![Staurosporine](https://upload.wikimedia.org/wikipedia/commons/0/07/Staurosporine.svg)
Indolocarbazoles (ICZs) are a class of compounds that are under current study due to their potential as anti-cancer drugs and the prospective number of derivatives and uses found from the basic backbone alone. First isolated in 1977,
a wide range of structures and derivatives have been found or developed throughout the world. Due to the extensive number of structures available, this review will focus on the more important groups here while covering their occurrence, biological activity, biosynthesis, and laboratory synthesis.
Chemical classification
Indolocarbazoles belong to the
alkaloid
Alkaloids are a class of basic, naturally occurring organic compounds that contain at least one nitrogen atom. This group also includes some related compounds with neutral and even weakly acidic properties. Some synthetic compounds of similar ...
sub-class of bisindoles. The most frequently isolated indolocarbazoles are Indolo(2,3-a)carbazoles; the most common subgroup of the Indolo(2,3-a)carbazoles are the Indolo(2,3-a)pyrrole(3,4-c)carbazoles. These can be divided into two major classes - halogenated (chlorinated) with a fully oxidized C-7 carbon with only one indole nitrogen containing a β-glycosidic bond and the second class consists of both indole nitrogen glycosylated, non-halogenated, and a fully reduced C-7 carbon.
Occurrence
The first isolated ICZ, dubbed
staurosporine
Staurosporine (antibiotic AM-2282 or STS) is a natural product originally isolated in 1977 from the bacterium '' Streptomyces staurosporeus''.
It was the first of over 50 alkaloids to be isolated with this type of bis-indole chemical structure. Th ...
(STA) was in 1977 from a culture of ''Streptomyces staurosporeus'' found in a soil sample from Iwate Prefecture, Japan. The proper
stereochemistry
Stereochemistry, a subdiscipline of chemistry, involves the study of the relative spatial arrangement of atoms that form the structure of molecules and their manipulation. The study of stereochemistry focuses on the relationships between stereois ...
was not proven until 1994. Over the course of the next decade, further study of the compound showed some fungal inhibition, hypotensive activity, and most importantly, a broad protein kinase inhibitor. The next landmark discovery came with the detection of
rebeccamycin
Rebeccamycin (NSC 655649) is a weak topoisomerase I inhibitor isolated from ''Nocardia sp.'' It is structurally similar to staurosporine, but does not show any inhibitory activity against protein kinases. It shows significant antitumor properties ...
(REB) in a sample of ''Lechevalieria aerocolonigenes'', again in soil, but this time in a sample from
Panama
Panama ( , ; es, link=no, Panamá ), officially the Republic of Panama ( es, República de Panamá), is a transcontinental country spanning the southern part of North America and the northern part of South America. It is bordered by Cos ...
. REB was found to act against
leukemia
Leukemia ( also spelled leukaemia and pronounced ) is a group of blood cancers that usually begin in the bone marrow and result in high numbers of abnormal blood cells. These blood cells are not fully developed and are called ''blasts'' or ' ...
and
melanoma
Melanoma, also redundantly known as malignant melanoma, is a type of skin cancer that develops from the pigment-producing cells known as melanocytes. Melanomas typically occur in the skin, but may rarely occur in the mouth, intestines, or eye ( ...
in mice, and also against human
adenocarcinoma
Adenocarcinoma (; plural adenocarcinomas or adenocarcinomata ) (AC) is a type of cancerous tumor that can occur in several parts of the body. It is defined as neoplasia of epithelial tissue that has glandular origin, glandular characteristics, or ...
cells.
Since 1977, ICZs have been discovered all over the world in actinomycetes, bacteria commonly found in soil. Numerous forms have tested positive for anti-tumor activity, such as 7-hydroxy-STA and 7-oxo-STA2. Some of the strains from which ICZ compounds have been found are Actinomadura melliaura in Bristol Cove,
San Diego County, California
San Diego County (), officially the County of San Diego, is a county in the southwestern corner of the U.S. state of California. As of the 2020 census, the population was 3,298,634, making it California's second-most populous county and the fi ...
, ''
Streptomyces hygroscopicus'' in
Numazu Prefecture, Japan, Micromonospora sp. L-31-CLO-002 from
Fuerteventura Island, Canary Islands, Spain, and Actinomadura sp. Strain 007 from Jiaozhou Bay,
Shandong
Shandong ( , ; ; alternately romanized as Shantung) is a coastal province of the People's Republic of China and is part of the East China region.
Shandong has played a major role in Chinese history since the beginning of Chinese civilizati ...
, China. The wide distribution of the various strains that produce these compounds is not surprising due to the number of properties these compounds can take on with limited functionalization on the species's part.
In addition to
actinomycetes
The Actinomycetales is an order of Actinomycetota. A member of the order is often called an actinomycete. Actinomycetales are generally gram-positive and anaerobic and have mycelia in a filamentous and branching growth pattern. Some actinomycete ...
