Manzamines

Abstract

Manzamines are a unique class of β-carboline marine alkaloids with an unusual tetra- or pentacyclic system. These alkaloids have shown a variety of bioactivities against infectious diseases, cancer and inflammatory diseases. The greatest potential for the manzamine alkaloids appears to be against malaria, with improved potency relative to chloroquine and artemisinin. Over 80 manzamine-related alkaloids have been isolated from more than 16 species of marine sponges belonging to five families distributed from the Red Sea to Indonesia, which suggests a possible microbial origin for manzamine alkaloids. The current review summarizes marine literature, focusing on the biological activities of manzamines, the possible microbial origin of this class of compounds and the Red Sea as a possible source of manzamines from biosynthetic gene clusters of Red Sea microbes.

Keywords: :

• alkaloids
• antimalarial
• biosynthetic gene clusters
• manzamines
• marine sponges
• microbial origin
• Red Sea biota

Introducing Manzamine Alkaloids

Manzamines are a unique class of polycyclic alkaloids identified from marine sponges in the late 1980s.¹ These compounds possess a fused and bridged unique 5-, 6-, 6-, 8-, 13-membered heterocyclic ring system coupled to a β-carboline moiety through an apparent Pictet-Spengler reaction with the aldehyde known as ircinal A shown in the biogenetic pathway proposed by Baldwin and Whitehead.² Manzamine A, the prototype of this group, was first reported as the hydrochloride salt by Higa’s group from an Okinawan sponge Haliclona sp in 1986.¹

Following manzamine A, over 80 β-carboline-containing manzamine alkaloids and manzamine-related alkaloids have been isolated from marine sponges over the past two decades and the discovery of additional manzamine producing sponges seems highly likely. Wide structural variation has been reported, including the complex manzamine dimers kauluamine³ and neo-kauluamine,⁴ manadomanzamines A and B with unprecedented ring arrangements⁵ and 12,34-Oxamanzamines with a novel ring system generated through a new ether bridge formed between carbons 12 and 34 of the typical manzamine structure⁶ (Fig. 1).

Figure 1. Representative members of manzamine family of alkaloids.

Table 1 presents a survey of the β-carboline-containing manzamine and manzamine-related alkaloids discovered to date. Following this survey, it now appears that manzamines and related alkaloids are restricted to eight genera (Haliclona, Reniera, Amphimedon, Cribrochalina, Acanthostrongylophora, Petrosia, Hyrtios, Ircinia) within five families (Chalinidae, Niphatidae, Petrosiidae, Thorectidae, Irciniidae) in two orders (Haplosclerida, Dictyoceratida). These sponges have been collected from Okinawa, Philippines, Indonesia, Red Sea, Italy, South Africa and Papua New Guinea. Species producing the highest yields of manzamines are those in the genera Amphimedon sp^12,42,46 and Acanthostrongylophora,^5,6,38,47 which to date have yielded the greatest number of β-carboline-containing manzamine and manzamine-related alkaloids.

