Metabolites from Marine Sponges and Their Potential to Treat Malarial Protozoan Parasites Infection: A Systematic Review

Malaria is an infectious disease caused by protozoan parasites of the Plasmodium genus through the bite of female Anopheles mosquitoes, affecting 228 million people and causing 415 thousand deaths in 2018. Artemisinin-based combination therapies (ACTs) are the most recommended treatment for malaria; however, the emergence of multidrug resistance has unfortunately limited their effects and challenged the field. In this context, the ocean and its rich biodiversity have emerged as a very promising resource of bioactive compounds and secondary metabolites from different marine organisms. This systematic review of the literature focuses on the advances achieved in the search for new antimalarials from marine sponges, which are ancient organisms that developed defense mechanisms in a hostile environment. The principal inclusion criterion for analysis was articles with compounds with IC50 below 10 µM or 10 µg/mL against P. falciparum culture. The secondary metabolites identified include alkaloids, terpenoids, polyketides endoperoxides and glycosphingolipids. The structural features of active compounds selected in this review may be an interesting scaffold to inspire synthetic development of new antimalarials for selectively targeting parasite cell metabolism.


Introduction
Human malaria is an infectious disease caused by single-celled protozoan parasites of the Plasmodium genus (P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi) through the bite of female Anopheles mosquitoes [1]. It affected 228 million people in 2018, and nearly half of the world's population is still at risk for this disease [2]. Symptoms can range from being mild to very severe, causing chronic illness, physical disability, death and a huge health burden, especially to the most vulnerable populations.
Antimalarials based in quinolines scaffolds (i.e., chloroquine, mefloquine, amodiaquine, and piperaquine) possess a complex mechanism of action. One well-studied mechanism involves compromising the detoxification of hemoglobin degradation with heme polymerization for hemozoin crystal formation in digestive vacuole by protonated forms of quinolones [3]. It was noted that some strains of P. falciparum triggered resistance to protonated drugs due to a genetic mutation in the transporter (PfCRT) and could lead to antimalarial drug extrusion from the organelle [3].
Artemisinin-based combination therapies (ACTs) are the most recommended treatment for uncomplicated P. falciparum malaria, while artesunate is considered the most effective antimalarial drug for severe cases [4], with several biochemical processes reported as targets in parasite cells [3,5]. Despite the safety and efficiency that have been proven for the use of these drugs, the emergence of multidrug resistance has unfortunately limited their effects and challenged the field [6]. The resistance to ACTs is already spreading from from low micromolar to low nanomolar range, and the species Xestospongia sp showed the best bioactive potential, from which the compound Saringosterol was extracted, which had an IC 50 of 0.25 nM. The individual IC 50 for each extracted compound is reported in Table 1. The IC 50 value units in µg/mL and ng/mL were converted to µM and nM for data comparison, and then some of compounds in Table 1, which IC 50 was below 10 µg/mL became higher than 10 µM (see Section 3.2.2, exclusion criteria), as were compounds 10 and 11 [21], 52 [22], 2 and 3 [23], 99 [24]. A summary of the studies is presented in Table 1. The articles analyzed were published from 1992 to 2019 in different countries. The antimalarial activity was assessed in vitro using Plasmodium falciparum culture [20] for quantification of cell viability over 24-      To assess the study quality, we used the GRADE method [55]. The 36 studies analyzed were categorized as moderate quality (17) because (i) there were no controls in the experiments; (ii) the toxicity of the compounds was not assessed in parallel, which made it impossible to determine the selectivity of compounds; (iii) all compounds analyzed presented a high cytotoxicity, which demonstrates the unspecified use against P. falciparum; (iv) the methods used to measure the antiplasmodial activity were not described. A total of 19 studies were classified as being of high quality (Table S1).
After this detailed review of the articles reporting the activity of compounds from marine sponges, we made a brief survey of data in the literature to compare the number of articles published reporting the activity of marine organisms with the number of articles published reporting the activity of extracts from plants. To do so, the following combinations of keywords were used: "new antimalarials and plants" or "new antimalarials and marine" and selected the works published in the last 10 years. Figure 2 represents the number of studies reporting antiplasmodial activity of new compounds found. The search for new compounds from marine sources is still uncommon compared to the search for natural products from plants. Other recent reviews have also reported this comparison, which reinforces the importance of seeking new products from marine sources, especially considering that the diverse nature of metabolites produced by these alternative sources presents a compelling case for intensive exploration [56].
1 determine the selectivity of compounds; (iii) all compounds analyzed presented a high cytotoxicity, which demonstrates the unspecified use against P. falciparum; (iv) the methods used to measure the antiplasmodial activity were not described. A total of 19 studies were classified as being of high quality (Table S1).
After this detailed review of the articles reporting the activity of compounds from marine sponges, we made a brief survey of data in the literature to compare the number of articles published reporting the activity of marine organisms with the number of articles published reporting the activity of extracts from plants. To do so, the following combinations of keywords were used: "new antimalarials and plants" or "new antimalarials and marine" and selected the works published in the last 10 years. Figure 2 represents the number of studies reporting antiplasmodial activity of new compounds found. The search for new compounds from marine sources is still uncommon compared to the search for natural products from plants. Other recent reviews have also reported this comparison, which reinforces the importance of seeking new products from marine sources, especially considering that the diverse nature of metabolites produced by these alternative sources presents a compelling case for intensive exploration [56].