, ICZs have been found in
slime mold
Slime mold or slime mould is an informal name given to several kinds of unrelated eukaryotic organisms with a life cycle that includes a free-living single-celled stage and the formation of spores. Spores are often produced in macroscopic mu ...
s (myxomycetes), blue-green algae (
cyanobacteria
Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blu ...
, and marine invertebrates. Like the ones derived from actinomycetes, the ones found in myxomycetes cover an expansive range of derivatives and functionalizations. Two of the more important ones to date have been Arcyriacyanin A, which was found to inhibit a panel of human cancer cells by effecting PKC and protein tyrosine kinase, and lycogalic acid dimethyl ester A (found in Tokushima, Japan from ''
Lycogala epidendrum
''Lycogala epidendrum'', commonly known as wolf's milk or groening's slime, is a cosmopolitan species of myxogastrid amoeba which is often mistaken for a fungus. The aethalia, or fruiting bodies, occur either scattered or in groups on damp rott ...
''), which showed strong antiviral activity. A few of the strains of
myxomycete
Myxogastria/Myxogastrea (myxogastrids, International Code of Zoological Nomenclature, ICZN) or Myxomycetes (International Code of Nomenclature for algae, fungi, and plants, ICN), is a Class (biology), class of slime molds that contains 5 o ...
s studied are
Arcyria
''Arcyria'' is a genus of Amoebozoa in the family Arcyriaceae.Cooke, M.C. (1877) Contributions to Mycologica Britannica. The Myxomycetes of Great Britain: 69 (1877)[Kochi Prefecture
Kochi (), also known as Cochin ( ) (List of renamed Indian cities and states#Kerala, the official name until 1996) is a major port city on the Malabar Coast of India bordering the Laccadive Sea, which is a part of the Arabian Sea. It is part ...]
, Japan.
Three species of cyanobacteria has been found to produce ICZ compounds. ''
Nostoc
''Nostoc'', also known as star jelly, troll’s butter, spit of moon, fallen star, witch's butter (not to be confused with the fungi commonly known as witches' butter), and witch’s jelly, is the most common genus of cyanobacteria found in var ...
sphaericum'' from Manoa Hawaii, ''
Tolypothrix tjipanasensis'' from
Vero Beach, Florida
Vero Beach is a city in and the seat of Indian River County, Florida, United States. Vero Beach is the second most populous city in Indian River County. Abundant in beaches and wildlife, Vero Beach is located on Florida's Treasure Coast. It is thi ...
, and ''
Fischerella
''Fischerella'' is a genus of cyanobacteria belonging to the family Hapalosiphonaceae.
The genus was first described by M. Gomont in 1895.
The genus has cosmopolitan distribution.
The genus name of ''Deightoniella'' is in honour of Christia ...
ambigua'' strain 108b from
Leggingen, Switzerland. An interesting note on the first two is that many of the ICZ derived from them do not have the annelated pyrrolo
,4-cunit.
The final major group in which ICZs are found are various marine invertebrates. Three species of
tunicate
A tunicate is a marine invertebrate animal, a member of the subphylum Tunicata (). It is part of the Chordata, a phylum which includes all animals with dorsal nerve cords and notochords (including vertebrates). The subphylum was at one time ca ...
, one
mollusk
Mollusca is the second-largest phylum of invertebrate animals after the Arthropoda, the members of which are known as molluscs or mollusks (). Around 85,000 extant species of molluscs are recognized. The number of fossil species is e ...
, one
flatworm
The flatworms, flat worms, Platyhelminthes, or platyhelminths (from the Greek πλατύ, ''platy'', meaning "flat" and ἕλμινς (root: ἑλμινθ-), ''helminth-'', meaning "worm") are a phylum of relatively simple bilaterian, unsegment ...
, and one
sponge
Sponges, the members of the phylum Porifera (; meaning 'pore bearer'), are a basal animal clade as a sister of the diploblasts. They are multicellular organisms that have bodies full of pores and channels allowing water to circulate through t ...
have been discovered in places ranging from Micronesia to New Zealand. Testing for further invertebrate production is ongoing by both genetic and phylum-based studies.
Biological activity
Indolocarbazoles have been found to exhibit a wide range of activities, which makes their range of presence in nature unsurprising. Because of this variety, the following section will examine their modes of action in bacterial and mammalian cells independently, with special attention paid to cancer cell effects.
The general modes of action found in mammalian cells are inhibition of
protein kinase
A protein kinase is a kinase which selectively modifies other proteins by covalently adding phosphates to them (phosphorylation) as opposed to kinases which modify lipids, carbohydrates, or other molecules. Phosphorylation usually results in a fu ...
s, inhibition of eukaryotic
DNA topoisomerase
DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues i ...
, and intercalative binding to DNA. The number of protein kinases thought to exist in the human genome exceeds six hundred, making a nanomolar inhibitor such as STA extremely useful for both treatment of various diseases and study of protein kinases in a variety of functions. Since this discovery, a vast effort has been undergone to make highly specific STA and REB derivatives.
One of the major lessons learned from initial research on STA was the development of the
pharmacophore
300px, An example of a pharmacophore model.
