Nature as Source of Medicines

3.06 Nature as Source of Medicines; Novel Drugs
from Nature; Screening for Antitumor Activity
Gordon M. Cragg and David J. Newman, NCI-Frederick, Frederick, MD, USA
ª 2010 Elsevier Ltd. All rights reserved.
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References
Introduction
The Role of Traditional Medicine in Drug Discovery
Nature’s Continuing Role in Drug Discovery
Why Nature?
The Origin of Natural Products
Classical Natural Sources: Untapped Potential
Microorganisms: Unexplored Potential
Improved culturing procedures
Genomic mining and the metagenome
Extremophiles
Endophytes
Marine microbes
Microbial symbionts
Biomass Acquisition and International Collaboration
Multidisciplinary Collaboration – An Essential Factor
Combinatorial biosynthesis
Total synthesis
Diversity-oriented synthesis, privileged structures, and combinatorial chemistry
Nature as a Source of Molecular and Mechanistic Diversity in Cancer Chemotherapy
Antitumor Screening
Molecular target assays
Cell-based assays
In vivo assays
Tubulin Interactive Agents
Inhibitors of Topoisomerases I and II
Inhibitors of Histone Deacetylases
Protein Kinase Inhibitors
Flavopiridol
Bryostatins
Adenine derivatives
Indigo and the indirubins
Protein Folds and Inhibitors of Kinases and Phosphatases
Inhibitors of Heat Shock Protein 90
Ansamycins: geldanamycin derivatives
Non-ansamycin inhibitors
Proteasome Inhibitors
DNA Interactive Agents (Non-Topoisomerases I and II Inhibitors)
Caspase Activation and Apoptosis Induction
Hypoxia Inducible Factor
Miscellaneous Target Inhibitors
Summary and Future Prospects
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136 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
3.06.1 Introduction
Throughout the ages humans have relied on nature to cater for their basic needs, not the least of which are
medicines for the treatment of a wide spectrum of diseases. Plants, in particular, have formed the basis of
sophisticated traditional medicine systems, with the earliest records, dating from around 2600 BCE, documenting the uses of approximately 1000 plant-derived substances in Mesopotamia, many of which are still used
today for the treatment of ailments ranging from coughs and colds to parasitic infections and inflammation.1
Egyptian medicine dates from about 2900 BC, but the best-known record is the ‘Ebers Papyrus’ dating from
1500 BCE, documenting over 700 drugs, mostly of plant origin. The Chinese materia medica has been
extensively documented over the centuries,2 with the first record dating from about 1100 BCE (Wu Shi Er
Bing Fang, containing 52 prescriptions), followed by works such as the Shennong Herbal (100 BCE; 365
drugs) and the Tang Herbal (CE 659; 850 drugs). Likewise, documentation of the Indian ayurvedic system dates
from before 1000 BCE (Charaka; Sushruta and Samhitas with 341 and 516 drugs, respectively).3,4
The Greeks and Romans contributed substantially to the rational development of the use of herbal drugs in
the ancient Western world. Dioscorides, a Greek physician (CE 100), accurately recorded the collection,
storage, and use of medicinal herbs during his travels with Roman armies throughout the then ‘known world’,
while Galen (CE 130–200), a practitioner and teacher of pharmacy and medicine in Rome, was well known for
his complex prescriptions and formulae used in compounding drugs. The Arabs, however, preserved much of
the Greco-Roman expertise during the Dark and Middle Ages (fifth to twelfth centuries), and expanded it to
include the use of their own resources, together with Chinese and Indian herbs unknown to the Greco-Roman
world. A comprehensive review of the history of medicine may be found on the website of the National Library
of Medicine (NLM), US National Institutes of Health (NIH), at http://www.nlm.nih.gov/hmd/index.html.
3.06.1.1
The Role of Traditional Medicine in Drug Discovery
Plant-based systems continue to play an essential role in health care, and their use by different cultures has
been extensively documented.5,6 The World Health Organization (WHO) has estimated that approximately
65% of the world’s population rely mainly on plant-derived traditional medicines for their primary health care,
while plant products also play an important role in the health care systems of the remaining population, mainly
residing in developed countries.7 A survey of plant-derived pure compounds used as drugs in countries hosting
WHO-Traditional Medicine Centers indicated that, of 122 compounds identified, 80% were used for the same
or related ethnomedical purposes and were derived from only 94 plant species.7,8 Probably the best example of
ethnomedicine’s role in guiding drug discovery and development is that of the antimalarial drugs, particularly
quinine and artemisinin. Malaria remains one of the greatest health challenges confronting humankind, and the
search for better drugs, both in terms of efficacy and cost, is a global health imperative. The isolation of
the antimalarial drug, quinine, from the bark of Cinchona species (e.g., Cinchona officinalis), was reported in 1820
by the French pharmacists, Caventou and Pelletier.9 The bark had long been used by indigenous groups in the
Amazon region for the treatment of fevers and was first introduced into Europe in the early 1600s for
the treatment of malaria. Quinine formed the basis for the synthesis of the commonly used antimalarial
drugs, chloroquine and mefloquine, which largely replaced quinine in the mid-twentieth century, but with
the emergence of resistance to both these drugs in many tropical regions, another plant long used in the
treatment of fevers in traditional Chinese medicine (TCM), Artemisia annua (Quinhaosu), gained prominence.10
The discovery of artemisinin (1; Figure 1) by Chinese scientists in 1971 provided an exciting new natural
product lead compound,11 and artemisinin analogues are now used for the treatment of malaria in many
countries.12 Many analogues of artemisinin have been prepared in attempts to improve its activity and utility,12
and two of the most promising of these are the totally synthetic analogue OZ277 (2; Figure 1),13 and the
dimeric analogue (3; Figure 1). Single doses of the latter compound were shown to cure malaria-infected mice,
while corresponding treatments with artemisinin were much less effective.14
Although plants have a long history of use in the treatment of cancer,15 many of the claims for the efficacy of
such treatment should be viewed with some skepticism because cancer, as a specific disease entity, is likely to be
poorly defined in terms of folklore and traditional medicine.16
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137
Figure 1 Natural antimalarial agents and analogues.
3.06.2 Nature’s Continuing Role in Drug Discovery
The authors have reviewed the continuing valuable contributions of nature as a source of potential chemotherapeutic agents.17 In our paper, we analyzed the sources of new drugs over the period from January 1981 to June
2006, and classified these compounds as N (an unmodified natural product); ND (a modified natural product);
S (a synthetic compound with no natural product conception); S ; S /NM (a synthetic compound with a natural
product pharmacophore; S /NM indicating competitive inhibition); and S/NM (a synthetic compound showing competitive inhibition of the natural product substrate). This analysis indicated that while 66% of the 974
small molecules, new chemical entities (NCEs), are formally synthetic, 17% correspond to synthetic molecules
containing pharmacophores derived directly from natural products classified as S and S/NM. Furthermore,
12% are actually modeled on a natural product inhibitor of the molecular target of interest, or mimic
(i.e., competitively inhibit) the endogenous substrate of the active site, such as ATP (S/NM). Thus, only
37% of the 974 NCEs can be classified as truly synthetic (i.e., devoid of natural inspiration) in origin (S)
(Figure 2). Considering disease categories, close to 70% of anti-infectives (antibacterial, antifungal,
antiparasitic, and antiviral) are naturally derived or inspired (N; ND; S ; S /NM; S/NM), while in the cancer
treatment area 77.8% are in this category, with the figure being 63% if the S/NM category is excluded.
3.06.3 Why Nature?
3.06.3.1
The Origin of Natural Products
While the contributions of natural secondary metabolites to modern medicine are abundantly clear, the reasons
for the production of these inherently biologically active compounds by organisms are still debated. Initially
they were regarded as waste products, but it seems reasonable to assume that, in many instances, the production
S*
5%
N
6%
S*/NM
12%
ND
28%
S/NM
12%
S
37%
Figure 2 Small molecule new chemical entities, January 1981 to June 2006, by source (N ¼ 974).
138 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Figure 3 Secondary metabolites in chemical defense and quorum sensing.
of these complex and often toxic chemicals has evolved over aeons as a means of chemical defense by essentially
stationary organisms such as plants and many marine invertebrates, against predation and consumption
(e.g., herbivory). For instance, pupae of the coccinellid beetle, Epilachna borealis, appear to exert a chemical
defensive mechanism against predators through the secretion of droplets from their glandular hairs containing a
library of hundreds of large-ring (up to 98 members) macrocyclic polyamines, with the simplest example
having the generic formula shown (4; Figure 3).18 These libraries are built up from three simple
2-hydroxyethylamino-alkanoic acid precursors and are clear evidence that combinatorial chemistry has been
pioneered and widely used in nature for the synthesis of biologically active compound libraries.
Microorganisms are reported to produce and excrete antimicrobial toxins as a means of killing sensitive
strains of the same or related species.19 This is similar to allelopathy in which plants release toxic compounds in
order to suppress the growth of neighboring plants.20,21 Bacteria also control their density of population growth
and the so-called biofilm formation through a cell-to-cell signaling mechanism known as quorum sensing
involving the excretion of quorum-sensing compounds. The best studied of these are the acyl homoserine
lactones (AHLs), with the compounds from Vibrio fischeri being examples; these include N-3-oxohexanoyll-homoserine lactone (5; Figure 3) and a previously unidentified furanone boronate diester that appears to be a
universal signal (6; Figure 3), and they signal the activation of genes promoting virulence, spore formation,
biofilm formation, and other phenomena.22,23
Natural products may be used for purposes of both predation and defense. Thus, species of the cone snail
genus, Conus, stun their prey before capturing by the injection of venom composed of combinatorial libraries of
several hundred peptides24 and the venom may also be used for defense against predators. One component of
this mixture has been developed as Ziconotide, a nonnarcotic analgesic that is currently marketed as Prialt.25
3.06.3.2
Classical Natural Sources: Untapped Potential
Despite the intensive investigation of terrestrial flora, it is estimated that only 6% of the approximately 300 000
species (some estimates are as high as 500 000 species) of higher plants have been systematically investigated,
pharmacologically, and only some 15% phytochemically.8,26,27 The potential of the marine environment as a
source of novel drugs remains virtually unexplored,28,29 and until recently, the investigation of the marine
environment had largely been restricted to tropical and subtropical regions; however, the exploration is being
expanded to colder regions. The isolation of novel pyrido-pyrrolo-pyrimidine derivatives, the variolins (e.g.,
variolin B: 7; Figure 4), from the Antarctic sponge, Kirkpatrickia variolosa, was reported in 1994,30,31 followed by
their total synthesis in 2003,32 while the isolation of a cytotoxic macrolide palmerolide A (8; Figure 4) from an
Antarctic tunicate has recently been reported,33 with total synthesis leading to a revision of the original
structure.34
The selective and reproducible production of bioactive compounds has been induced through exposure of
the roots of hydroponically grown plants to chemical elicitors,35 while feeding of seedlings with derivatives of
selected biosynthetic precursors can lead to the production of nonnatural analogues of the natural metabolites.
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Figure 4 Natural products from novel sources.
