Marine sponge compounds with antiplasmodial properties: Focus on in vitro study against Plasmodium falciparum

Malaria continues to be a major cause of morbidity and mortality in many tropical countries. The lack of progress in drug discovery and the spread of drug resistance become the reason behind this. Porifera (sponges) is a potential source of novel bioactive compounds to provide future drugs against malaria. In this review, we summarized 243 isolated molecules belonging to 35 different genera that active against Plasmodium falciparum from published paper until March 2019. The molecules were classified into potent, good, moderate, low, and inactive based on their IC50, and among observed bioactive metabolites, there were 57 marine sponge molecules reported to act as potent antiplasmodium against various strains of P. falciparum including drug resistance and nondrug resistance. Table 2 represents the list of isolated compounds with “potent” antimalarial activity. The class of the listed compounds includes manzamine alkaloid, guanidine alkaloids, bispyrroloiminoquinone alkaloids, pyrroloiminoquinone alkaloids, ingamine alkaloids, bromotyrosine alkaloids, sesquiterpenoids, diterpene formamides, aminoimidazole, β-galactosylceramides, β-lactam, meroterpene, trisoxazole macrolides, peroxides, thiazine alkaloids, and sterols. With this up-to-date review, we attempt to present new perspectives for the rational discovery of novel sponge metabolites that can be used as lead compounds in antimalarial drug development.


INTRODUCTION
Malaria is the most life-threatening and infectious disease caused by Plasmodium parasites such as Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae. Among those protozoans, P. falciparum is considered to be responsible for most severe diseases and most fatal cases. The World Health Organization (2018) stated in the year of 2017 that more than 99% of estimated malaria cases in the WHO African Region followed by the WHO regions of the Western Pacific (71.9%), the Eastern Mediterranean (69%), and Southeast Asia (62.8%) were caused by this most prevalent malaria parasite. In the same period, the WHO reported approximately 219 million cases of malaria occurred worldwide including 435,000 deaths.
Nowadays, malaria continues to be a major cause of morbidity and mortality in tropical countries. It is further aggravated by an increase in a number of multidrug-resistant strains of Plasmodium accompanied by a lack of progress in the development of vaccines and drug discovery. As a consequence, the search of new agent that actives against malaria becomes urgent needs (Antony and Parija 2016;Burrows et al., 2011;Cui et al., 2015;Dondorp et al., 2000;Noedl et al., 2008).
Marine ecosystems are the largest part of the biosphere. More than 70% of the Earth's surface is covered by water, and several theories believe that the life on earth originated from the ocean. In certain marine ecosystems such as coral reefs or the deep-sea floor, scientists estimate that the diversity of marine biota is even greater than the biota inhabiting tropical rainforests. Many immotile or slow-moving marine invertebrates, which usually do not have physical protection such as shells or thorns, will produce secondary metabolites as a form of defense mechanism from the environment and other creatures in the ocean (Ebada et al., 2008). These compounds attract the attention of researchers from various fields such as chemistry, pharmacology, biology, and ecology. This statement is supported by the fact that the number of new bioactive constituents isolated from marine biota has been increasing in the past three decades (332 compounds were isolated in 1984, and 1490 new compounds were isolated in 2017) (Blunt et al., 2016;Carroll et al., 2019).
From the perspective of drug discovery, a marine sponge is one of the invertebrate organisms which is interesting to be explored due to its potency producing new compounds (Anjum et al., 2016). The lack of physical defense of sponges resulting in secondary metabolites is estimated to vary depending on their habitats. Metabolite compounds isolated from sponges are highly diverse such as alkaloids, esters, fatty acids, glycosides, ketones, lipids, macrolides, peptides, peroxides, quinones, terpenoids, and polyketides and have shown many biological activities, in which one of them is antimalaria (Blunt et al., 2016;Carroll et al., 2019). These kinds of compounds have been found to interfere with pathogenesis at many distinct points; therefore, this can be beneficial in developing selective antimalarial drugs (Sipkema et al., 2005) The aim of this review is to summarize compounds isolated from marine sponges which exhibit in vitro antiplasmodial properties, to identify the compounds with potent activity based on their IC 50 values, and to highlight the most important functional groups of the compounds related to their potent activity against various strains of P. falciparum. One of the advantages of an in vitro study is that the study could thoroughly illustrate an effect of structural features of tested compounds to their activity with no interference from other factors such as biological system which can be found on in vivo study. Therefore, it can be used to generate more potent derivatives of the compounds to develop selective antimalaria drugs that work in blood-stage P. falciparum.