Classes of Compounds Found in Marine Sponge Extracts
The compounds isolated from marine sponges presented in the articles analyzed with antiplasmodial effect belong to alkaloids, terpenes and polyketides class of secondary metabolites. Most of the compounds with potential activity against Plasmodium sp. are alkaloids (69% of 259), followed by terpenoids (17%) and polyketides endoperoxides (13%) (Figure 3). There are also reports of P l a n t s S p o n g e B a c t e r i a F u n g i

Classes of Compounds Found in Marine Sponge Extracts
The compounds isolated from marine sponges presented in the articles analyzed with antiplasmodial effect belong to alkaloids, terpenes and polyketides class of secondary metabolites. Most of the compounds with potential activity against Plasmodium sp. are alkaloids (69% of 259), followed by terpenoids (17%) and polyketides endoperoxides (13%) (Figure 3). There are also reports of glycosphingolipids (GSL) from sponges able to inhibit the malaria parasite as well. The structures with their potency are described, and some of them present the known mechanism of action, which are discussed below.
2 glycosphingolipids (GSL) from sponges able to inhibit the malaria parasite as well. The structures with their potency are described, and some of them present the known mechanism of action, which are discussed below.
A bioguided fractionation of Pacific marine sponge Acanthostrongylophora ingens (Petrosiidae family) using in vitro assay with P. falciparum yielded the isolation of manzamine alkaloids (12-15)
Derivatives of plakortin named gracilioetheres A-C from Agelas gracilis were isolated from a bioassay-guided approach from an active extract using P. falciparum assay in vitro, highlighting gracilioether B (106) with a IC 50 value of 1.41 µM and moderate cytoxicity [46].

Glycosphingolipids
Glycosphingolipids (GSL) are glycolipids with sugar moiety well known for the immunomodulating activity, and they have been identified in marine sponges from Agelas and Axinyssa genus [35,64]. Although there are few reports of GSL from marine sponges with antimalarial potential, Farokhi and co-workers [35] isolated a GSL with antiplasmodial activity in the low micromolar range (IC 50 of 0.53 µM) and with low cytotoxic effect. The active mixture of GSL consists of different carbon chain lengths named axidjiferoside-A, -B and -C (107) from Axinyssa djiferi (Dictyonellidae family).

Mechanisms of Action of the New Compounds Found in Marine Sponge Extracts
We explore the mechanism of action of each class in the literature among other cell models to present a possible mechanism involved in the inhibition of Plasmodium development ( Figure 6) because of the absence of this information in many articles described in Table 1.
The alkaloids are the largest group of compounds mentioned in this review; however, they contain a significant number of molecules (17%) with unknown mechanisms. Some alkaloid compounds can be related with inhibition of signaling pathways, and induction of apoptosis and changes in gene expression are also indicated (14-37%) [23,[65][66][67][68][69][70][71][72][73][74][75][76][77]. Alkaloids could present oxidant and antioxidant effects depending on the biosynthetic precursor. For example, bromothyrosine derivatives can induce apoptosis by the formation of reactive oxygen species or selective inhibition of histone deacetylases in eukaryotic cell lines [65]. This effect can be also observed with a marine metabolite (Psammaplin A) and analogues, resulting in disruption of the epigenetic cell control and compromising the gene expression and cell survival [73,78].