Thus, the production of nonnatural terpene indole alkaloids related to the vinca alkaloids has been reported
through the feeding of seedlings of Catharanthus roseus with various tryptamine analogues.36
3.06.3.3
Microorganisms: Unexplored Potential
Until recently, the inability to cultivate most naturally occurring microorganisms has severely limited the study
of natural microbial ecosystems, and it has been estimated that much less than 1% of microorganisms seen
microscopically have been cultivated. Yet, despite this limitation, the number of highly effective microbially
derived chemotherapeutic agents discovered and developed thus far has been highly impressive. Given the
observation that ‘‘a handful of soil contains billions of microbial organisms,’’37 and the assertion that
‘‘the workings of the biosphere depend absolutely on the activities of the microbial world,’’38 the microbial
universe clearly presents a vast untapped resource for drug discovery. In addition, substantial advances in the
understanding of the gene clusters encoding multimodular enzymes involved in the biosynthesis of a multitude
of microbial secondary metabolites, such as polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs), has enabled the sequencing and detailed analysis of the genomes of long-studied microbes such
as Streptomyces avermitilis. These studies have revealed the presence of additional PKS and NRPS clusters
resulting in the discovery of novel secondary metabolites not detected in standard fermentation and isolation
processes.39 Such genome mining has been used in the discovery of a novel peptide, coelichelin, from the soil
bacterium, Streptomyces coelicolor,40 and this concept is further expanded in the discussion in Section 3.06.3.3.2.
3.06.3.3.1
Improved culturing procedures
Recent developments of procedures for cultivating and identifying microorganisms are aiding microbiologists
in their assessment of the earth’s full range of microbial diversity. For example, the use of ‘nutrient-sparse’
media simulating the original natural environment enables the massive parallel cultivation of gel-encapsulated
single cells (gel microdroplets (GMDs)) derived from microbes separated from environmental samples
(seawater and soil).41 This has permitted ‘‘the simultaneous and relatively noncompetitive growth of both
slow- and fast-growing microorganisms,’’ thereby preventing the overgrowth by fast-growing ‘microbial weeds’,
and leading to the identification of previously undetected species (using 16S rRNA gene sequencing), as well as
the culturing and scale-up cultivation of previously uncultivated microbes. Coupled with the recent report of
the sequencing of the marine actinomycete, Salinospora tropica, where it was found that approximately 10% of
the genome coded for potential secondary metabolites,42 and the recent paper on cultivation of Gram-positive
marine microbes,43 the potential for discovery of novel agents is immense.
3.06.3.3.2
Genomic mining and the metagenome
Despite improvement in culturing techniques, greater than 99% of microscopically observed microbes still defy
culture. Extraction of nucleic acids (the metagenome) from environmental samples, however, permits the identification of uncultured microorganisms through the isolation and sequencing of ribosomal RNA or rDNA (genes
encoding for rRNA). Samples from soils and seawater are currently being investigated,44,45 and whole-genome
shotgun sequencing of environmental-pooled DNA obtained from water samples collected in the Sargasso Sea off
140 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
the coast of Bermuda by the Venter group, indicated the presence of at least 1800 genomic species, which included
148 previously unknown bacterial phylotypes.45 Venter and his coworkers46 are also examining microbial communities in water samples collected by the Sorcerer II Global Ocean Sampling (GOS) expedition, and their data
predict more than six million proteins, nearly twice the number of proteins present in current databases, with some
of the predicted proteins bearing no similarity to any currently known proteins, and therefore representing new
families. These methods may be applied to other habitats, such as the microflora of insects47 and marine animals,48
and there is a recent report of an ‘Air Genome Project’ being launched in Manhattan where samples of air are being
analyzed for the content of DNA from bacteria, fungi, and other microbes.49 The cloning and understanding of the
novel genes discovered through these processes, and the heterologous expression of gene clusters encoding
the enzymes involved in biosynthetic pathways in viable host organisms, such as Escherichia coli, should permit
the production of novel metabolites produced from as yet uncultured microbes.
The enormous unexplored potential of microbial diversity and the strategy of genome mining were briefly
mentioned in the introduction of Section 3.06.3.3. As a result of the rapid evolution of genomic sequencing and
the ever-dropping costs of performing such studies, the amount of genomic information is ever increasing,
resulting in the potential for the expression of previously unrecognized metabolites. A recent review discusses
the general aspects of genomics in natural product research.50 It has now become evident, initially through the
pioneering work of Hopwood, that the genome of the Streptomycetes and by extension, Actinomycetes in
general, contain large numbers of previously unrecognized secondary metabolite clusters. An excellent
example is the investigation of the genome of the well-known vancomycin producer, Amycolatopsis orientalis
(ATCC 43491), which resulted in the isolation of the novel antibiotic ECO-0501 (9; Figure 5), which was
only found by using the genomic sequence to predict the molecular weight, and then looking for the molecule
directly by high performance liquid chromatography–mass spectrometry (HPLC–MS). The compound had a
very similar biological profile to vancomycin but was masked by this compound.51 Many more examples of the
value of this type of investigation have been provided in two recent reviews,52,53 which give up-to-date
information on the manifold structures that can be found by expression of environmental DNA.
Figure 5 New compounds from genome mining.
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The presence of potential gene products controlling metabolite production has been predicted in a recently
reported genomic analysis of the fungus Aspergillus nidulans, which not only suggested the presence of clustered
secondary metabolite genes having the potential to generate up to 27 polyketides, 14 nonribosomal peptides
(NRPs), 1 terpene, and 2 indole alkaloids, but also identified the potential controller of expression of these
clusters.54 This was demonstrated by expressing terrequinone A (10; Figure 5), a compound not previously
reported from this species.54 Similar predictions can be made from Aspergillus fumigatus and Aspergillus oryzae as a
result of the analysis of the potential number of secondary metabolite clusters in these fungi.54 A recent review
expands the discussion on control of secondary metabolites in fungi.55
Even the myxobacteria have now yielded to genomic analyses, and the identification and utilization of ChiR,
the gene-controlling production of chivosazol (11; Figure 5), an extremely potent eukaryotic antibiotic, has
been reported.56 This paper also deals with the major problem in secondary metabolite expression, whether in
homologous or heterologous hosts, which is the identification and application of the transcriptional control
mechanisms involved.
3.06.3.3.3
Extremophiles
Extremophilic microbes (extremophiles) abound in extreme habitats. These include acidophiles (acidic sulfurous hot springs), alkalophiles (alkaline lakes), halophiles (salt lakes), piezo (baro-) and (hyper)thermophiles
(deep-sea vents),57–61 and psychrophiles (Arctic and Antarctic waters, alpine lakes).62 Thus far, investigations
have centered on the isolation of thermophilic and hyperthermophilic enzymes (extremozymes),63–67 but there
is little doubt that these extreme environments will also yield novel bioactive chemotypes. Abandoned
mine-waste disposal sites have yielded unusual acidophiles, which thrive in the acidic, metal-rich waters,
polluted environments that are generally toxic to most prokaryotic and eukaryotic organisms.68 The novel
sesquiterpenoid and polyketide-terpenoid metabolites, berkeleydione (12; Figure 6) and berkeleytrione
(13; Figure 6) showing activity against metalloproteinase-3 and caspase-1, activities relevant to cancer,
Huntington’s disease, and other diseases, have been isolated from Penicillium species found in the surface waters
of Berkeley Pit Lake in Montana.68–70
3.06.3.3.4
Endophytes
As indicated in Sections 3.06.1 and 3.06.3.2, plants have been relatively extensively studied as sources of
bioactive metabolites, but the endophytic microbes that reside in the tissues between living plant cells have
received little attention. Relationships between endophytes and their host plants may vary from symbiotic to
pathogenic, and studies have revealed an interesting realm of novel chemistry.71–73 A wide range of new
bioactive molecules have been discovered, including peptide antibiotics, the coronamycins, isolated from a
Streptomyces species associated with an epiphytic vine (Monastera species) found in the Peruvian Amazon,74 and
the cytotoxic aspochalasins I, J, and K (14–16, respectively; Figure 7), isolated from endophytes of plants from
the southwestern desert regions of the United States.75
Of particular significance has been the production of various important anticancer agents in small quantities
from endophytic fungi isolated from plants. Examples (Figure 7) are Taxol (17) from Taxomyces76 and many
Pestalotiopsis species,77 as well as camptothecin (18),78,79 podophyllotoxin, an epimer of the precursor to the
anticancer drug, etoposide (19),80,81 vinblastine (20),82 and vincristine (21)83,84 from endophytic fungi isolated
Figure 6 New compounds from extreme environments.
142 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Figure 7 Natural products from endophytes.
from the original source plants. It has been demonstrated that these compounds are not artifacts, and so the
identification of the gene/gene product controlling metabolite production by these microbes could provide an
entry into greatly increased production of key bioactive natural products.
3.06.3.3.5
Marine microbes
Deep-ocean sediments are proving to be a valuable source of new actinomycete bacteria that are unique to the
marine environment.42 Use of a combination of culture and phylogenetic approaches has led to the description
of the first truly marine actinomycete genus named Salinospora,43,85 and its members are proving to be
ubiquitous, being found in concentrations of up to 104 per milliliter in sediments on tropical ocean bottoms
and in more shallow waters, as well as appearing on the surfaces of numerous marine plants and animals. On
culturing using the appropriate selective isolation techniques, significant antibiotic and cytotoxic activity has
been observed, and has resulted in the isolation of a potent cytotoxin, salinosporamide A (22; Figure 8), a very
potent proteasome inhibitor (IC50 ¼ 1.3 nmol l1),86 currently in Phase I clinical trials. More recently, the
isolation and cultivation of another new actinomycete genus, named Marinispora, has been reported, and novel
macrolides called marinomycins have been isolated. Marinomycins A (23; Figure 8) to D show potent activity
against drug-resistant bacterial pathogens and some melanomas.87 Recent publications by the Fenical group on
the novel and diverse chemistry of these new microbial genera include the isolation of potential chemopreventive agents, saliniketals A and B from Salinispora arenicola,88 while two new cyclic peptides,
thalassospiramides A and B, possessing immunosuppressive activity have been isolated from a new member
of the marine -proteobacterium Thalassospira.89
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Figure 8 Examples of novel microbial natural products.
3.06.3.3.6
Microbial symbionts
Evidence is mounting indicating that many bioactive compounds isolated from various macroorganisms are
actually metabolites synthesized by symbiotic bacteria.90 These include the anticancer compounds, the
maytansanoids (24; Figure 8), originally isolated from several plant genera of the Celastraceae family,91 and
the pederins (25; Figure 8), isolated from beetles of genera Paederus and Paederidus as well as from several
marine sponges.92–94 In addition, a range of antitumor agents isolated from marine organisms closely resemble
bacterial metabolites.28
An interesting example of a complex symbiotic–pathogenic relationship involving a bacterium–fungus–
plant interaction has been discovered in the case of rice seedling blight. The toxic metabolite, rhizoxin
(26; Figure 8), originally isolated from the contaminating Rhizopus fungus, has actually been found to be
produced by an endosymbiotic Burkholderia bacterial species.95 Rhizoxin exhibits potent antitumor activity, but
its further development as an anticancer drug has been precluded by toxicity problems. Thus, in addition to
144 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
offering potentially new avenues for pest control, this unexpected finding has enabled the isolation of rhizoxin
as well as rhizoxin analogues through the cultivation of the bacterium independently of the fungal host. This
may have significant implications in the development of agents with improved pharmacological properties.