METHOD
A systematic search was accomplished to find all publications related to the theme until March 2019 in PubMed and Google Scholar. The keywords used to search the articles were "Plasmodium falciparum, sponge, antimalarial" or "Plasmodium falciparum, sponge, antiplasmodial." The data included in the review were primary articles in English about in vitro antimalarial study of pure compounds isolated from marine sponges against P. falciparum as shown in Table 1. The articles obtained were then removed if they are review articles, conference articles, and thesis, and there are no data available to be retrieved. All the synthetic compounds derived from naturally occurring metabolites in sponge are not mentioned in this review. Variables assessed in this review include sponge species/genus, isolated compound, strain of P. falciparum, region/country of origin, and effect on parasite growth inhibition.

EXPLORATION OF MARINE SPONGE METABOLITES FOR ANTIPLASMODIAL ASSAY
Among marine invertebrates, a sponge is the most dominant source for discovering natural products that have been used as lead compound to develop therapeutic drugs (Perdicaris et al., 2013). However, the study done in the investigation of marine sponge metabolites for antimalarial activity is relatively low compared to those of antitumor and anticancer. From literature published until March 2019, we included 50 primary articles for the review (Table 1). We identified that 35 different genera have been studied for their antiplasmodial activities and found that the most frequently studied genera were genus Agelas, Plakortis, and Xestospongia from different locations. Although many bioactive compounds have been isolated from marine sponges (Blunt et al., 2016;Carroll et al., 2019), the evaluation of their antiplasmodial activity is still relatively low. Figure 1 shows the number of studies that have been done on the examination of in vitro antiplasmodium of isolated compounds from marine sponge.
Overall, the number of publications from year to year shows fluctuation pattern. The highest number of the published papers was in the year of 2010 with 10 articles, followed by six publications in 2009 and 2012. In regard to the number of publications from 2013 to March 2019, it seemed to be stuck at one to three studies each year. This indicates that exploration trend of marine sponge metabolites for antiplasmodial activity diminished from 31 published papers during the period of 1992-2010 to 21 publications during the period of 2011-March 2019. One of the reasons behind the trend is that many scientists are interested in microbiological sample investigations for marine natural product exploration including bacteria and fungi sponge associated, making the detriment of sponge-derived compounds (Carroll et al., 2019;Thomas et al., 2010).
Various ecological studies have shown that secondary metabolites produced by sponges often serve defensive purposes to protect them from threats such as predator attacks, microbial infections, biofouling, and overgrowth by other sessile organisms (Paul and Puglisi, 2004;Paul et al., 2006). Therefore, compounds isolated from the same sponge species are more likely to be different if their habitat is distinct due to the ecological response (Mani et al., 2012). Moreover, a review done by Qaralleh (2016) found out that among 27 species of genus Neopetrosia, there are only nine species which have been chemically studied thus far. These facts disclose significant opportunities to do the chemical constituent exploration from not only genus Neopetrosia but also the other genus. In terms of collection site of the sponges, Australia, Bahamas, Indonesia, and Thailand were the most explored site so far for the search of compounds which exhibit in vitro antiplasmodium (P. falciparum strains). Other sponges were collected from Turkey, Vanuatu, Madagascar, Caledonia, Fiji, China, Japan, Alaska, Jamaica, Solomon Island, Puerto Rico, Papua New Guinea, and others (Table 1).   (20)

CLASSIFICATION OF ANTIPLASMODIAL ACTIVITY OF ISOLATED COMPOUND FROM SPONGES
In this review, we give an overview of the bioactive metabolites recently isolated from marine sponges that have shown activity in in vitro study against P. falciparum. To compare the IC 50 values, the units in µg/ml and nM were converted to µM. All the isolated compounds were then classified based on their IC 50 values by following the definition of Batista et al. (2009), who grouped compounds into potent activity: IC 50 < 1 μM, good activity: IC 50 of 1-20 μM, moderate activity: IC 50 of 20-100 μM, low activity: IC 50 of 100-200 μM, and inactive: IC 50 >200 μM (Batista et al., 2009). To be noted, the mechanism of the in vitro continuous cultures of P. falciparum approach is only related to the inhibition of growth in erythrocytic stages of the parasite (Chin et al., 1979). Consequently, this IC 50 -based classification would exclude compounds that may have other specific mechanism of action. It would be wise to re-evaluate "not active compounds" with other assay or holistic approach such as the reverse pharmacology technique (Simoes-Pires et al., 2014).
As shown in Figure 2, among observed bioactive metabolites, there were 57 different compounds that have potent activity, 101 with good activity, and 26 compounds with moderate activity against various strains of P. falciparum. Some of the compounds could not be classified because, in the highest tested concentration, their activity was low or inactive and some reports use inhibition concentration instead of IC 50 , making it incomparable. In regard to the dependency of IC 50 to plasmodium strains, it seems that antiplasmodial activity of some isolated compounds did not depend on chloroquine/drug sensitivity of the strain Longeon et al., 2010;Mani et al., 2012).

