Novel Antiretroviral Structures from Marine Organisms

In spite of significant advancements and success in antiretroviral therapies directed against HIV infection, there is no cure for HIV, which scan persist in a human body in its latent form and become reactivated under favorable conditions. Therefore, novel antiretroviral drugs with different modes of actions are still a major focus for researchers. In particular, novel lead structures are being sought from natural sources. So far, a number of compounds from marine organisms have been identified as promising therapeutics for HIV infection. Therefore, in this paper, we provide an overview of marine natural products that were first identified in the period between 2013 and 2018 that could be potentially used, or further optimized, as novel antiretroviral agents. This pipeline includes the systematization of antiretroviral activities for several categories of marine structures including chitosan and its derivatives, sulfated polysaccharides, lectins, bromotyrosine derivatives, peptides, alkaloids, diterpenes, phlorotannins, and xanthones as well as adjuvants to the HAART therapy such as fish oil. We critically discuss the structures and activities of the most promising new marine anti-HIV compounds.


Introduction
Human immunodeficiency virus (HIV) infections pose a global challenge given that in 2017, according to the World Health Organization data, 36.9 million people were living with HIV and additional 1.8 million people were becoming newly infected globally (Table 1). HIV targets immune cells and impairs the human defense against pneumonia, tuberculosis, and shingles as well as certain types of cancer [1]. The most advanced stage of HIV infection is the Acquired Immunodeficiency Syndrome (AIDS), which can take from two to 15 years to develop, depending on the individual [2]. HIV has two viral forms: HIV-1 (the most common form that accounts for around 95% of all infections worldwide) and HIV-2 (relatively uncommon and less infectious). HIV-1 consists of groups M, N, O, and P with at least nine genetically distinct subtypes of HIV-1 within group M (A, B, C, D, F, G, H, J, and K). Additionally, different subtypes can combine genetic material to form a hybrid virus known as the 'circulating recombinant form' (CRFs) (Figure 1). HIV-2 consists of eight known groups (A to H). Of these, only groups A and B are pandemic. The HIV-2 mechanism is not clearly defined and neither is its difference from HIV-1. However, the transmission rate is much lower in HIV-2 than in HIV-1. HIV-2 is estimated to be more than 55% genetically distinct from HIV-1.  HIV has two viral forms: HIV-1 (the most common form that accounts for around 95% of all infections worldwide) and HIV-2 (relatively uncommon and less infectious). HIV-1 consists of groups M, N, O, and P with at least nine genetically distinct subtypes of HIV-1 within group M (A, B, C, D, F, G, H, J, and K). Additionally, different subtypes can combine genetic material to form a hybrid virus known as the 'circulating recombinant form' (CRFs) (Figure 1). HIV-2 consists of eight known groups (A to H). Of these, only groups A and B are pandemic. The HIV-2 mechanism is not clearly defined and neither is its difference from HIV-1. However, the transmission rate is much lower in HIV-2 than in HIV-1. HIV-2 is estimated to be more than 55% genetically distinct from HIV-1.