3.06.3.4
Biomass Acquisition and International Collaboration
The acquisition of biomass has changed significantly from the days when drug companies and others routinely
collected organisms with little thought of ownership by, or benefit sharing with, the source country. Today,
thanks to the Convention on Biodiversity (CBD) and similar documents and agreements such as the US
National Cancer Institute’s (NCI) Letter of Collection (LOC: http://ttc.nci.nih.gov/forms/), all ethical biomass
acquisitions now include provisions for the source country to be compensated in some way for the use of its
biomass. It should be noted that the LOC predated the CBD by 3 years; its terms, as a minimum, must be
adhered to by any investigator who has his or her collections funded by the NCI/NIH. Such recompense to
the source country is best provided through formal agreements with government organizations and collectors in
the source country, with such agreements providing not only for reimbursement of collecting expenses but also
for further benefits (often in the form of milestone and/or royalty payments) in the event that a drug is
developed from a collected sample. Agreements often include terms related to the training of source country
scientists and transfer of technologies involved in the early drug discovery process. Recognition of the role
played by indigenous peoples through the stewardship of resources in their region and/or the sharing of their
ethnopharmacological information in guiding the selection of materials for collection is important in determining the distribution of such benefits. There have been sample legal agreements96 and discussions as to methods
used by various groups published in the last few years.97–99
All samples collected, irrespective of type of source, must if at all possible be fully identified to genus and
species. Such identification is usually possible for all plant species, but it is not always possible for microbes and
marine organisms. Voucher specimens should be provided to an appropriate depository in the source country as
well as to a similar operation in the home country of the collector. The use of traditional knowledge in the drug
discovery process has been briefly discussed in Section 3.06.1.1. The selection of plant samples using such
knowledge, the ethnobotanical/ethnopharmacological approach, usually involves the selection of plants that
have a documented (written or oral) use by traditional healers and has the advantage of tapping into the
empirical knowledge developed over centuries of use by large numbers of people. In addition, the bioactive
constituents may be considered as having had a form of continuing clinical trial in man. The benefits of this
approach have been extolled in several relatively recent articles,100–102 but a weakness of the ethnobotanical
approach has always been that it is slow, requiring careful interviewing of traditional healers by skilled
scientists, including ethnobotanists, anthropologists, trained physicians, and pharmacologists. In addition, the
quoted medicinal activity in the collected plant(s) may not be detectable, given the particular assays used by the
screening laboratory. The highest possibility of success of the ethnobotanical approaches lies in studies related
to overt diseases/conditions such as parasitic infections, fungal sores, and contraception and conception to
name a few. In such cases, there are adequate controls, even on the same patient. Where there does not yet
appear to be any successful relationship is in diseases such as cancer and AIDS-related conditions, where
extensive testing of the patient is required for an accurate diagnosis.16
3.06.3.5
Multidisciplinary Collaboration – An Essential Factor
The probability that a directly isolated natural product (e.g., adriamycin or taxanes in the antitumor area) will
be the drug used for the treatment of a given disease in the future is relatively low. In many instances, however,
these natural molecules can serve as lead compounds that can be optimized through the application of
methodologies such as combinatorial biosynthesis and/or combinatorial chemistry to give products suitable
for drug development. In addition, novel methods of total chemical syntheses of the natural molecules can yield
intermediates possessing equal or superior preclinical activity to that observed for the natural product, and that
can be optimized for drug development using medicinal or combinatorial chemistry approaches. Of course, all
these approaches require suitable biological assays for evaluation of the optimization products, and thus a truly
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145
multidisciplinary, collaborative approach is required for effective drug development. That these ideas are not
just pipe dreams can be seen in the following examples.
3.06.3.5.1
Combinatorial biosynthesis
The substantial advances made in the understanding of the role of multifunctional PKS enzymes in bacterial
aromatic polyketide biosynthesis have led to the identification of many such enzymes, together with their
encoding genes.103–106 The same applies to NRPSs responsible for the biosynthesis of NRPs.105 The rapid
developments in the analysis of microbial genomes have enabled the identification of a multitude of gene
clusters encoding for polyketides, NRPs, and hybrid polyketide–NRP metabolites, and have provided the tools
for engineering the biosynthesis of novel ‘nonnatural’ natural products through gene shuffling, domain
deletions, and mutations.105,107 Results of the application of these combinatorial biosynthetic techniques to
the production of novel analogues of anticancer agents, such as the anthracyclines, ansamitocins, epothilones,
enediynes, and aminocoumarins, have recently been reviewed by Shen et al.108
The efficient scale-up production of epothilone D exemplifies the power of this technique. Epothilone D,
the des-epoxy precursor of epothilone B, was the most active of the epothilone series isolated from the
myxobacterium, Sorangium cellulosum, and entered clinical trials as a potential anticancer agent but has now
been discontinued in favor of a congener, 9,10-didehydroepothilone D. The polyketide gene cluster producing
epothilone B has been isolated and sequenced from two S. cellulosum strains,109,110 and the epoxidation of
epothilone D to epothilone B has been shown to be due to the last gene in the cluster, epoK, encoding a
cytochrome P-450. Heterologous expression of the gene cluster minus the epoK in Myxococcus xanthus resulted in
large-scale production of crystalline epothilone D.111
3.06.3.5.2
Total synthesis
The total synthesis of complex natural products has long posed challenges to the top synthetic chemistry
groups worldwide and has led to dramatic advances in the field of organic chemistry.112 As eloquently stated by
Nicolaou and his coauthors, ‘‘Today, natural product total synthesis is associated with prudent and tasteful
selection of challenging and preferably biologically important target molecules; the discovery and invention of
new synthetic strategies and technologies; and explorations in chemical biology through molecular design and
mechanistic studies. Future strides in the field are likely to be aided by advances in the isolation and
characterization of novel molecular targets from nature, the availability of new reagents and synthetic methods,
and information and automation technologies.’’112
In some instances, as noted in Section 3.06.3.2 regarding the cytotoxic macrolide palmerolide A (8; Figure 4),
total synthesis has led to a revision of the original published structure;34 another notable example is that of the
marine-derived antitumor compound, diazonamide A (27; Figure 9).113 Significant strides have been made in
the synthesis and structural modification of drugs that are difficult to isolate in sufficient quantities for
development. Adequate supply can be a serious limiting factor in the preclinical and clinical development of
some naturally derived drugs, and the focus of many top synthetic groups on devising economically feasible
synthetic strategies is a very welcome development for both clinicians conducting clinical trials and patient
populations. An excellent example is the marine-derived anticancer agent discodermolide (28; Figure 9),
where total synthesis provided sufficient quantities for thorough clinical trials, but unfortunately, these have
now been terminated due to the lack of objective responses and toxicity.114,115
The process of total synthesis can often lead to the identification of a substructural portion of the molecule
bearing the essential features necessary for activity (the pharmacophore), and, in some instances, this has resulted
in the synthesis of simpler analogues having similar or better activity than the natural product itself. One of the
most notable examples is that of the marine-derived antitumor agent, halichondrin B (29; Figure 9), where total
synthetic studies revealed that the right-hand half of the molecule retained all or most of the potency of the
parent compound, and the analogue, E7389 (Eribulin) (30; Figure 9), is currently in Phase III clinical trials.116
In some instances, clinical trials of the original natural product may fail, but totally synthetic analogues
continue to be developed. Thus, while clinical trials of the marine-derived anticancer agents, dolastatin 10 and
dolastatin 15, have been terminated, the synthetic analogues of dolastatin 10 (31; Figure 9), TZT-1027
(auristatin PE or soblidotin) and ILX651 (synthadotin or tasidotin) based on dolastatin 15, are in Phase II
clinical trials.117
146 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Figure 9 Products of total synthesis.
3.06.3.5.3 Diversity-oriented synthesis, privileged structures, and combinatorial
chemistry
While there are claims that combinatorial chemistry is generating new leads,118 the declining numbers of new
NCEs119 indicate that the use of de novo combinatorial chemistry approaches to drug discovery over the past
decade have been disappointing, with some of the earlier libraries being described as ‘‘poorly designed,
impractically large, and structurally simplistic.’’118 As stated in this article, ‘‘an initial emphasis on creating
mixtures of very large numbers of compounds has largely given way in industry to a more measured approach
based on arrays of fewer, well-characterized compounds’’ with ‘‘a particularly strong move toward the synthesis
of complex natural product-like compounds – molecules that bear a close structural resemblance to approved
natural product-based drugs.’’ The importance of the use of natural product-like scaffolds for generating
meaningful combinatorial libraries has been further emphasized in a recent article entitled ‘‘Rescuing
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
147
Combichem diversity-oriented synthesis (DOS) aims to pick up where traditional combinatorial chemistry left
off.’’120 In this article it is stated that ‘‘the natural product-like compounds produced in DOS have a much better
shot at interacting with the desired molecular targets and exhibiting interesting biological activity.’’
The synthesis of natural product-like libraries is exemplified by the work of the Schreiber group who have
combined the simultaneous reaction of maximal combinations of sets of natural product-like core structures
(latent intermediates) with peripheral groups (skeletal information elements) in the synthesis of libraries of over
1000 compounds bearing significant structural and chiral diversity.121,122 Through detailed analyses of active
natural product skeletons, relatively simple key precursor molecules may be identified, which form the building
blocks for use in combinatorial synthetic schemes, thereby enabling structure–activity relationships (SARs) to
be probed. The generation of small libraries, built through the solid-phase synthesis of molecules such as
epothilone A (32; Figure 10), dysidiolide (33; Figure 10), galanthamine (34; Figure 10), and psammaplin
(35; Figure 10), has been reviewed.123–129
Use of an active natural product as the central scaffold can also be applied to the generation of large numbers
of analogues for structure–activity studies, the so-called parallel synthetic approach, and is exemplified by the
syntheses around the sarcodictyin (36; Figure 10) scaffold.130 The importance of natural products as leads for
combinatorial synthesis is further illustrated by the concept of ‘‘privileged structures,’’131 and this approach has
been successfully developed by several groups.129,132–134 In one such case, a search of the literature yielded
nearly 4000 2,2-dimethyl-2H-benzopyran moieties (37; Figure 10), with another 8000 structures identified
Figure 10 Diversity-oriented and parallel synthesis and privileged structures.
148 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
through the inclusion of a slight modification of the search. Application of solid-phase synthetic methods led to
the identification and subsequent optimization of benzopyrans with a cyanostilbene substitution
(38; Figure 10) that are effective against vancomycin-resistance bacteria.132–134
3.06.4 Nature as a Source of Molecular and Mechanistic Diversity in Cancer
Chemotherapy
A list of all anticancer drugs currently in clinical use and classified according to their source is given in Table 1.
Readers are referred to the authors’ 2007 review17 for detailed references. In the sections below, after briefly
reviewing the various methodologies used in antitumor screening, we have provided an overview of the
chemotherapeutic agents currently in clinical use or development for the treatment of cancer. Our discussion
of these agents is divided into subsections based on their mechanisms of action. The discussions are brief since
other chapters in this series will be dealing in more detail with many of the agents mentioned. Information on
ongoing clinical trials may be found at http://www.clinicaltrials.gov/, and readers are referred to this site for
details.