Table 1. Marine sponges yielding manzamines and related alkaloids

Taxonomy				Collection localities	Alkaloids		References
					β-Carboline Manzamines	Manzamine-related marine alkaloids
Order HAPLOSCLERIDA
	Family CHALINIDAE
		Genus Haliclona
			Haliclona spp	Manzamo and Amitori Bay (Iriomote Island), Okinawa	Manzamines A, B, C, D and Y		1,7,8
			Haliclona tulearensis	Sodwana Bay, South Africa		Haliclorensin	9
		Genus Reniera
			Reniera sp	Capo Miseno, Naples, Italy		Misenine	10
			Reniera sarai	Pulitzeri-Finali Bay of Naples		Saraine A	11
	Family NIPHATIDAE
		Genus Amphimedon
			Amphimedon spp	Kerama Islands, Okinawa	Manzamines A, B, C, D, G, H, L, M and Y, Keramamine, 3,4-dihydromanzamine A, 3,4-dihydro-6-hydroxy-manzamine A, 3,4-dihydromanzamine J, 1,2,3,4-tetrahydromanzamine B, Ma'eganedin	Nakadomarin A, Keramaphidin B, Ircinals A and B, Ircinols A and B, Keramaphidin C	12-24
		Genus Cribrochalina
			Cribochalina sp	Madang, Papua New Guinea	8-hydroxy-2-N-methyl- 1,2,3,4-tetrahydromanzamine A		25
	Family PETROSIIDAE
		Genus Acanthostrongylophora
			Xestospongia spp	Miyako Island and Amitori Bay (Iriomote Island), Okinawa	Manzamines A, B, C, D, E, F and X, Xestomanzamines A and B, Xestocyclamines A and B	Xestocyclamine A and B	26-30
			Prianos sp	Manado Bay, Sulawesi, Indonesia	Kauluamine		31
			Xestospongia ashmorica	Mindoro Island, Philippines	Manzamines A, E, F and J, 6-deoxy-manzamine X, manzamine A N-oxide, manzamine J N-oxide, 3,4-dihydromanzamine A N-oxide		32
			Xestospongia ingens	Papua New Guinea		Ingamines A and B, Ingenamine, Ingenamines B, C, D, E and F, Madangamines A, B, C, D and E	33-37
			Petrosiidae sp	Manado Bay, Sulawesi, Indonesia	ent-manzamine E, ent-8- hydroxymanzamine A, neo- kauluamine ent-12,34-Oxamanzamines E and F, 12,34-oxamanzamine A, manadomanzamines A and B, 32,33-dihydro-31-hydroxymanzamine A, 32,33-dihydro-6,31-dihydroxymanzamine A, 32,33-dihydro-6-hydroxymanzamine A-35-one		4-6,38,39
			Pachypellina sp	Manado Bay, Sulawesi, Indonesia	Manzamines A and G		40
			Pellina sp	Kerama islands, Okinawa	Manzamines A and E		41
		Genus Xestospongia de Laubenfels
			Xestospongia exigua	Papua New Guinea		Motuporamines A, B and C	42
		Genus Petrosia
			Petrosia contignata	Thiele Milne Bay, Papua New Guinea	8-hydroxy-1,2,3,4-tetrahydromanzamine A, 8-hydroxy-2-N-methyl- 1,2,3,4-tetrahydromanzamine A		25
Order DICTYOCERATIDA
	Family THORECTIDAE
		Genus Hyrtios
			Hyrtios erecta	Red Sea	Hyrtiomanzamine		43
	Family IRCINIIDAE
		Genus Ircinia
			Ircinia sp	Kise, Okinawa	Manzamine A, B, D, E, H and J	Ircinals A and B	12,14,44
Undetermined				Palau	epi-manzamine D, 2-N-methyl-epi-manzamine D		45

Biological Activity

The manzamine alkaloids have shown a diverse range of bioactivities including: cytotoxicity,^{1,13,26,27,31,32,44,48-50} antimicrobial,^4,39,41 insecticidal,^32,50 anti-inflammatory⁵¹ properties, and have shown activity against HIV and AIDS opportunistic infections.⁵² To date, the greatest potential for the manzamine alkaloids appears to be against malaria with manzamine A, (-)-8-hydroxymanzamine A, as well as neo-kauluamine¹³ showing improved activity over the clinically used drugs chloroquine and artemisinin in animal models. Recently, the manzamines have been patented for their immunosuppressant activity, which makes them candidates for use in organ transplant and autoimmune diseases.⁵³

The extraordinary biological activity of manzamines has stimulated great interest in finding a way to ensure a steady supply of these drug-leads for clinical trials and applications.

A simple method for kilogram-scale preparation of manzamines has been developed where the fresh sponge was extracted with acetone, and then a total alkaloid fraction was obtained using an acid-base partition scheme followed by fractionation using a silica gel vacuum liquid chromatography procedure. To overcome insufficiencies of this method, a model large-scale supercritical fluid chromatography (SFC) system for producing manzamine alkaloids was established.⁵⁴ As with any natural source of a potential medicine, a large-scale collection of the source marine sponge can be difficult due to scarcity of the invertebrate, and can also have negative environmental consequences. In addition, natural supplies can fluctuate, either seasonally or due to environmental changes.

