.180 Notably, some of thebenzolactone enamides, including (−)-salicylihalamide A 113,were shown to inhibit mammalian V-ATPases with unprecedentedselectivity.181 Conversely, a few years later several marine-derivedpolyketide macrolide lactams were identified with selectivitytowards non-mammalian V-ATPases, again using the NCI cell-60 antitumour profiling. The macrolides, including chondropsin A114, were shown to inhibit fungal V-ATPase at IC50 values of 0.04–0.7 lM. This observed selectivity adds impetus to the possibilityof developing selective V-ATPase modulators.182 Finally, thecytotoxic macrolactone archazolid A 115 from the myxobacteriumArchangium gephyra,183,184 and palmerolide A 116 from the Arctictunicate Synoicum adareanum,185 were also shown to inhibit V-ATPase in the nanomolar range, whilst macrolactone iejimalide A117 from Eudistoma cf. rigida displayed potent inhibitory effectsagainst yeast V-ATPase.186

11 Conclusion

From this review several points are generally evident. Firstly, forthe majority of significant targets within the cell there exists atleast one cognate natural product ligand. Secondly, whilst somenatural product groups, e.g. polyketides and terpenoids, appear


to be most well represented, on the whole most structural classesof natural products have been shown in some way to interact andmodulate some function of the cell. Thus no specific cellular targetsappear resistant to modulation through interaction with naturalproducts, and no major group of natural products seem to existthat do not bind to or interact with cellular targets. It is difficult todetermine if the specific interactions that have been demonstratedbetween natural products and their cellular targets are the directconsequence of evolutionary selection or coincidence. Indeed, itis probably a combination of both. Nevertheless, as evidencedin this review, natural products do offer a privileged startingpoint in the search for highly specific and potent modulators ofbiological function. Not only are natural products of value inpharmaceutical drug development, but they are also increasinglyproving indispensable in chemical genomics efforts, aimed atprobing the individual functions of all gene products in thewhole cell. In combination with increasingly sophisticated high-throughput screening methods, new natural products possessingnovel biological activities continue to be uncovered at an increas-ing rate, even from traditional sources. Furthermore, it is predictedthat genome mining, novel heterologous expression systems andallied technologies will further accelerate this discovery process,providing access to the vast majority of natural product structuraldiversity that remains largely untapped in the environment. Nodoubt these emerging technologies will have a profound impacton the application of natural products in medical and biochemicalresearch.

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