Table 1 All anticancer drugs (1940s to December 2007) (organized alphabetically by generic name
within source)
Generic name
Year introduced
Referencea
131I-chTNT
H-101
Aldesleukin
Alemtuzumab
Bevacizumab
Celmoleukin
Cetuximab
Denileukin diftitox
Interferon alfa2a
Interferon alfa2b
Interferon, gamma-1a
Interleukin-2
Mobenakin
Nimotuzumab
Panitumumab
Pegaspargase
Rexin-gb
Rituximab
Tasonermin
Teceleukin
Tositumomab
Trastuzumab
Aclarubicin
Actinomycin D
Angiotensin II
Arglabin
Asparaginase
Bleomycin
Carzinophilin
Chromomycin A3
Daunomycin
Doxorubicin
Leucovorin
Masoprocol
Mithramycin
2007
2005
1992
2001
2004
1992
2003
1999
1986
1986
1992
1989
1999
2006
2006
1994
2007
1997
1999
1992
2003
1998
1981
1964
1994
1999
1969
1966
1954
1961
1967
1966
1950
1992
1961
I 393351
DNP 19
ARMC 25
DNP 15
ARMC 40
DNP 06
ARMC 39
ARMC 35
I 204503
I 165805
ARMC 28
ARMC 25
ARMC 35
DNP 20
DNP 20
ARMC 30
I 34631
DNP 11
ARMC 35
DNP 06
ARMC 39
DNP 12
I 090013
FDA
ARMC 30
ARMC 35
FDA
FDA
Japan Antibiotics
Japan Antibiotics
FDA
FDA
FDA
ARMC 28
FDA
Page
46
314
38
450
102
346
338
332
314
345
29
28
306
25
349
102
364
35
296
335
333
Source
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
N
N
N
N
N
N
N
N
N
N
N
N
N
(Continued )
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Table 1
(Continued)
Generic name
Year introduced
Referencea
Mitomycin C
Neocarzinostatin
Paclitaxel
Palictaxel nanoparticlesc
Paclitaxel nanoparticlesd
Pentostatin
Peplomycin
Sarkomycin
Solamargine (aka BEC)
Trabectedin
Streptozocin
Testosterone
Vinblastine
Vincristine
Kunecatechins
Sinecatechins
Alitretinoin
Amrubicin hcl
Belotecan hydrocholoride
Calusterone
Cladribine
Cytarabine ocfosfate
Dexamethasone
Docetaxel
Dromostanolone
Elliptinium acetate
Epirubicin HCI
Estramustine
Ethinyl estradiol
Etoposide
Exemestane
Fluoxymesterone
Formestane
Fosfestrol
Fulvestrant
Gemtuzumab ozogamicin
Goserelin acetate
Hexyl aminolevulinate
Histrelin
Hydroxyprogesterone
Idarubicin hydrochloride
Irinotecan hydrochloride
Ixabepilone
Leuprolide
Medroxyprogesterone acetate
Megesterol acetate
Methylprednisolone
Methyltestosterone
Miltefosine
Mitobronitol
Nadrolone phenylpropionate
Norethindrone acetate
Pirarubicin
Prednisolone
Prednisone
Temsirolimus
Teniposide
Testolactone
1956
1976
1993
2005
2007
1992
1981
1954
1987
2007
Pre-1977
Pre-1970
1965
1963
2006
2007
1999
2002
2004
1973
1993
1993
1958
1995
1961
1983
1984
1980
Pre-1970
1980
1999
Pre-1970
1993
Pre-1977
2002
2000
1987
2004
2004
Pre-1970
1990
1994
2007
1984
1958
1971
1955
1974
1993
1979
1959
Pre-1977
1988
Pre-1977
Pre-1970
2007
1967
1969
FDA
Japan Antibiotics
ARMC 29
DNP 19
I 422122
ARMC 28
I 090889
FDA
DNP 03
I 139221
FDA
FDA
DNP 20
I 283701
ARMC 35
ARMC 38
ARMC 40
FDA
ARMC 29
ARMC 29
FDA
ARMC 31
FDA
I 091123
ARMC 20
FDA
Page
342
45
334
25
24
333
349
449
335
335
341
318
FDA
DNP 13
46
ARMC 29
337
ARMC 38
DNP 14
ARMC 23
I 300211
I 109865
357
23
336
ARMC 26
ARMC 30
I 293356
ARMC 20
FDA
FDA
FDA
FDA
ARMC 29
FDA
FDA
303
301
ARMC 24
309
218793
FDA
FDA
319
340
Source
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NBe
NBe
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
(Continued )
149
150 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Table 1
(Continued)
Generic name
Year introduced
Referencea
Page
Source
Topotecan hcl
Triamcinolone
Triptorelin
Valrubicin
Vapreotide acetate
Vindesine
Vinorelbine
Zinostatin stimalamer
Amsacrine
Arsenic trioxide
Bisantrene hydrochloride
Busulfan
Carboplatin
Carmustine (BCNU)
Chlorambucil
Chlortrianisene
Cis-diamminedichloroplatinum
Cyclophosphamide
Dacarbazine
Diethylstilbestrol
Flutamide
Fotemustine
Heptaplatin /SK-2053R
Hexamethylmelamine
Hydroxyurea
Ifosfamide
Lenalidomide
Levamisole
Lobaplatin
Lomustine (CCNU)
Lonidamine
Mechlorethanamine
Melphalan
Mitotane
Nedaplatin
Nilutamide
Nimustine hydrochloride
Oxaliplatin
Pamidronate
Pipobroman
Porfimer sodium
Procarbazine
Ranimustine
Razoxane
Semustine (MCCNU)
Sobuzoxane
Sorafenib mesylate
Thiotepa
Triethylenemelamine
Zoledronic acid
Anastrozole
Bicalutamide
Bortezomib
Camostat mesylate
Dasatinib
Erlotinib hydrochloride
Fadrozole hcl
Gefitinib
1996
1958
1986
1999
2003
1979
1989
1994
1987
2000
1990
1954
1986
1977
1956
Pre-1981
1979
1957
1975
Pre-1970
1983
1989
1999
1979
1968
1976
2005
Pre-1981
1998
1976
1987
1958
1961
1970
1995
1987
Pre-1981
1996
1987
1966
1993
1969
1987
Pre-1977
Pre-1977
1994
2005
1959
Pre-1981
2000
1995
1995
2003
1985
2006
2004
1995
2002
ARMC 32
FDA
I 090485
ARMC 35
I 135014
FDA
ARMC 25
ARMC 30
ARMC 23
DNP 14
ARMC 26
FDA
ARMC 22
FDA
FDA
BOYD
FDA
FDA
FDA
320
ND
ND
ND
ND
ND
ND
ND
ND
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S/NM
S/NM
S/NM
S/NM
S/NM
S/NM
S/NM
S/NM
350
320
313
327
23
300
318
ARMC 19
ARMC 25
ARMC 35
FDA
FDA
FDA
DNP 19
Boyd
DNP 12
FDA
ARMC 23
FDA
FDA
FDA
ARMC 31
ARMC 23
Boyd
ARMC 32
ARMC 23
FDA
ARMC 29
FDA
ARMC 23
318
313
348
ARMC 30
DNP 19
FDA
Boyd
DNP 14
ARMC 31
ARMC 31
ARMC 39
ARMC 21
DNP 20
ARMC 40
ARMC 31
ARMC 38
310
45
45
35
337
347
338
313
326
343
341
24
338
338
345
325
27
454
342
358
(Continued )
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Table 1
151
(Continued)
Generic name
Year introduced
Referencea
Page
Source
Imatinib mesilate
Lapatinib ditosylate
Letrozole
Nafoxidine
Nilotinib hydrochloride
Sunitinib maleate
Tamoxifen
Toremifene
Aminoglutethimide
Azacytidine
Capecitabine
Carmofur
Clofarabine
Cytosine arabinoside
Decitabine
Doxifluridine
Enocitabine
Floxuridine
Fludarabine phosphate
Fluorouracil
Ftorafur
Gemcitabine hcl
Mercaptopurine
Methotrexate
Mitoxantrone HCI
Nelarabine
Thioguanine
Uracil mustard
Abarelix
Bexarotene
Pemetrexed
Raltitrexed
Tamibarotene
Temozolomide
Vorinostat
Bcg live
Hpv vaccine (Merck)
Hpv vaccine (GSK)
Melanoma theraccine
2001
2007
1996
Pre-1977
DNP 15
I 301036
ARMC 32
38
S/NM
S/NM
S/NM
S/NM
S/NM
S/NM
S/NM
S/NM
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S /NM
S /NM
S /NM
S /NM
S /NM
S /NM
S /NM
V
V
V
V
2006
1973
1989
1981
Pre-1977
1998
1981
2005
1969
2006
1987
1983
1971
1991
1962
1972
1995
1953
1954
1984
2005
1966
1966
2004
2000
2004
1996
2005
1999
2006
1990
2006
2007
2001
I 386178
DNP 20
FDA
ARMC 25 319
FDA
ARMC 34
FDA
DNP 19
FDA
DNP 20
ARMC 23
ARMC 19
FDA
ARMC 27
FDA
FDA
ARMC 31
FDA
FDA
ARMC 20
DNP 19
FDA
FDA
ARMC 40
DNP 14
ARMC 40
ARMC 32
DNP 19
ARMC 35
DNP 20
DNP 04
DNP 20
I 309201
DNP 15
311
27
319
44
27
332
318
327
344
321
45
446
23
463
315
45
350
27
104
26
38
a
Refer to Newman and Cragg17 for decoding the reference citations in this column.
No generic name; this is the trade name.
c
Abraxane (entirely different from below in particle source and approved in USA).
d
Nanoxel (entirely different from above in particle source and approved in India).
e
NB is a new classification (natural product/botanical); these agents are for genital warts but approved for sale with a
disease indication.
b
3.06.4.1
Antitumor Screening
As mentioned in Section 3.06.3.5, the successful development of effective new drugs requires suitable assays to
guide, not only the discovery of a bioactive lead but also the evaluation of products developed through
optimization of the lead. Thus, a given organism provides the investigator with a complex library of unique
bioactive constituents, analogous to the library of crude synthetic products initially produced by combinatorial
chemistry techniques; the two approaches can be seen as complementary to each other, with each providing
access to (initially) different lead structures. The task of the natural products researcher is to select those
compounds of pharmacological interest through bioassay-guided fractionation of the ‘natural combinatorial
152 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
libraries’ produced by the extraction of organisms, and then to collaborate in the optimization and development
of the lead natural product structure as discussed in Section 3.06.3.5 above. Fortunately, the means to do this
efficiently are now at hand.
3.06.4.1.1
Molecular target assays
Structural diversity is not the only reason why natural products are of interest to drug development. Natural
products frequently possess highly selective and specific biological activities based on mechanisms of action. A
striking illustration is the influence of natural products on many of the molecular processes operative in cell
cycle progression, and details may be found at the website of the Roscoff Biological Station (http://
www.sb-roscoff.fr), which covers diagrams originally published by Meijer135 on natural products and the cell
cycle, with a modified version shown in Figure 11.
In the early days of natural products research, new compounds were simply isolated at random, or at best by
the use of simple broad-based bioactivity screens based on antimicrobial or cytotoxic activities. Although these
screens did result in the isolation of many bioactive compounds,136 they were considered to be too nonspecific
for the next generation of drugs. Fortunately, a large number of robust and specific biochemical- and
genetics-based screens using transformed cells, a key regulatory intermediate in a biochemical or genetic
pathway, or a receptor–ligand interaction (often derived from the explosion in genomic information since the
middle 1990s), are now in routine use. These screens will permit the more precise detection of bioactive
compounds in the complex matrices that are natural product extracts.
