The discovery of medically useful natural products has heralded
hitherto unimagined possibilities in the chemotherapy of human
and animal diseases.
It is well known that important medical compounds, such as
penicillin, cyclosporine A and lovastatin, were only developed as
drugs once their key properties were recognized, more than 10 years
after their initial discovery.4 Similarly, in the case of staurosporines,
their crucial protein kinase inhibitory properties were only identified a
decade or so after their initial discovery.
In 1986, 9 years after the isolation of staurosporine from a streptomyces,
the related natural indolocarbazole products, staurosporine and
K252, were shown to be nanomolar inhibitors of protein kinases, offering
tremendous promise for drug development. The reports led many
pharmaceutical companies to begin searching for selective protein kinase
inhibitors through natural product screening and chemical synthesis,
with the result that, during the 1990s, protein kinases became the second
most important drug target after G-protein-coupled receptors.
In parallel with the development of indolocarbazoles as
anticancer drugs targeting protein kinases, mammalian DNA
topoisomerase I was shown to be a new target for indolocarbazoles
by Yamashita et al. Thereafter, many antitumor indolocarbazoles
have been synthesized, as DNA topoisomerases were known to be
targets for antitumor drugs such as camptothecin and VP-16.
DNA topoisomerases alter DNA topology by transiently breaking
and re-sealing one strand of DNA through a covalent protein–DNA
intermediate. In 1996, it was shown that topoisomerase I has an
intrinsic protein kinase activity (Topo I kinase) required for phosphorylation
of the SR (serine arginine-rich) protein required for
splicing.
The action of indolocarbazole derivatives on topoisomerase
indicated that these compounds may selectively interact with ATPbinding
sites of not only protein kinases but also other proteins. As an
example of this, during the current decade, it was shown that ABCG2,
an ABC transporter with importance in drug resistance, oral drug
absorption and stem cell biology, could be a key new target for
indolocarbazoles.
This review outlines the pivotal pioneering studies relating to the
discovery, biosynthesis and biological activities of natural indolocarbazole
products.
PRODUCING ORGANISM
Staurosporine was discovered in 1977 in a culture of an actinomycete
(Streptomyces strain AM-2282T) while screening for microbial alkaloids
using chemical detection methods11. The strain AM-2282T
(NRRL 11184, ATCC 55006) has been renamed through repeated
revisions of the taxonomy of soil Actinomyces as Streptomyces staurosporeus
AM-2282T in 1977, Saccharothrix aerocolonigenes subsp.
staurosporea AM-2282T in 199512 and Lentzea albida in 2002. Over
the past 30 years, staurosporine and related natural indolocarbazole
compounds have been isolated from several actinomycetes (including
Streptomyces, Saccharothrix, Lentzea, Lechevalieria, Nocardia, Nocardiopsis,
Nonomuraea, Actinomadura and Micromonospora) as well as
from myxomycetes (slime molds) and cyanobacteria (Figure 1).
Staurosporine derivatives have also been isolated from marine
invertebrates, such as sponges, tunicates, bryozoans and mollusks.
However, it remains unknown whether invertebrates actually have
genes for indolocarbazole biosynthesis, as many natural products from
marine invertebrates are produced by associated microorganisms.14
Interestingly, half of the 14 indolocarbazole-producing strains
deposited in the global culture collection have been isolated from
Japanese soils. In the 1980 s, fermentation broths of 5163 new Japanese
soil isolates were tested and five Streptomyces were found to produce
staurosporine, together with new analogs (UCN-01 and UCN-02
(stereo-isomers of 7-hydroxy staurosporines)). In other words, ca
0.1% of newly isolated soil actinomycetes were shown to produce
staurosporine using a fixed culture condition.15
In 1993, staurosporine and K252a were shown to inhibit in vitro
phosphorylation of crude extracts from Streptomyces griseus and also
from a staurosporine-producing Streptomyces sp.16 Although staurosporine
did not show significant antibacterial activity, it was shown to
affect cell differentiation processes in Streptomycetes, such as pigment
production and spore formation, depending on the AfsK family
serine/threonine protein kinases involved. Later, on the basis of
genome sequence analysis of Streptomyces avermitilis in 200117
and Streptomyces coelicolor in 2002,18 it was revealed that more than
30 protein kinase genes are coded in these organisms. Further research
is needed to determine the exact role and impact of staurosporine on
differentiation of producing strains and microorganisms in soil.