Ideally, an efficient chemical synthesis of the desired natural product could be achieved; however the structural complexity of these natural products makes them challenging targets for total synthesis.^55,56 Synthetic studies of the manzamines have been reported by a number of groups using five different strategies (Fig. 2).⁵⁷ These include biomimetic, radical cyclisation and photochemical, ionic cyclisation, intermolecular Diels-Alder type cycloadditions, and intramolecular Diels-Alder cycloadditions approaches. Cycloadditions play a preeminent role in these synthetic studies, but alternative strategies based for instance, on [2+2] cycloaddition or Mannich cyclisations are also quite efficient. Biomimetic syntheses, however, still at an early stage, may prove to be versatile giving rise not only to manzamine type alkaloids, but also to related alkaloids in this series.⁵⁷

Figure 2. Different strategies used for total synthesis of Manzamine A.

All reported syntheses to date represent inefficient multistep approaches that cannot meet the demands of pre-clinical and clinical development. Identification of a simpler, more easily synthesized structure which retains the biological activity is another option, but the best scenario would be to have a supply of the natural product that can be generated inexpensively and reproducibly in the lab under controlled conditions.⁵⁸

A Microbial Source for Manzamines

The isolation of manzamine alkaloids from a growing number of sponge genera indicates the possible existence of a sponge-associated microorganism as the actual biosynthetic source for the manzamine alkaloids.^2,26-28 This possibility was proposed by Kobayashi et al. in 1995²⁸ after six species of sponges were known to contain manzamines and appears even more likely now that an even greater diversity of sponges are known to yield manzamines. This possibility warrants careful investigation as the pharmaceutical potential of manzamine alkaloids continues to grow. If these compounds are actually produced by symbiotic bacteria within the sponge, isolation and culture of the producing bacteria may provide an efficient method for production of the compound in fermentation systems. This could ensure a steady supply of a particular manzamine in the high likelihood that one of these drug-leads will advance into clinical trials and applications. Certainly, if it could be shown that manzamines are microbial products, the pharmaceutical and biotechnology community would express a greater interest in this group of compounds and sponge metabolites in general.

Sustainable sourcing for development has been a strong justification for thorough microbiological evaluation of manzamine-producing sponges. In addition, evidence that sponge-associated microbes may play a significant role in the bioconversion of manzamines to the growing number of alkaloids found in manzamine producing sponges is provided by the biotransformation of 8-hydroxymanzamine A and its entantiomer to manzamine A and ent-12,34-oxamazamine F, respectively.^6,38,47

The culturable microbial communities associated with two Acanthostrongylophra species were investigated to obtain isolates that could be examined for manzamine production.^5,6,38,47 A full molecular community analysis of the entire bacterial community (both culturable and unculturable) was completed for one of these species. This molecular approach is essential to explore the full diversity of microbes associated with sponges since typically less than 1% of the bacteria present are culturable by conventional approaches. Culturable isolates of heterotrophic bacteria were obtained from both species and unequivocally identified by 16S rRNA gene sequence analysis.

Diversity of Red Sea Biota

Our research groups at Suez Canal University (SCU) have been deeply involved in screening for production of biologically active compounds in microbes associated with marine macrobiota. We have isolated many different marine bacterial and fungal strains producing antimicrobial compounds and maintained the marine bacteria culture collection. A total of 453 marine bacterial isolates were obtained in pure culture from Red Sea sponges and cryopreserved. Based on colony morphology, 110 of these isolates were classified as putative actinomycetes. Both putative actinomycetes (grown on actinomycete selective media) and heterotrophic bacteria were screened for biologically active alkaloid production. Out of the 453 bacterial isolates tested, 95 isolates were positive for alkaloid production. A total of 25 of these isolates were putative actinomycetes. The culturable and unculturable microbial community associated with two manzamine-producing Red Sea Demosponges, Hyrtios erectus and Amphimedon sp were investigated and phylogenetic analysis revealed that the organisms isolated were from diverse phylogenetic groups, including Proteobacteria, Actinobacteria, Firmicutes, and Acidobacteria.^59-61 Thirty five heterotrophic bacterial isolates, including actinomycetes and α-proteobacteria, were isolated in a preliminary effort to identify a possible microbial origin for these compounds. These isolates are currently under investigations for manzamine production. In addition, our work provides an excellent resource of several candidate bacteria for production of novel pharmaceutically important compounds.