One interesting feature of such screens has increased the attractiveness of natural products to the pharmaceutical industry. The screens themselves are all highly automated and high-throughput (upward of 50 000
Trabectedin
Nitrogen mustards
Nitrosoureas
Mitomycin C
Hydroxyurea
Cytarabine
Antifolates
5-Fluorouracil
6-Mercaptopurine
Wortmannin
Caffeine
Fumagillin,TNP-470
PRIMA-1, pifithrin α
UCN-01, SB-218078
Debromohymenialdisine
Isogranulatimide
Menadione (K3)
(R)-roscovitine (CYC202)
Paullones, indirubins
p53/MDM2
ATM/ATR
Chk1
Nucleotide excision
Chk2
Repair
Vinca alkaloids
PD0166285 Taxol/taxotere
Halichondrin
CDC25
HMGA
Spongistatin
FK317
S
CDK1 Wee1
Rhizoxin
Camptothecin
Topoisomerase i
Aurora
Cryptophycin
Pin1
Tubulin
Podophyllotoxin,doxorubicin Topoisomerase ii
Sarcodictyin
M Polymerization/
Etoposide, mitoxantrone
CDK2
Eleutherobin
depolymerization
Epothilones
Cdc7
(R)-Roscovitine (CYC 202)
Discodermolide
CDK4
Kinesin Eg5
Paullones, indirubins
Indibulin
ODC/SAMDC
Actin
G1
Dolastatin
GSK-3
Flavopiridol
Pin1
Combretastatin
AhR
Polyamine analogues
Monastrol Eribulin
MEK1/Erk - 1/2
G0
Cytochalasins
Raf
Paullones, indirubins
ROCK
Latrunculin A
Farnesyl
transferase
DF203
Scytophycins
Tyrosine kinases
PD98059, U0126
Dolastatin 11
Proteasome
PS-341
Jaspamide
Sorafenib*
Choline kinase
CT-2584
Y-27632
mTOR/FRAP
Rapamycin
Tipifarnib Gleevec
Bryostatin, PKC412
PKC
Lonafarnib iressa
HSP90
Geldanamycin, 17-AAG
erlotinib
Cytosolic phospholipase A2
ATK, MAFP
Histone deacetylase
Trichostatin, FK228
Phospholipase D
Hexadecylphosphocholine
Phosphatases
Okadaic acid, fostreicin, calyculin A
DNA synthesis
Plk1
G2
Figure 11 Natural products and the cell cycle. Modified from L. Meijer, Le cycle de division cellulaire et sa régulation.
Oncologie 2003, 5 (7–8), 311–326. Copyright Springer-Verlag 2003. Reprinted with permission.
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
153
assay points per day in a number of cases), and the resultant screening capacity at many companies is
significantly larger than the potential input from in-house chemical libraries. Since screening capacity is no
longer the rate-limiting step, some major pharmaceutical companies became very interested in screening
natural products (either as crude extracts or as prefractionated ‘peak libraries’) as a low-cost means of
discovering novel lead compounds. A good illustration is the discovery at Merck Research Laboratories of a
new antibiotic, through the testing of a library of 250 000 natural product extracts in a custom-designed assay
involving an engineered strain of Staphylococcus aureus incorporating the fatty acid synthase pathway enzyme,
FabF.137 Platensimycin is a selective FabF inhibitor and exhibits in vitro activity against several drug-resistant
bacteria. Such promise has also spawned small companies such as Merlion Pharmaceuticals in Singapore, which
has a library of many thousands of natural product extracts and a smaller number of pure natural products
derived from a variety of sources, which it exposes to validated drug targets provided by pharmaceutical
companies, with the goal of generating drug leads.138 Most of the screens used are proprietary and the published
information is rare, although general summaries of this approach have been published.139
High-throughput assays are becoming less expensive, and such assays are moving from the industrial or
industrial–academic consortium-based groups to academia in general, with specific expression systems being
employed as targets for natural product lead discovery.140 The application of new techniques, including new
fluorescent assays, NMR, affinity chromatography, and DNA microarrays, has led to significant advances in the
effectiveness of high-throughput screening.141,142
3.06.4.1.2
Cell-based assays
As mentioned in Section 3.06.4.1.1, the advent of new and robust high-throughput screens has had, and
continues to have, a major impact on natural products research in the pharmaceutical industry. While some
of the molecular target screens alluded to in Section 3.06.4.1.1 may involve use of transformed cells, the NCI’s
60 cell line cytotoxicity screen for antitumor agents represents a more traditional cell-based screen. It has been
described in detail,143 and although this is not a true receptor-based screen, it has now been developed into a
system whereby a large number of molecular targets within the cell lines may be identified by informatics
techniques, and refinements are continuing. Information as to the current status of the screens involved can be
obtained from the following URL: http://dtp.nci.nih.gov/. An assay based on differential susceptibility to
genetically modified yeast strains has been described144 and has led to many screens based upon genetically
modified yeasts, but at times, the low permeability of the unmodified yeast cell wall to chemical compounds has
been overlooked. Thus, data from such screens, particularly those designed with gene deletions, must be
carefully scrutinized since a large number are based upon hosts without a modified cell wall. In addition, there
are simple but robust assays that can be used by workers in academia who do not have access to, or may not
need, high-throughput screens. Examples are the brine shrimp and potato disc assays.145,146
3.06.4.1.3
In vivo assays
Once the bioactive component has been obtained in pure form, either as a novel structure or as a known
compound exhibiting previously unreported activity, then it must be assessed in a series of biological assays to
determine its efficacy, potency, toxicity, and pharmacokinetics. These assays will help to determine the priority
of the compound’s spectrum of activity within the portfolio of compounds that a group may be assessing as
suitable for advanced development as either drug candidates or leads thereto. Knowledge of its putative
mechanism of action (MOA) at this stage can also be a valuable discriminator in the prioritizing process.
The NCI uses the Hollow Fiber Assay147 as a relatively inexpensive in vivo prescreen to prioritize
compounds for testing in the more definitive human tumor xenograft models.148 Information on the in vivo
assays currently used by the NCI, including a detailed description of the protocol used in the Hollow Fiber
Assay, can be obtained from the following URL: http://dtp.nci.nih.gov/.
3.06.4.2
Tubulin Interactive Agents
The majority of the tubulin interactive agents (TIAs) in development through to 2003, from preclinical studies
up to clinical use, have been discussed in detail in a 2004 review by the authors,149 and also more recently
(2005) in the book Anticancer Agents from Natural Products.150 The TIAs covered in that volume include taxanes
154 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Figure 12 Tubulin interactive agents.
(Taxol, 17; Figure 7),151 epothilones (Epo A, 32; Figure 10),152 and discodermolide (28; Figure 9),153 which
act as promoters of polymerization of tubulin heterodimers to microtubules, leading to mitotic arrest through
suppression of dynamic changes in microtubule functions. Other chapters are devoted to combretastatins
(e.g., CA-4 phosphate, 39; Figure 12),154 vinca alkaloids (20, 21; Figure 7),155 maytansanoids
(24; Figure 8),91 dolastatins (e.g., dolastatin 10, 31; Figure 9),117 halichondrins (29; Figure 9),116 and hemiasterlins (e.g., hemiasterlin A, 40; Figure 12),156 which act through inhibition of tubulin heterodimer
polymerization. The coverage also includes agents derived from or synthetically modeled on those initial
structures in order to develop drug candidates with improved solubilities, pharmacodynamics, or metabolic
patterns, compared with the original natural products. Besides this review and the book chapters cited, the
interested reader should consult relevant chapters in this series, together with the references given therein, for a
discussion of the multiplicity of structures that have been developed from natural product lead compounds.
Another detailed discussion of the marine-derived TIAs mentioned above (discodermolide, dolastatins,
halichondrins, hemiasterlins) is presented in a review of natural products from marine invertebrates and
microbes as modulators of antitumor targets.157 Other agents discussed in this review include dictyostatin,
diazonamide (27; Figure 9), eleutherobin and laulimalide (41; Figure 12), which all act in a similar manner to
the taxanes.
While most TIAs act either as reversible inhibitors or promoters of tubulin heterodimer polymerization as
mentioned above, pironetin (42; Figure 12), derived from a Streptomyces species, is the only TIA identified so far
that acts through covalent binding to the -tubulin chain.158 The binding occurs at Lys352, an amino acid
located at the entrance of a small pocket in -tubulin that faces the -tubulin of the next dimer.159 No
derivatives have yet been reported as candidate leads.
3.06.4.3
Inhibitors of Topoisomerases I and II
In early 2004 these authors reviewed new developments in the field of topoisomerase inhibitors in a special
issue of the Journal of Natural Products honoring Drs. Monroe Wall and Mansukh Wani, the codiscoverers of
both Taxol and camptothecin.160 The history of camptothecin (18; Figure 7) is presented in this review, and
although the majority of new topoisomerase I inhibitors are based on the camptothecin pharmacophore,161 the
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155
Figure 13 Topoisomerase and HDAC inhibitors.
protein kinase inhibitor staurosporine (43; Figure 13) is also a topoisomerase inhibitor and various derivatives
of the basic staurosporine scaffold inhibit both topoisomerases I and II.162 The anthracyclines are another class
of important drugs that act via inhibition of topoisomerase II, with doxorubicin (44; Figure 13) being a prime
example of the many members of this class.163 It should be pointed out, however, that almost all of the clinically
useful compounds of this chemical class were developed as a result of their cytotoxic activities and without
prior knowledge of this MOA.163 Likewise, the clinically active podophyllotoxin derivative, etoposide
156 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
(19; Figure 7), was developed by the then Sandoz company through modification of epi-podophyllotoxin
without prior knowledge of the mechanism.164 It is interesting to note that podophyllotoxin acts as an inhibitor
of tubulin polymerization, whereas etoposide acts on topoisomerase II. Although etoposide is a commonly used
anticancer drug, acquired drug resistance and poor water solubility remain serious problems, and extensive
research is being devoted to the production of a new generation of clinical trial candidates.164
A subsequent paper has reviewed the anticancer activity of some new topoisomerase inhibitors, which
include 6 topoisomerase I, 12 topoisomerase II, and 6 dual topoisomerase inhibitors, many of which are
derivatives of natural products.165 A second paper166 has reported on an analogue, AK-37 (45; Figure 13), of
a marine-derived pyridoacridine, which stabilizes the topoisomerase I cleavable complex in a manner comparable to that of 9-nitro-camptothecin, which is currently in Phase III clinical trials for the treatment of pancreatic
cancer in combination with gemcitabine. For those interested in reading further, the wide variety of structures
and activities of pyridoacridines has been reviewed.167
3.06.4.4
Inhibitors of Histone Deacetylases
The role of histone deacetylases (HDACs) in the regulation of gene expression, oncogenic transformation,
cellular differentiation, and the promotion of angiogenesis is discussed by the authors157 and references cited
therein. Suffice it to say, the inhibition of HDAC activity can exert a significant role in suppression of the
neoplastic process.