BIOSYNTHESIS OF STAUROSPORINE
Biosynthetic studies carried out in the 1980s and 1990s using isotopelabeled
precursors showed that the indolocarbazole structure of
staurosporine is derived from two molecules of tryptophan, and
that the sugar moiety is derived from glucose and methionine.
Cloning of the biosynthetic genes of staurosporine was triggered in
2000 by identification of the ngt gene encoding N-glycosyltransferase.
Ohuchi et al.19 heterologously expressed the ngt transferase gene from
Lechevalieria aerocolonigenes, a rebeccamycin producer, in Streptomyces
lividans and showed that ngt is responsible for N-glycosylation of the
indolocarbazole chromophore. Starting from the ngt gene, whole
biosynthetic gene clusters of staurosporine and rebeccamycin have
been cloned by Onaka et al.20,21 and Sanchez et al.22 To date, structures
of the accumulated products from 18 single-gene disruption mutants
of staurosporine and rebeccamycin biosynthesis gene clusters have
been identified (Figure 2).
Studies of these accumulated products and the gene function
predicted by the amino-acid sequence database searches have revealed
the biosynthetic pathway of staurosporine and rebeccamycin.14,23
(Figure 3).
In staurosporine biosynthesis, staO initiates synthesis by catalyzing
L-tryptophan to the imine form of indole-3-pyruvic acid (IPA imine)
and staD, and then catalyzes the coupling of two IPA imines to yield
chromopyrrolic acid. Formation of the indolocarbazole core of
staurosporine is catalyzed by staP, which converts chromopyrrolic
acid into three indolocarbazole compounds, staurosporine aglycone
(K252c), 7-hydroxy-K252c and acryriaflavin A, by intramolecular C–C
bond formation and oxidative decarboxylation. Crystallography of
P450 staP revealed that a heme of staP removes two electrons from the
indole ring to generate an indole cation radical, and intramolecular
radical coupling then forms the C–C bond to yield the indolocarbazole
core.24 The presence of staC predominantly directs the formation
of K252c. staG catalyzes N-glycosidic bond formation between N-13
and C-6ў and then staN, a P450 homolog, catalyzes an additional
C–N bond formation between N-12 and C-2ў. These two enzymes
convert K252c to 3ў-O-demethyl, 4ў-N-demethyl-staurosporine
through holyrine A and holyrine B.
The genes involved in the main pathway of indolocarbazole
structure formation in staurosporine and rebeccamycin showed striking
similarity between staO, staD, staP, staC and staG, and rebO, rebD,
rebP, rebC and rebG, respectively. The formation of chromopyrrolic
acid or 11,11-dichlorochromopyrrolic acid, key intermediates of
indolocarbazole biosynthesis, is catalyzed by staD or rebD. It is
noteworthy that the staD family includes only two homologs, rebD
and VioB, which are involved in violacein biosynthesis. The staD
family is a new type of hemoprotein with a novel structure and
function.
N-glycosidic bond formation between the N-12 and C-1ў positions
is catalyzed by staG or rebG N-glycosyltransferases. rebG is the same
gene that was cloned in 2000 by Ohuchi et al. as ngt, which can
catalyze the N-glycosylation of the indolocarbazole chromophore.
In the staurosporine structure, there exists an additional, unusual
C–N bond between the N-13 and C-6ў positions. Onaka et al. showed
through gene disruption and bioconversion experiments that staN, a
P450 homolog, is responsible for this unusual C-N bond formation.
StaN was the first example used to show that the P450 homolog is
involved in N-glycosidic bond formation. Deletion of staG abolished
glycosylation and led to accumulation of K252c, whereas deletion of
staN resulted in the production of holyrine A. Salas et al. also showed
the function of staN in C–N bond formation by heterologous
expression of the staN gene.
hitherto unimagined possibilities in the chemotherapy of human
and animal diseases.