FUNCTIONAL GROUP IN POTENT ANTIPLASMODIAL ACTIVITY
Some marine isonitriles show various biological activities such as antimalarial, antitubercular, antifouling, and antiplasmodial effect. Marine isonitriles differ from terrestrial isonitriles in terms of their biosynthetic pathways. Most of the marine compounds containing isonitrile were derived from terpenoid, whereas terrestrial isonitriles originate from α-amino acids (Emsermann  (1) is a sesquiterpene derived from chloroform fraction of sponge Acanthella klethra containing isonitrile group which appears to be crucial for activity since the corresponding isothiocyanate derivative compound 2 (moderate activity) is less active than 1 (potent activity) (Angerhofer et al., 1992). The eudesmane compounds 3 and 4 which contain isothiocyanate still showed good antiplasmodial activity, whereas the reversal of the stereochemical configuration between 4 and 5 exhibits a significant change on their antiplasmodial effect (see Figure 3). The manzamines are a group of marine alkaloids characterized by a fused and bridged tetra-or pentacyclic ring system attached to a β-carboline moiety. Since manzamine was isolated in different genus of sponges, it is thought that manzamine is actually produced by associated microorganism. An interesting review had been done by Fattorusso and Taglialatela-Scafati (2009) who described the key role of the eight member rings as well as other functional groups that affect the antiplasmodial activity of manzamines; therefore, we will not discuss it in this review.
Sponge Cymbastela hooperi sp. nov. described by Soest et al. (1996) (20) were new diterpene formamides which were isolated from C. hooperi (Figure 3). Compound 18 is a unique molecule since it contains both formamide and isonitrile functionalities where such a feature is rarely found in natural product. Based on its IC 50 against P. falciparum FCR3F86, this substituent is classified into potent (Wright and Lang-Unnasch, 2009). The lack of isonitrile in the structure of 19 decreases the activity to be moderate. This finding is supported by the activity of compound 1 that possesses isonitrile too (Angerhofer et al., 1992).
Psammaplysin H (21) derived from sponge genus Pseudoceratina is also included in the potent activity group against P. falciparum 3D7 with IC 50 0.41 µM. This activity is more likely caused by the presence of quaternary amine in the R group at C-20 (see Figure 3). However, the secondary amine at the same position in psammaplysin F (22) reduced antimalarial activity 4-fold lower than compound 21. In addition, when the alkyl amine is substituted with a urea at C-20 in Psammaplysin G (23), the activity decreased to have IC 50 5.99 µM (Xu et al., 2011). Consistently, the loss of amine substituent in psammaplysin K (24) dispelled the antiplasmodial activity (Mudianta et al., 2012).

CONCLUSION
Data presented in the review indicate that marine sponges could be used as sources for lead compounds in drug discovery program including the development of non-resistance antimalarial drugs in this case. The summarized "potent" isolated compounds highlight the most promising candidates which include manzamine alkaloids, guanidine alkaloids, bispyrroloiminoquinone alkaloid, pyrroloiminoquinone alkaloids, ingamine alkaloids, sesquiterpenoids, diterpene formamides, aminoimidazole, β-galactosyl ceramides, β-lactam, meroterpene, trisoxazole macrolides, peroxides, thiazine alkaloids, bromotyrosine alkaloids, and sterols. A holistic approach for their pharmacological evaluation is still needed since in vitro P. falciparum assay could only evaluate a specific mechanism of action for antiplasmodium. To reproduce the compounds for their further evaluation, the possibility of bioengineering or/and bacterial fermentation could be worth.

ACKNOWLEDGMENT
The author would like to acknowledge the funding support from UGM No: 3040/UN1/DITLIT/DIT-LIT/LT/2019.