HDAC inhibitors have been described as tripartite: an enzyme-binding group, frequently aromatic; a
hydrophobic spacer group; and an inhibitor group.168–170 Trichostatin A (TSA) (46; Figure 13) clearly
demonstrates such a system, with the structure mimicking the Lys side chain of the substrate (the ‘linker’),
the inhibitory end being the zinc-chelating hydroxamic acid, and the aromatic enzyme-binding group being the
4-dimethylaminobenzoyl group. This molecule, together with its congeners (trichostatins B, C, and D), was first
isolated as an antifungal agent,171 and approximately a decade later they were found to have potent
differentiation-inducing and antiproliferative activities in Friend erythroleukemia cells. Subsequently, TSA
demonstrated potent in vitro and in vivo inhibition (nanomolar range) of class I and class II HDACs, with a slight
selectivity for HDAC1 and HDAC6 compared to HDAC4. The S enantiomer of TSA was inactive, and neither
enantiomer had any activity against the class III enzymes. The full MOA has not yet been elucidated, but a
large series of effects were observed in signal transduction systems, including induction of apoptosis when
healthy and tumor cells from many different sources were treated with this agent.172
Identification of the basic structural features of TSA and its initial activities led to research on the synthesis
of compounds that were more stable and had improved water solubility. Early research with hexamethylene
bisacetamide (HMBA) (47; Figure 13), belonging to a family of molecules known as hybrid polar compounds
(HPCs), demonstrated that it induced hyperacetylation of histone H4 in healthy keratinocytes, as well as in
squamous cell carcinoma derived from these cells, but did not inhibit their growth in vitro and induced a wide
variety of other pathway modulations.173 The high doses of HMBA required for in vivo activity resulted in
toxicity and led to cessation of development, but these results, combined with a knowledge of the basic
structure of TSA, led to the development of a series of second-generation HPCs, which were tested as
HDAC inhibitors. The lead compound from these studies, suberoylanilide hydroxamic acid (SAHA) (48;
Figure 13), was approved in 2006 as vorinostat (Zolinza) and still is currently in over 40 clinical trials (phases I,
II, and III), either as a single agent or in combination with other agents, against a variety of refractory tumors,
both solid and leukemic in nature, including a Phase II study of an oral formulation.174 Efforts to resolve the
problems of low yields of (R)-TSA from natural sources and difficulties in achieving its total synthesis have
resulted a simple four-step strategy being devised for the synthesis of achiral amide analogues of the natural
product. The analogues consisted of a hydroxamate function, a benzamide and an aliphatic spacer, with
maximal inhibitory activity being observed with a five-carbon linker chain.175 The resulting lead compound
was 6-(4-dimethylaminobenzoyl)-aminocaproic acid hydroxamide (49; Figure 13), and although the antitumor
and cell transduction activities of these compounds have been reported, no in vivo data have yet
been published.176
The natural product trapoxin (50; Figure 13) was reported to be an irreversible inhibitor of HDACs in 1993,
but in contrast to TSA, it was found to demonstrate some selectivity against class I and class II HDACs,
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inhibiting HDAC1 and HDAC4 but not HDAC6.177 Combination of structural features of trapoxin, TSA, and
another potent HDAC inhibitor, the marine natural product psammaplin A (35; Figure 10), resulted in the
de novo synthesis of NVP-LAQ-824 (dacinostat) (51; Figure 13), which inhibits HDAC and the proliferation of
cancer cell lines at low nanomolar concentrations; it showed efficacy in a number of solid tumor xenograft
models, advancing to Phase I clinical trials in 2002178,179 but was discontinued by Novartis in 2005. The full
history of its evolution has been reviewed.180
The microbially derived depsipeptide, FR-901228 (romidepsin) (52; Figure 13), which was originally
identified as a result of its potent antitumor activity, is now known to be active in signal transduction as a
result of its HDAC activity,181 and it is currently in Phase I and Phase II clinical trials.
3.06.4.5
Protein Kinase Inhibitors
Several agents that have advanced into clinical trials or commercial use in recent years have either been derived
directly from nature or incorporate key structural features from natural products. Thus, the development of
Gleevec can be traced back to ATP mimicry, with its history briefly reviewed by Newman et al.,182 and the
history of Iressa is similar.
3.06.4.5.1
Flavopiridol
The flavone, flavopiridol (Alvocidib) (53; Figure 14), is totally synthetic, but its novel structure is based on the
natural product rohitukine (54; Figure 14), isolated from Dysoxylum binectariferum. It was originally considered
to be an inhibitor of cyclin-dependent kinases (CDKs) (the regulators of the G2 to M transition in the cell
cycle), and has entered into Phase I and then Phase II clinical trials against a broad range of tumors.183 Like the
olomucine (55; Figure 14) derivative, roscovitine (selicicib) (56; Figure 14), it has now been reported to be a
very potent inhibitor of CDK-7 and CDK-9, the kinases primarily responsible for promoting RNAP II (RNA
polymerase II) activity, thus involving these agents in the transcription process. The molecular targets/
interactions involved in the transcription processes and flavopiridol interactions have been reviewed.184,185
Currently (early 2008), several single-agent Phase I and Phase II clinical trials against leukemias, lymphomas,
and solid tumors are active, while over 10 Phase I and Phase II trials are active in combination with other
anticancer agents.
3.06.4.5.2
Bryostatins
The bryostatins are a class of highly oxygenated macrolides, and the multiyear program that culminated in the
isolation and purification of (currently) 20 bryostatin structures, has been well documented by a number of
authors over the years.186–193 These reviews may be consulted for the experimental details that indicated that
the bryostatins, and in particular, bryostatin 1 (57; Figure 14), which has been the focus of preclinical and
clinical studies, have signal transduction activities, and details on the clinical trials of bryostatin 1 are reviewed
in Newman.193
While the total synthesis of bryostatin 1 is not a feasible process for the production of this agent, three of the
naturally occurring bryostatins, bryostatins 7,194 2,195 and 3,196 have been synthesized, and their syntheses and
the syntheses of other partial bryostatin structures, including bryostatin 1, have been reviewed;190,192,197 these
reviews should be consulted for specific details of reaction schemes and comparisons of routes. None of these
methods, however, are viable for the large-scale production of any of the bryostatins for further development.
However, analytical studies by the Wender group of the potential binding site of the phorbol esters on protein
kinase C (PKC) as a guide to the design of simpler analogue of these agents198 were expanded to bryostatin 1,199
and led to the production of simpler bryostatin analogues known colloquially as ‘bryologues’ that maintained
the putative binding sites at the oxygen atoms at C1 (ketone), C19 (hydroxyl), and C26 (hydroxyl). These
molecules (58, 59; Figure 14) demonstrated nanomolar binding constants when measured in displacement
assays of tritiated phorbol esters, with the figures being in the same general range as bryostatin 1, and had
activities in in vitro cell line assays close to those demonstrated by bryostatin 1 itself.200–203 Introduction of a
second lactone gave a compound (60; Figure 14) with 8 nmol l1 binding affinity and an ED50 of 113 nmol l1
against P388,204 and the use of different fatty acid esters gave compounds exhibiting binding affinities for PKC
isozymes in the 7–232 nmol l1 range depending upon the fatty acid used.205 A further simple modification
158 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
involving removal of a methyl group in the C26 side chain gave a compound (61; Figure 14) with the binding
affinity to PKC at the picomolar level,206 and demonstrating greater potency than bryostatin 1 in in vitro cell
line assays. Improved syntheses of the bryologues might well permit further exploration of these
analogues.207,208
3.06.4.5.3
Adenine derivatives
The observation that substituted purines, particularly 6-dimethylamino-purine (6-DMAP) (62; Figure 14) and
isopentenyladenine (63; Figure 14), from Castanea species, showed weak inhibition of CDK1/cyclin B led to
the search for other purine-derived compounds.209 Another plant secondary metabolite originally isolated from
the cotyledons of the radish, and subsequently named olomucine (55; Figure 14), demonstrated an improved
Figure 14 (Continued)
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159
Figure 14 Protein kinase inhibitors
efficacy (IC50 ¼ 7 mol l1) and selectivity for CDKs, and to some extent, MAP kinases, by direct competition
with ATP. Olomucine, which earlier had been synthesized,210 disproved the existing dogma that no specific
kinase inhibitors could be found for ATP-binding sites since they would be swamped by the presence of excess
of ATP. Further development of this series using combinatorial chemistry techniques led to roscovitine
(56; Figure 14), and finally to purvalanol A (64; Figure 14) and purvalanol B (65; Figure 14). The purvalanols
demonstrated improved potency, with IC50 values in the range of 4–40 nmol l1, compared to 450 nmol l1 for
roscovitine.211 The R-isomer of roscovitine is currently in Phase II clinical trials in Europe. Although some
beneficial effects are observed with signal transduction inhibitors (STIs) alone, complete or partial responses
tend only to be demonstrated when sequential treatments of STI/cytotoxin are used, so also with R-roscovitine,
sequential treatment with cytotoxins is being used and/or considered.
3.06.4.5.4
Indigo and the indirubins
Hydroxylation of indigo in the 3-position, presumably by a suitable cytochrome P-450, gives a product that is
tautomeric with the 3-keto analogue, indoxyl (66; Figure 14), and various levels of oxidation then lead to a
mixture of indigo, indirubin (67; Figure 14) and their isomers, which is commonly used as the source of indigo
dyestuffs, a mixture obtained from the plant Isatis tinctoria found to contain an indigo precursor.212 Although
usually considered to be plant products, indigo and the indirubins have been reported from four nominally
independent sources: a variety of plants,212 a number of marine mollusks, usually belonging to the Muricidae
family of gastropods,213 natural or recombinant bacteria,214 and human urine.215
The indirubins have been identified as the major active components of the TCM formulation known as
Danggui Longhui Wan, which has been used for many years to treat chronic myelogenous leukemia (CML) in
China.216
Of importance from both a natural product and a pharmacological perspective, the indirubins were
recognized as being inhibitors of several CDKs and potent inhibitors of glycogen synthase kinase-3
(GSK-3).217 Included in this study were 6-bromoindirubin (68; Figure 14), first isolated from nature from
the mollusk Hexaplex trunculus,209 and its chemically modified oxime derivative BIO (69; Figure 14), and these
160 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
two compounds demonstrated an at least fivefold specificity versus CDK1/cyclin B and/or CDK/p25, and
significantly greater specificity against a wide range of other kinases. Significantly, GSK-3 is also an important
target in both Alzheimer’s disease and type 2 diabetes, and although indole derivatives have not been reported
as being associated with pharmacological intervention in these specific disease areas, their potential must be
considered quite high. The treatment potential for inhibitors of GSK-3, including a listing of other natural
product-related structures serving as possible inhibitors in these disease states, has recently been reviewed.218
Using the same basic suite of compounds, it was demonstrated that indirubins serve as ligands for the ‘orphan
receptor’ known as the aryl hydrocarbon receptor (AhR).219 No other natural ligands have yet been identified
for AhR, even though, contrary to earlier beliefs, it has existed for over 450 million years. Indole-containing
compounds, however, had been suggested as natural ligands for AhR slightly earlier.220 Full details of the
chemistry involved, and SARs established using X-ray crystallography and molecular modeling techniques,
have been published.221
Among other natural products with indirubin-like kinase inhibitory activities are the meridianins (e.g.,
meridianin A) (70; Figure 14), a group of halogenated indole derivatives that are closely related to the base
structures of variolin B (7; Figure 4), the psammopemmins (e.g., psammopemmin A) (71; Figure 14) and
discodermindol (72; Figure 14). Variolin B, the psammopemmins and discodermindol were isolated from
sponges, whereas the meridianins were isolated from the ascidian Aplidium meridianum.222
3.06.4.6
Protein Folds and Inhibitors of Kinases and Phosphatases
Significant effort has been, and continues to be, devoted to the development of novel kinase inhibitors through
the ‘‘fitting of structures to the ATP-binding sites,’’ and this approach has been quite successful in producing
structures for clinical trials.223 However, the Waldman group has successfully developed a variation on this
theme in which, rather than initially concentrating on the specifics of the ATP-binding site, they have used two
other fundamental premises to search for kinase, and other enzyme inhibitors. First, they considered that
biologically active natural products are viable, biologically validated starting points for library design, permitting the discovery of lead compounds possessing an enhanced probability of success if included in
high-throughput screening;125,127 and second, that although estimates of the number proteins in humans
range between 100 000 and 450 000, the number of topologically distinct shapes, defined as protein folds, is
actually much lower, with estimates of 600–8000.224 Thus, if an inhibitor of a specific protein fold from nature
could be found, then it could be used as a prototype for the development of closely related structures that may
inhibit proteins with similar folds, and even allow for the discovery of specificity. These concepts are
fundamentally similar to the privileged structure concept mentioned in Section 3.06.3.5.3, but the Waldmann
approach has the added dimension of using protein folding patterns as the basis for subsequent screens.