It is well known that important medical compounds, such as
penicillin, cyclosporine A and lovastatin, were only developed as
drugs once their key properties were recognized, more than 10 years
after their initial discovery.4 Similarly, in the case of staurosporines,
their crucial protein kinase inhibitory properties were only identified a
decade or so after their initial discovery.
In 1986, 9 years after the isolation of staurosporine from a streptomyces,
the related natural indolocarbazole products, staurosporine and
K252, were shown to be nanomolar inhibitors of protein kinases, offering
tremendous promise for drug development. The reports led many
pharmaceutical companies to begin searching for selective protein kinase
inhibitors through natural product screening and chemical synthesis,
with the result that, during the 1990s, protein kinases became the second
most important drug target after G-protein-coupled receptors.
In parallel with the development of indolocarbazoles as
anticancer drugs targeting protein kinases, mammalian DNA
topoisomerase I was shown to be a new target for indolocarbazoles
by Yamashita et al. Thereafter, many antitumor indolocarbazoles
have been synthesized, as DNA topoisomerases were known to be
targets for antitumor drugs such as camptothecin and VP-16.
DNA topoisomerases alter DNA topology by transiently breaking
and re-sealing one strand of DNA through a covalent protein–DNA
intermediate. In 1996, it was shown that topoisomerase I has an
intrinsic protein kinase activity (Topo I kinase) required for phosphorylation
of the SR (serine arginine-rich) protein required for
splicing.
The action of indolocarbazole derivatives on topoisomerase
indicated that these compounds may selectively interact with ATPbinding
sites of not only protein kinases but also other proteins. As an
example of this, during the current decade, it was shown that ABCG2,
an ABC transporter with importance in drug resistance, oral drug
absorption and stem cell biology, could be a key new target for
indolocarbazoles.
This review outlines the pivotal pioneering studies relating to the
discovery, biosynthesis and biological activities of natural indolocarbazole
products.
PRODUCING ORGANISM
Staurosporine was discovered in 1977 in a culture of an actinomycete
(Streptomyces strain AM-2282T) while screening for microbial alkaloids
using chemical detection methods11. The strain AM-2282T
(NRRL 11184, ATCC 55006) has been renamed through repeated
revisions of the taxonomy of soil Actinomyces as Streptomyces staurosporeus
AM-2282T in 1977, Saccharothrix aerocolonigenes subsp.
staurosporea AM-2282T in 199512 and Lentzea albida in 2002. Over
the past 30 years, staurosporine and related natural indolocarbazole
compounds have been isolated from several actinomycetes (including
Streptomyces, Saccharothrix, Lentzea, Lechevalieria, Nocardia, Nocardiopsis,
Nonomuraea, Actinomadura and Micromonospora) as well as
from myxomycetes (slime molds) and cyanobacteria (Figure 1).
Staurosporine derivatives have also been isolated from marine
invertebrates, such as sponges, tunicates, bryozoans and mollusks.
However, it remains unknown whether invertebrates actually have
genes for indolocarbazole biosynthesis, as many natural products from
marine invertebrates are produced by associated microorganisms.14
Interestingly, half of the 14 indolocarbazole-producing strains
deposited in the global culture collection have been isolated from
Japanese soils. In the 1980 s, fermentation broths of 5163 new Japanese
soil isolates were tested and five Streptomyces were found to produce
staurosporine, together with new analogs (UCN-01 and UCN-02
(stereo-isomers of 7-hydroxy staurosporines)). In other words, ca
0.1% of newly isolated soil actinomycetes were shown to produce
staurosporine using a fixed culture condition.15
In 1993, staurosporine and K252a were shown to inhibit in vitro
phosphorylation of crude extracts from Streptomyces griseus and also
from a staurosporine-producing Streptomyces sp.16 Although staurosporine
did not show significant antibacterial activity, it was shown to
affect cell differentiation processes in Streptomycetes, such as pigment
production and spore formation, depending on the AfsK family
serine/threonine protein kinases involved. Later, on the basis of
genome sequence analysis of Streptomyces avermitilis in 200117
and Streptomyces coelicolor in 2002,18 it was revealed that more than
30 protein kinase genes are coded in these organisms. Further research
is needed to determine the exact role and impact of staurosporine on
differentiation of producing strains and microorganisms in soil.