A pharmacophore is an abstract description of molecular features that are necessary for molecular recognition of a ligand by a biological macromolecule. IUPAC defines a pharmacophore to be "an ensemble o ...
model for a protein kinase inhibitor in which a bidentate hydrogen donating system flanked by various hydrophobic groups inserts into the binding site. The information derived from this original pharmacophore has led to the synthesis of highly specific inhibitors against a number of protein kinases, including PKC, cyclin-dependent kinases, G-protein coupled receptor kinases, tyrosine kinase, and cytomegalovirus pUL97 protein.
Topoisomerase
DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues i ...
I and
II cleave and relegate one and two sides of a DNA strand, respectively, and are consequently vital parts of cell reproduction. Studies have found that in REB-like structures, the imide function of the pyrrole segment acts to interact with Topoisomerase I, the main carbon backbone acts as an intercalative inhibitor, and the sugar moiety undergoes DNA groove binding.
The latter two actually act in unison due to the three-dimensional structure of a glycosylated REB molecule.
The Top1 inhibitor section binds to cleavable DNA-Top1 complexes so as to prevent the relegation step. Because of this, sensitivity is based on quantity of Top1 present, making cells undergoing constant reproduction and growth (namely tumor cells) most vulnerable.
At this point, bacterial inhibition of Top1 has not been found using ICZs. Because of this, it is thought that most of the anti-cell growth function of ICZs comes from inhibition of various protein kinase groups and intercalative DNA binding.
Studies on ''
Streptomyces griseus
''Streptomyces griseus'' is a species of bacteria in the genus ''Streptomyces'' commonly found in soil. A few strains have been also reported from deep-sea sediments. It is a Gram-positive bacterium with high GC content. Along with most other s ...
'' with in vitro protein labelling have led to inhibition of a wide range of cellular functions. This led to the theory that there were several eukaryotic protein kinases present required for secondary metabolism.
Biosynthesis
Unfortunately, only biosynthesis of REB, STA, and K252a have been studied in depth. This section will emphasize the REB pathway due to how well studied it is. The pathway begins with the modification of L-
tryptophan
Tryptophan (symbol Trp or W)
is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α- carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
to 7-chloro-L-tryptophan. This is done by catalysis using RebH in vitro halogenation and RebF (a flavin reductase) to provide FADH2 for the halogenase. RebO (a tryptophan oxidase) then deaminates, after which it is further reacted with another one of itself and RebD (a heme containing oxidase). This forms the majority of the carbon backbone, which then undergoes decarboxylative ring closure using RebC and RebP. A
glycosylation
Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not al ...
occurs using RebG and NDP-D-glucose, which finally goes through methylation by RebM. These latter tailoring enzymes have been noted as permissive in terms of both aglycons/acceptors and glycosyl/alkyl donors. A parallel pathway has been put forth for the structurally related disaccharide-substituted indolocarbazole
AT2433, the aminopentose of which is also found appended to the 10-membered
enediyne
In organic chemistry, enediynes are organic compounds containing two triple bonds and one double bond.
Enediynes are most notable for their limited use as antitumor antibiotics (known as enediyne anticancer antibiotics). They are efficient at ...
calicheamicin.
Information for this pathway, along with those of K252a and STA, was derived from information on known genes, enzymes, and intermediates. The two types of studies done on these pathways are in vivo studies of gene disruption of L. aerocolonigenes or recombinant strains of S. albus.
The second type of experiment consisted of in vitro experiments done on cell extracts.
Synthesis
Laboratory synthesis of ICZs has been a topic of great interest since their discovery. Unfortunately, due to the somewhat complex nature of the molecule and the high level of reactivity of carbons on indole molecules, a facile high yield synthesis has yet to be found. Despite this, there have been many ways found to produce this compound in its various forms. Of special interest is one of the better REB syntheses, found in 1999. The process begins by producing 7-chloroindole-3-acetamide by treating 7-chloroindole with a series of reagents, shown farther down. This molecule is then glycosylated and reacted with methyl 7-chloroindole-3-glyoxylate to produce an intermediate that goes on to stabilize into the final product. While this process is one of the better ones to date, it is still work and time intensive, going through 12 total steps and only yielding 12%.
Further developments
Ever since the birth of ICZ research in the late seventies, the field has been burgeoning with continued advances in both technology and organic chemistry techniques. While only a handful of ICZ based compounds have made it past stage II clinical trials, the sheer variety that these molecules can take on leaves much still unexplored territory. Of particular recent interest in synthesis techniques is the use of
palladium
Palladium is a chemical element with the symbol Pd and atomic number 46. It is a rare and lustrous silvery-white metal discovered in 1803 by the English chemist William Hyde Wollaston. He named it after the asteroid Pallas, which was itself na ...
based catalysts, which have been found to be excellent activators for use in formation of carbon-carbon bonds.
References
External links
Rebeccamycin and AT2433 from Center for Pharmaceutical Research and Innovation
{{Chemotherapeutic agents
Protein kinase inhibitors
Experimental cancer drugs