The success of this approach was demonstrated by the derivation of inhibitors of Tie-2, insulin-like growth
factor 1 receptor (IGF-1R), and vascular endothelial growth factor receptors 2 and 3 (VEGFR-2 and
VEGFR-3), from the original discovery of the Her-2/Neu inhibitor, nakijiquinone C (73; Figure 15).
Derived from a marine sponge and first reported by Kobayashi et al.225 in 1995, nakijiquinone C was shown
to be an inhibitor of epidermal growth factor receptor (EGFR), c-ErbB2, and PKC, in addition to having
cytotoxic activity against L1210 and KB cell lines. Testing of a library of 74 compounds, built around the basic
nakijiquinone C structure, against a battery of kinases with similar protein domain folds, yielded seven new
inhibitors with low micromolar activity in vitro, including one VEGFR-2 inhibitor (74; Figure 15) and four
inhibitors of Tie-2 kinase (75–78; Figure 15), a protein intimately involved in angiogenesis, and for which, at
the beginning of the study, no inhibitors were known.124 During the study, the first natural product inhibitor of
Tie-2 kinase was reported226 (79; Figure 15) from the plant Acacia aulacocarpa, and a set of four papers from
another research group demonstrated the activity of synthetic pyrrolo[2,3-d]pyrimidines as inhibitors of the
same class of kinases.227–230 The details of the models used, the chemistry leading to the nakijiquinone-based
compounds, and the ribbon structures of the kinase domain of the insulin receptor, with the corresponding
homology domains of the as yet uncrystallized VEGFR-2 and Tie-2, have been fully reviewed.129,231
A similar approach has been used in the identification of phosphatase inhibitors. Postulating that the
-hydroxy-butenolide group of the marine-derived metabolite, dysidiolide (33; Figure 10), was the major
determinant of phosphatase activity, testing of a 147-member library built around this molecule yielded a
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Figure 15 Kinase, phosphatase, and Hsp90 inhibitors.
161
162 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
compound (80; Figure 15) 10-fold more potent (IC50 ¼ 350 nmol l1) than the parent against Cdc25A.125 In
addition, other members of the library were identified with low micromolar activities against the enzymes,
acetylcholinesterase and 11 -hydroxysteroid dehydrogenase type 1, which fall within the same ‘similarity
cluster’ as Cdc25A.232
3.06.4.7
Inhibitors of Heat Shock Protein 90
Heat shock protein 90 (Hsp90) is a chaperone protein that plays an important role in stabilizing the
conformation of many cell-signaling proteins and maintaining their function. In this respect, many oncogenic
proteins are more dependent on Hsp90 than their normal counterparts, and hence Hsp90 plays an important
role in maintaining transformation and increasing the survival and growth tendency of cancer cells. It has also
been shown to exist in an activated form in cancer cells while existing in a latent inactive form in normal cells,
thus making it an attractive target for chemotherapy in cancer and other diseases, such as neurodegenerative
diseases.233 Advances in the development of Hsp90 inhibitors have been reviewed.234,235
3.06.4.7.1
Ansamycins: geldanamycin derivatives
The development of the ansamycins leading to the 17-substituted analogues has been reviewed.236 This review
highlights the significant differences in the macrocyclic ring stereochemistries reported in the literature for
what is nominally the same molecule. These differences are not simply due to a complete stereochemical
inversion around the ring, where the relative stereochemistries are maintained, but are quite different renditions from different research groups and should be noted when referring to different papers.
17-Allylaminogeldanamycin (17-AAG; tanespimycin) (81; Figure 15) is in over 10 phases I, II, and III
clinical trials against leukemias, lymphomas, and solid tumors, either as a single agent or in combination with
other agents. The more soluble material, 17-dimethylamino-ethylaminogeldanamycin (17-DMAG) (82;
Figure 15), is in Phase I clinical trials as a single agent against solid tumors, while a number of other
17-substituted derivatives have been prepared as potential alternative candidates.237,238
Two apparent anomalies in the interactions of geldanamycin (GA) derivatives and radicicol (monorden)
(83; Figure 15) with Hsp90 have been under intensive study. The first anomaly is that, despite the fact that
both healthy and tumor cells require Hsp90 for cellular function, they respond differently to these drugs, and
the second is the fact that the affinity of these drugs for recombinant Hsp90 (rHsp90) is much lower than the
levels required for responses in tumor cell lysates. The higher binding affinity for Hsp90 in tumor lysates has
been attributed to the existence of other cochaperones in tumor cells that are not expressed in healthy cells, and
this effect was demonstrated by the addition of such proteins to rHsp90.239 In addition, X-ray crystal studies
have demonstrated that the structure of GA in the unbound form has a trans-configuration at the amide bond
between the benzoquinone and the rest of the ansa ring, whereas when bound to Hsp90, GA displays the
cis-configuration at this center.240 Similarly, Jez et al.241 reported that the closely related GA derivative
17-DMAG requires both a macrocyclic ring conformational change and a trans–cis isomerization of the
amide bond in order to bind to Hsp90. The tumor selectivity, however, is still a subject of investigation.242
3.06.4.7.2
Non-ansamycin inhibitors
Supply problems associated with GA derivatives and radicicol, together with GA toxicity problems, led
Chiosis et al. to propose the use of a simple substituted adenine derivative as a potential base molecule.
Significantly, the proposal was based on considering which particular substructures might provide ATP mimics
with improved binding characteristics, rather than computerized modeling. Thus, knowledge of the requirements
of the ATP-binding pocket of Hsp90, and demonstration that a small molecule could function as a cytostatic
agent,243 provided the intellectual stimulus for designing the purine-based PU class of compounds.244–246
Rational changes in the substituents in both rings and alteration of the length and rigidity of the linker gave rise
to PU24FCl (84; Figure 15),247 which, although not the most active in the series, was utilized to further
investigate Hsp90 inhibition in both healthy and tumor cells. The extensive effects exhibited by both healthy
and tumor tissues when exposed to the compound have been reported,248 and, as with 17-AAG and GA,
PU24FCl exhibited at least 10- (brain, pancreas, lung) to 50-fold (heart, kidney, liver) lower affinity for Hsp90s
from healthy tissues as compared to those from transformed cells. Later studies have shown that replacement of
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163
the methylene bridge with sulfur gives 8-arylsulfanyl adenine derivatives (e.g., 85; Figure 15) of greater
potency,249 while introduction of an ionizable amino group in the N(9) side chain improved both the water
solubility and potency of the compounds to give orally active agents (e.g., 86; Figure 15).250,251
3.06.4.8
Proteasome Inhibitors
The proteasome is a multienzyme complex involved in the ubiquitin–proteasome pathway control of cell-cycle
progression, in the termination of signal transduction cascades, and in the removal of mutant, damaged, and
misfolded proteins. As such, it is a promising therapeutic target, and the background to this aspect has been
reviewed.252–255 The synthetic dipeptidyl peptide boronate, bortezomib (Velcade) (87; Figure 16), is the first
clinical drug that uses this MOA,256–258 and the development of this compound, which is based upon a natural
product-derived structure that inhibited chymotrypsin, has been described by the original inventor.259
There are however a significant number of other compounds from nature, and their derivatives, that have
led to a greater understanding of the intricacies of this multienzyme complex. The 20S proteasome in mammals
has three closely linked proteolytic activities, which are termed trypsin-, chymotrypsin-, and caspase-like from
their substrate profiles, although the complex only acts as a concerted whole; individual activities are not
demonstrable. In fact, if the chymotrypsin-like activity is inhibited by a suitable compound then a large
reduction in the rate of protein degradation is observed, but if the sites corresponding to the other nominal
activities are modified, the overall rate of hydrolysis of proteins is not significantly changed. Owing to the
substrate specificity of chymotryptic sites, most inhibitors are hydrophobic, whereas in the case of the other two
active sites, their ‘peptide-based’ substrates/inhibitors tend to be charged. As a result, almost all of the
proteasome inhibitors tend to have chymotrypsin-like activities with some overlapping, but weaker, effects
on the other sites.
In 1991, the microbial metabolite lactacystin (88; Figure 16) was reported to induce neuritogenesis in
neuroblastoma cells,260 and this was followed by reports261,262 demonstrating that radio-labeled lactacystin
selectively modified the 5(X) subunit of the mammalian proteasome, and irreversibly blocked activity. In
subsequent studies, it was demonstrated263,264 that the actual inhibitor in vitro was the -lactone,
clastolactacystin- -lactone (89; Figure 16), and that this substance was formed spontaneously when lactacystin
was exposed to neutral aqueous media. The parent compound and other analogues have been synthesized,
and the authors suggested that clasto-lactacystin- -lactone should be named omuralide (89; Figure 16).265,266
The marine bacterial metabolite salinosporamide A (Section 3.06.3.3.5) (22; Figure 8) demonstrates activity as a
cytotoxic proteasome inhibitor86 and has been synthesized.267 Compared to omuralide, salinosporamide is
uniquely functionalized and has a cyclohexene ring replacing the isopropyl group found at the C(5)-position in
omuralide. The isopropyl group in omuralide is essential for the activity, so salinosporamide A might interact
with the 20S proteasome in a modified manner. This molecule is being developed by Nereus Pharmaceuticals,
and currently is in Phase I clinical trials against refractory lymphomas and myelomas, as well as various solid
tumors.
The epoxyketone microbial metabolites epoxomicin (90; Figure 16) and eponemycin (91; Figure 16)
exhibited cytotoxic activities as a result of proteasome inhibition,268,269 being the most selective proteasome
inhibitors reported to date. There are reports of other natural products active as proteasome inhibitors but with
different mechanisms to those above. Thus, the cyclic peptide TMC-95-A (92; Figure 16), isolated from
Apiospora montagnei, is a potent chymotrypsin-like inhibitor, but with activity against the other sites as well,270
apparently binding noncovalently to active sites through an array of hydrogen bonds. ()-Epigallocatechin
3-gallate (93; Figure 16) is a potent covalent inhibitor of the 20S proteasome, apparently due to acylation of the
active site threonines through threonine cleavage of the ester linkage in EGCG.271
3.06.4.9
DNA Interactive Agents (Non-Topoisomerases I and II Inhibitors)
The complex alkaloid ecteinascidin 743 (Et-743, Yondelis) (94; Figure 16), discovered from the colonial
tunicate Ecteinascidia turbinata,272,273 was found to have a unique MOA, binding to the minor groove of DNA
and interfering with cell division, the genetic transcription processes, and DNA repair machinery.274,275 There
has been a considerable number of reports published in the literature giving possibilities as to the MOA(s) of
164 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Figure 16 Proteasome inhibitors and DNA interactive agents.