BIOSYNTHESIS OF STAUROSPORINE
Biosynthetic studies carried out in the 1980s and 1990s using isotopelabeled
precursors showed that the indolocarbazole structure of
staurosporine is derived from two molecules of tryptophan, and
that the sugar moiety is derived from glucose and methionine.
Cloning of the biosynthetic genes of staurosporine was triggered in
2000 by identification of the ngt gene encoding N-glycosyltransferase.
Ohuchi et al.19 heterologously expressed the ngt transferase gene from
Lechevalieria aerocolonigenes, a rebeccamycin producer, in Streptomyces
lividans and showed that ngt is responsible for N-glycosylation of the
indolocarbazole chromophore. Starting from the ngt gene, whole
biosynthetic gene clusters of staurosporine and rebeccamycin have
been cloned by Onaka et al.20,21 and Sanchez et al.22 To date, structures
of the accumulated products from 18 single-gene disruption mutants
of staurosporine and rebeccamycin biosynthesis gene clusters have
been identified (Figure 2).
Studies of these accumulated products and the gene function
predicted by the amino-acid sequence database searches have revealed
the biosynthetic pathway of staurosporine and rebeccamycin.14,23
(Figure 3).
In staurosporine biosynthesis, staO initiates synthesis by catalyzing
L-tryptophan to the imine form of indole-3-pyruvic acid (IPA imine)
and staD, and then catalyzes the coupling of two IPA imines to yield
chromopyrrolic acid. Formation of the indolocarbazole core of
staurosporine is catalyzed by staP, which converts chromopyrrolic
acid into three indolocarbazole compounds, staurosporine aglycone
(K252c), 7-hydroxy-K252c and acryriaflavin A, by intramolecular C–C
bond formation and oxidative decarboxylation. Crystallography of
P450 staP revealed that a heme of staP removes two electrons from the
indole ring to generate an indole cation radical, and intramolecular
radical coupling then forms the C–C bond to yield the indolocarbazole
core.24 The presence of staC predominantly directs the formation
of K252c. staG catalyzes N-glycosidic bond formation between N-13
and C-6ў and then staN, a P450 homolog, catalyzes an additional
C–N bond formation between N-12 and C-2ў. These two enzymes
convert K252c to 3ў-O-demethyl, 4ў-N-demethyl-staurosporine
through holyrine A and holyrine B.
The genes involved in the main pathway of indolocarbazole
structure formation in staurosporine and rebeccamycin showed striking
similarity between staO, staD, staP, staC and staG, and rebO, rebD,
rebP, rebC and rebG, respectively. The formation of chromopyrrolic
acid or 11,11-dichlorochromopyrrolic acid, key intermediates of
indolocarbazole biosynthesis, is catalyzed by staD or rebD. It is
noteworthy that the staD family includes only two homologs, rebD
and VioB, which are involved in violacein biosynthesis. The staD
family is a new type of hemoprotein with a novel structure and
function.
N-glycosidic bond formation between the N-12 and C-1ў positions
is catalyzed by staG or rebG N-glycosyltransferases. rebG is the same
gene that was cloned in 2000 by Ohuchi et al. as ngt, which can
catalyze the N-glycosylation of the indolocarbazole chromophore.
In the staurosporine structure, there exists an additional, unusual
C–N bond between the N-13 and C-6ў positions. Onaka et al. showed
through gene disruption and bioconversion experiments that staN, a
P450 homolog, is responsible for this unusual C-N bond formation.
StaN was the first example used to show that the P450 homolog is
involved in N-glycosidic bond formation. Deletion of staG abolished
glycosylation and led to accumulation of K252c, whereas deletion of
staN resulted in the production of holyrine A. Salas et al. also showed
the function of staN in C–N bond formation by heterologous
expression of the staN gene.
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