Extracts
We explore the mechanism of action of each class in the literature among other cell models to present a possible mechanism involved in the inhibition of Plasmodium development ( Figure 6) because of the absence of this information in many articles described in Table 1. The alkaloids are the largest group of compounds mentioned in this review; however, they contain a significant number of molecules (17%) with unknown mechanisms. Some alkaloid compounds can be related with inhibition of signaling pathways, and induction of apoptosis and changes in gene expression are also indicated (14-37%) [23,[65][66][67][68][69][70][71][72][73][74][75][76][77]. Alkaloids could present oxidant and antioxidant effects depending on the biosynthetic precursor. For example, bromothyrosine derivatives can induce apoptosis by the formation of reactive oxygen species or selective inhibition of histone deacetylases in eukaryotic cell lines [65]. This effect can be also observed with a marine metabolite (Psammaplin A) and analogues, resulting in disruption of the Quinoline analogs have been extensively studied concerning their role as the cell targets in cancer, bacteria, virus, fungi and parasites. Some of its described mechanisms are related to key cellular processes (replication, transcription, protein metabolism, etc.) because of the interaction of quinolines compounds with DNA and inhibition of topoisomerase enzymes [79,80]. Endoplasmic reticulum stress, autophagy, and cell signaling with inhibition of several enzymes (i.e., N-acetyltransferase, cyclin dependent kinase, telomerase, caspase proteases) have also been observed [77]. The impairment of cell signaling and ionic homeostasis can be observed with the antagonist effect of voltage-dependent calcium channel by guanidine derivatives alkaloids [68] and Na + homeostasis by selective inhibition of Plasmodium falciparum P-type ATPase with indole-based natural alkaloids in a low micro-molar range [81,82]. Another important cell target is cytoskeleton filaments, which are essential for transport, cell division and organization. Some marine sponge compounds (trisoxazole-containing macrolides) can bind to F-actin subdomains by mimicking the interaction of actin-capping gelsolin family proteins, compromising the filament dynamics and leading to cell death [70,76]. The fatty acids biosynthesis is another important process for eukaryotic cells and is responsible for building membrane structures and energy metabolism. Pyrrole-imidazole alkaloids from marine sponge Agelas oroides present an inhibition effect at low micromolar range in Plasmodium falciparum enoyl-ACP reductase assay [23], which belongs to type II fatty acid pathway (FAS-II).
The second representative group is terpenes (43), which possess action related to oxidative stress and signaling pathways (30-34%) [49,50,83,84], as reported in normal and cancer cells lines, where ROS production was increased after a norterpene endoperoxide compound treatment [85]. A third group corresponds with the polyketides compounds (34), which have been shown to interact with Fe(II)heme, compromising the cell survival [86].
The available antimalarials (i.e., artemisinin) belong to the sesquiterpene group, and to some degree, the action mechanism of related sponge metabolites in Plasmodium was found to be consistent with that observed with artemisinin affecting the cell oxidative stress state and hemoglobin metabolism [3,81,87]. Hemoglobin metabolism as the principal parasite amino acid source in the host cell leads to the formation of toxic metabolites (reactive oxygen species-ROS and ferriprotoporphyrin IX). The unbalanced detoxification of these metabolites in parasite cytosol promoted by artemisinin or analogs affects many aspects of the cell physiology [81,87] as oxidative damage in different cell molecules. Some covalent protein interactions were identified with artemisinin in P. falciparum, indicating a broad action in cell metabolism, such as ornithine aminotransferase, pyruvate kinase, L-lactate dehydrogenase, spermidine synthase and S-adenosylmethionine synthetase [81]. In the same class of the endoperoxides, plakortin-related compounds from the sponge genus Plakortis bind to Fe(II) resulting in the formation of oxygen radicals and creates a cell-damaging environment for the parasite [86].
The current scenario of the development of new antimalarial drugs shows a promising molecule source from marine organisms such as sponges. However, these organisms have some weaknesses in discovering and developing antimalarial drugs: (i) the large amount of sponges' weight needed for each compound's identification and isolation; (ii) sponges are organisms' symbionts with sponge-specific microbiota (unicellular eukaryotes, bacteria, fungi, virus) [88], which increases the variability from each specimen and makes it very difficult to reproduce in laboratory cultivation for identifying the source of active compounds. However, due to the ancient relationship with the hostile environment, these organisms can present a large molecule library against pathogens, which would be useful for the development of synthetic derivatives and analogs with selective inhibition of human pathogens. The cost-accessible molecular strategies available in center facilities (i.e., highthroughput genome sequencing and mass spectrometry, molecular docking) could surpass these limitations to the identification of compounds from complex organisms. An upscaling number of articles on marine source compounds every year presenting molecules reveals its importance with different action mechanisms in eukaryotic cell physiology, as mentioned in this review.

Review Protocol
A systematic review of the literature was performed according to the SYRCLE guideline [89]. The following databases were consulted for this research: PubMed, Web of Science and Scopus. The search was carried out according to the orientations of PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis). To start the review, some descriptors of the MeSH (Medical Subject Headings) were defined: "Plasmodium falciparum", "P. falciparum", "antimalarial" and "sponge". In addition, two independent reviewers (J.R.P., A.C.C.A.) searched the databases, analyzing title and summary of the results, and identified them from the inclusion and exclusion criteria, and the selected studies were further reviewed during the full-text screening. 4. Computational studies that did not report in vitro biological activity.

Data Extraction
The analyzed data included the IC 50 value, which refers to the 50% growth inhibition of the parasite in vitro after incubation with different natural products extracted from marine sponges, according to the method applied to measure the antimalarial activity with the particular Plasmodium lab strain used. In addition, the sponge species, class and extraction location were also included in the analysis.

Types of Reported Results
Due to the heterogeneity of the primary studies, it was not possible to perform a meta-analysis. In order to compare the effect size (ES) of both techniques, we calculated the normalized average difference considering the values before and after the intervention. They were further classified as small (<0. 20), moderate (about 0.50) or large (>0.80), according to Cohen criteria.

Conclusions
In conclusion, marine sponge extracts represent a large arsenal of bioactive products with antimalarial potential. Different substances, such as alkaloids, endoperoxides (terpenes and polyketides), terpenoids and glycosphingolipids, have been isolated and identified in the extracts of different sponges around the globe. The structural features of active compounds can be an interesting core for synthetic development of new antimalarials for selectively targeting parasite cell metabolism. However, studies that aim to elucidate the mechanism of action of these new compounds are still scarce in the literature.