Et-743 when tumor cells are treated in vitro. A significant problem with some of the reports is that the
concentration(s) used in the experiments are often orders of magnitude greater than those that demonstrate
activity in vivo. These levels are in the low nanomolar to high picomolar range and thus care should be taken
when evaluating published work on the MOA of this compound.
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165
The issue of compound supply for advanced studies was solved by the development of a semisynthetic route
from the microbial product cyanosafracin B (95; Figure 16),276 and this and other aspects of the discovery and
development have been comprehensively reviewed.157,277 Under the name Yondelis, Et-743 has been granted
orphan drug designation in Europe and the United States, and was approved by the European Medicines
Agency (EMEA) in late September 2007 for the treatment of soft tissue sarcomas (STS).278 It is also in Phase II
and Phase III trials in ovarian, metastatic breast and prostate cancers, and pediatric sarcomas.
3.06.4.10
Caspase Activation and Apoptosis Induction
The relatively simple naphthoquinone -lapachol (96; Figure 17) is a well-known compound obtained from
the bark of the lapacho tree, Tabebuia avellanedae, and other species of the same genus that are native to South
America. -Lapachol and other plant components are extensively used as ethnobotanical treatments in the
Figure 17 Inhibitors of caspase activation, apoptosis induction, HIF, and miscellaneous targets.
166 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Amazonian region, and -lapachol was advanced to clinical status by the NCI in the 1970s. It was later
withdrawn due to unacceptable levels of toxicity, but its close relative -lapachone (97; Figure 17) has
demonstrated interesting molecular target activity, with one MOA being the induction of apoptosis in
transformed cells.279 Evidence of its involvement in transcription processes has been reported demonstrating
that the agent induced activation of caspase-3, inhibition of nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-B) and subsequent downregulation of bcl-2.280 Currently, -lapachone (ARQ501) is in
Phase II clinical trials in the United States for advanced solid tumors, and further information on the
background of these agents may be obtained from the 2004 review.281
3.06.4.11
Hypoxia Inducible Factor
Hypoxia inducible factor 1 (HIF-1) is composed of two subunits, an oxygen-sensitive inducible factor (HIF-1 )
and the constitutive HIF-1 (also known as AhR nuclear translocator (ARNT)), which may prove to be an
important target in diseases that have a hypoxic component such as cancer (where the interior of a tumor is
anoxic compared with the outer surfaces), heart disease, and/or stroke. The involvement of HIF proteins with a
variety of inhibitors (not necessarily direct inhibition, but alteration of transduction pathways upstream and
downstream) have been reviewed, and included in the review are well-known materials with natural product
‘backgrounds’, such as Taxol., vincristine, 2-methoxyestradiol, rapamycin, GA, quinocarmycin, and the IP3K
inhibitors, wortmannin and LY-294002.282
Of significance from a natural product perspective was the initial realization that inhibition of thioredoxin
reductase 1 (TRX-1) may act indirectly on HIF-1 . By comparing the NCI 60 human cancer cell line
cytotoxicity profile of a known TRX-1 inhibitor and Phase II clinical candidate, PX-12 (98; Figure 17), with
the profiles of a range of compounds in the NCI screening database, the fungal natural product pleurotin (99;
Figure 17) was identified as exhibiting a similar killing pattern to PX-12.283 Research on a focused combinatorial library of naphthoquinone acetals based upon palmarumycin CP1 (100; Figure 17), which included
diepoxins (e.g., diepoxin, 101; Figure 17) and deoxypreussomerins (e.g., deoxypreussomerin A, 102;
Figure 17), indicated that they possessed potent cytotoxicity, but their potential targets were unidentified at
that time.284 Palmarumycin CP1, however, was later shown to have inhibitory activity comparable to that of
pleurotin in the TRX-1 assay, with IC50 values in the range of 170–350 nmol l1, and it was demonstrated that
certain aspects of the base structure, in particular the enone system, were required for activity in this assay.285
Evidence for direct inhibition of HIF-1 by both pleurotin and PX-12, helped to demonstrate that the
cytotoxicity of these compounds, and hence palmarumycin CP1, was likely due to HIF-1 interactions.286
Further palmarumycins isolated from extracts of the fermentation broth of an unidentified ascomycete from
Costa Rica failed to show activity in the assays used, but provided important SAR information.287 This
information in turn led to further modifications of the base structure, yielding the simple analogues S-11
(103; Figure 17) and S-12 (104; Figure 17), which exhibited biological activities comparable to pleurotin in
both the thioredoxin enzyme (TRX-1) system and (most importantly) in the cytotoxicity assays.287 Thus, a
fairly complex interaction of results from several different research groups has led to promising candidates for
further biological studies, including in vivo experiments that are planned and will be reported in due course.
3.06.4.12
Miscellaneous Target Inhibitors
There are a number of agents particularly from marine sources, whose initial molecular targets have been
identified although it is highly probable that over the next few years, these initial targets will be refined as
methods and other information becomes available.
One such compound, aplidine, is an agent with multiple targets. Formally, dehydrodidemnin B (105;
Figure 17), it was first reported in a patent and then referred to in a 1996 paper on SARs among the
didemnins.288 In 1996, the antitumor potential was reported by PharmaMar scientists and the total synthesis
was reported in a patent application in 2000 and the patent was issued in 2002. The compound was advanced to
Phase I clinical trials in 1999 under the trade name of Aplidin for the treatment of both solid tumors and
non-Hodgkin’s lymphoma and published details through early 2004 are given in Newman and Cragg28 together
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
167
with discussion as to the mechanisms of action that might be relevant. Details of the progress of this drug
through preclinical and clinical development have been reviewed.28,277,289
It should be noted that the clinical trials of the very close aplidine analogue didemnin B (106; Figure 17)
were discontinued because of the toxicities observed, including significant immunosuppression. In contrast,
evidence for the lack of myelosuppression by aplidine was reported using a murine competitive repopulating
model as the test system,290 and no hematological toxicity has been observed clinically.277 It is very interesting
both chemically and pharmacologically that the removal of two hydrogen atoms, that is, conversion of the lactyl
side chain to a pyruvyl side chain, appears to significantly alter the toxicity profile, as this is the only formal
change in the molecule when compared to didemnin B. However, the comments on dosage regimens should be
taken into account when such comparisons are made in the future.291
3.06.5 Summary and Future Prospects
Nature has been a source of medicinal products for millennia, and during the past century many useful drugs
have been developed from natural sources, particularly plants. It is clear that nature will continue to be a major
source of new drug leads. The drug potential of the marine environment remains relatively unexplored, but it is
becoming increasingly evident that the realm of microorganisms offers a vast untapped potential. With the
advent of genetic techniques that permit the isolation and expression of biosynthetic cassettes, microbes and
their marine invertebrate hosts may well be the new frontier for natural products lead discovery. Plant
endophytes also offer an exciting new resource, and research continues to reveal that many of the important
drugs originally thought to be produced by plants are actually products of endophytic microbes residing in the
tissues between living plant cells. This has been further accentuated by the recent report of the isolation of
hypericin from an endophytic fungus from Hypericum perforatum.292 Effective drug development will depend on
multidisciplinary collaboration embracing natural product lead discovery and optimization through the
application of total and DOS and combinatorial chemistry and biochemistry, combined with good biology.
The impressive number of anticancer drugs that are derived from natural sources are discussed in terms of their
mechanisms of action, and as can be seen from these discussions, natural products from all sources still have the
potential to lead chemists of all types into areas of drug discovery and development that would never have been
considered if the ‘privileged structures from nature’ had not been isolated, purified, and used as probes of
cellular and molecular mechanisms. In spite of the discussions in the early to late 1990s concerning the vast
potential of combinatorial chemistry as a discovery tool, it is now quite evident that this technique, except in
the very special cases of peptides and nucleosides (which are actually ‘privileged structures’ in their own right),
is not the panacea that it was thought to be. However, the application of combinatorial synthetic methodology
as a means to elaborate around a skeleton from a privileged structure demonstrates that the use of both
techniques will lead to novel agents having potential as drug entities in many disease states.293
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174 Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
Biographical Sketches
Gordon M. Cragg completed his undergraduate training in chemistry at Rhodes University,
South Africa, and his D. Phil. (organic chemistry) from Oxford University in 1963. After 2 years
of postdoctoral research at the University of California, Los Angeles, he returned to South Africa
to join the Council for Scientific and Industrial Research. In 1966, he joined the chemistry
department at the University of South Africa. He was transferred to the University of Cape
Town in 1972. In 1979, he returned to the United States to join the Cancer Research Institute at
Arizona State University. In 1985, he moved to the National Cancer Institute (NCI) in Bethesda,
Maryland, and was appointed chief of the Natural Products Branch in 1989. He retired in
December, 2004, and is currently serving as an NIH Special Volunteer. His major interests lie in
the discovery of novel natural product agents for the treatment of cancer and AIDS, with an
emphasis on multidisciplinary and international collaboration. He has given over 100 invited
talks at conferences in many countries worldwide and has been awarded NIH Merit Awards for
his contributions to the development of taxol (1991), leadership in establishing international
collaborative research in biodiversity and natural products drug discovery (2004), and contributions to developing and teaching NIH technology transfer courses (2004). He was the president
of the American Society of Pharmacognosy (1998–99) and was elected to honorary membership
of the society in 2003. In November, 2006, he was awarded the William L. Brown Award for
Plant Genetic Resources by the Missouri Botanical Garden at a 2-day symposium entitled
‘Realizing Nature’s Potential: The Once and Future King of Drug Discovery’ held in his honor.
The Missouri Botanical Garden also named a recently discovered Madagascar plant in his honor,
Ludia craggiana. He has established collaborations between the NCI and organizations in many
countries promoting drug discovery from their natural resources. He has published over 150
papers related to these interests.
David J. Newman is the current chief of the Natural Products Branch (NPB) in the
Developmental Therapeutics Program at the National Cancer Institute in Frederick, MD.
Nature as Source of Medicines; Novel Drugs from Nature; Screening for Antitumor Activity
He was born in Grays, Essex, UK in 1939. In 1963, he received his M.Sc. in synthetic organic
chemistry from the University of Liverpool working under Professor George Kenner, FRS,
on pyrrole and porphyrin syntheses. Following time as a synthetic chemist at Ilford, Ltd., he
joined the ARC’s Unit of Nitrogen Fixation at the University of London and then Sussex, as a
research assistant in metallo-organic chemistry with Professor J. Chatt, FRS, transferring to
the microbial biochemistry group in early 1966 as a graduate student under Professor John
Postgate, FRS, and was awarded a D. Phil. in 1968 for the work on microbial electron
transport proteins from Desulfovibrio. Following a move to the United States in September
1968, he did 2 years as a postdoc at the biochemistry department of the University of Georgia
working on protein sequencing of Desulfovibrio ferredoxins, and then in 1970 joined SK&F in
Philadelphia as a biological chemist. At SK&F, most work was related to biological chemistry
and antibiotic discovery, and he left SK&F in 1985 when the antibiotic group was dissolved.
For the next 6 years he worked in marine and microbial discovery programs (Air Products,
SeaPharm, and Lederle) and then in 1991, joined the NPB as a chemist responsible for marine
and microbial collection programs. He was given the NIH Merit Award in 2003 for this work
and following Gordon Cragg’s retirement from the position of chief, NPB at the end of 2004,
he was acting chief until appointed chief in late 2006. He has been the author or coauthor of
over 110 papers, reviews, book chapters (and an editor, with Gordon Cragg and David
Kingston of Anticancer Agents from Natural Products), and holds 18 patents.
175