Biosynthetic Potentials of Red Sea Biota

Recent studies have revealed that sponge-associated microbes are often the producers of biologically active secondary metabolites^62,63 and, as such, there is the potential for cultivation of the producing microorganism and isolation of the bioactive compound. However, if the producing microbe is unculturable, biotechnology can be used to isolate and clone the natural product’s biosynthetic genes and produce the compound in a heterologous host.⁶⁴ This has been accomplished for several compounds, including the polyketide antibiotic erythromycin^65,66 and the cytotoxic polyketide-non-ribosomal peptide hybrids epothilone^67-69 and yersiniabactin.⁷⁰ The modular nature of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) make these product biosynthetic enzymes amenable to cloning and heterologous production. NRPSs are multimodular enzymes in which each module is responsible for the addition of a single amino acid. These enzymes produce nonribosomal peptides (NRPs) by a thiotemplate mechanism. NRPSs can incorporate proteinogenic and non-protein amino acids, as well as carboxy and hydroxyl acids. Further modifications, such as N-methylation, epimerization, cyclization and heterocyclic ring formation, can also be performed by these enzymes.⁷¹ NRPSs occur in a wide range of organisms, including bacteria, fungi, plants and marine organisms. These compounds often possess desirable pharmaceutical characteristics, and have thus been used for many applications, including use as siderophores, antibiotics, immunosuppressants and anticancer drugs (Dirk Konz 1999). So far, the majority of NRPSs and their products have been characterized in bacteria, especially Bacillus and Streptomyces species.

In a recent study,⁷² 12 PKS gene fragments were PCR amplified from Aspergillus sp isolated from Sharm El Sheik, Red Sea, Egypt using degenerative primer pairs. Sequence analysis reveals that the amplified PCR gene fragments show similarity to highly reduced (HR-PKS), six fragments, partially reduced (PR-PKSs) (also called methylsalicylic acid synthase (MSAS)-type, two fragments, and non-reducing (NR) PKSs (also called WA-type) PKS gene fragments in the database. Also, we report the characterization of a nonribosomal peptide synthetase (NRPS) gene in Aspergillus sp isolated from Sharm El Sheik, Egypt.

In addition, PKS gene fragments were PCR amplified from Negombata magnifica derived bacteria obtained from Red Sea (unpublished data). The KS domains were diverge from KSs from well-characterized clusters such as erythromycin,⁷³ pikromycin,⁷⁴ and epothilone,^75,76 which is structurally very similar to latrunculin.

Future Directions

This review sheds the light on manzamines as unique and viable leads against infectious diseases, cancer, and inflammatory diseases. If local and international organizations provide better funding for manzmaine research, manzamines would give antimalarial drug discovery and vaccine development a decisive boosts and optimistically, signal the demise of a scourge that has plagued humankind with such impunity. The isolation of these alkaloids from a wide range of sponge genera has led to the speculation that these intriguing structures maybe of microbial origin. The Red Sea biodiversity, although represents an excellent source for drug discovery and development. The lack of funding requires international funding. In the absence of a legal formula to protect the right of Egypt as the country of origin, authorities will block the funding and the research. We strongly advocate expanding the exploration of Red Sea biota as an untapped source of novel microorganisms, which may serve as the leads and scaffolds for elaboration into desperately needed efficacious drugs for a multitude of diseases.

The diversity of alkaloid biosynthesis gene clusters hinders their exploration in microbial biota. In future, mining the Red Sea microbial genomes for biosynthetic gene clusters could booster the discovery of new classes of manzamine related compounds.