Chitosan and Its Derivatives
Chitosan (2, Figure 2), a natural marine byproduct, is a poly-cationic linear polysaccharide derived from chitin (1, Figure 2) after partial deacetylation. Chitin is a structural element in the exoskeleton of mainly shrimps and crabs and is mainly composed of the randomly distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. It has been previously shown that this compound can exhibit a large scale of different bioactivities and can also be used as a carrier for anti-HIV drugs [31]. Chitosan is loaded with saquinavir, an anti-HIV drug with a protease inhibitory activity, which showed better cell targeting efficiency than saquinavir alone [32]. Furthermore, trimethyl chitosan has improved Atripla, an anti-HIV drug consisting of efavirenz, emtricitabine, and tenofovir disoproxil fumarate, anti-HIV 1 activity, and has allowed it to be used in lower concentrations [33]. The antiretroviral activity is manifested in the chitosan-specific cationic nature that allows the formation of electrostatic complexes or multilayer structures with other negatively charged polymers [34]. Karagozlu et al. reported about new QMW-COS and WMQ-COS oligomers with anti-HIV activities. These oligomers are conjugates of chitosan and the Gln-Met-Trp peptide, which were constructed as a continuation of the authors' previous research, in which a high potency of synthetically constructed chitosan oligomers was confirmed in anti-HIV therapy. More specifically, it was shown that these oligomers suppress syncytium formation, which occurs as a fusion of infected cells with neighboring cells, induced by HIV in a dose-dependent manner. However, the authors also noticed that after a certain period, the number of syncytia once again increased, suggesting that the cells should be re-treated with QMW-COS and WMQ-COS oligomers to maintain the primary therapeutically-relevant effect. The inhibition of the HIV-1 induced lytic effect, determined by the cell viability assay, showed that IC 50 for QMW-COS was 48.14 µg/mL and was almost identical for WMQ-COS, 48.01 µg/mL. These oligomers effectively reduced the HIV load but showed no effects on HIV-1 RT and protease in vitro. Higher dosages were also required for the reduction in the HIV-1IIIB p24 antigen production assessed by the ELISA assay and the HIV-1 RTMDR p24 antigen production. The highest difference between the compounds was reflected in IC 50 values obtained from studies on the virus-induced luciferase activity in infected cells, where QMW-COS had a higher potency in comparison with WMQ-COS. Lastly, the authors determined the effects of oligomers on the interaction between gp41 and CD4 by using the CD4-gp41 ELISA assay, whereby both oligomers showed high potency. The effect of these oligomers was highest when they were applied immediately upon the HIV-1 infection of cells, indicating that they should be used as a potential treatment in the early stages of HIV infection, probably at the entry stage [31].
Molecules 2019, 24, x FOR PEER REVIEW 6 of 40 applied immediately upon the HIV-1 infection of cells, indicating that they should be used as a potential treatment in the early stages of HIV infection, probably at the entry stage [31].

Sulfated Polysaccharides
Sulfated polysaccharides (SP) are the most studied class of antiviral polysaccharides that are structural components of the alga cell wall where they play both the storage and structural role. They are an important source of galactans, commercially known as agar and carrageenan in red alga (Rhodophyta), fucans (fucoidan, sargassan, ascophyllan, and glucuronoxylofucan) in brown alga (Phaeophyta), and ulvans-sulfated heteropolysaccharides that contain galactose, xylose, arabinose, mannose, glucuronic acid, or glucose [35][36][37]. Many studies indicate that, in marine algae, sulfated polysaccharides facilitate water and ion retention in extracellular matrices, which is an important mechanism for coping with desiccation and osmotic stress in a highly salted environment [38][39][40]. The antiviral activity of this group of compounds is mainly connected to the degree of sulfation, constituent sugars, molecular weight, conformation, and dynamic stereochemistry [41,42] . The effect of counter cation should also be considered as an important factor in observed biological activity.
The antagonizing effect of the negatively charged sulfated polysaccharides on the HIV-1 entry into cells may be due to 1) their binding onto the positively charged V3 domain of gp120, thereby preventing the virus attachment to the cell surface [43][44][45] or 2) the masking of the docking sites of gp120 for sCD4 on the surface of T lymphocytes, thereby disrupting the CD4-gp120 interaction [46][47][48] and subsequently inhibiting the expression of the viral antigen and the activity of the viral reverse transcriptase [49,50].

Heparan Sulfate
Heparinoid polysaccharides can interact with the positive-charge regions of cell-surface glycoproteins, leading to a shielding effect on these regions, which prevents the binding of viruses to the cell surface [51]. The sulfated polysaccharides content in marine mollusks is high in comparison with the bovine mucosal heparin (73.5%) and the porcine mucosal heparin (72.8%) [52]. The acidic sulfate groups on heparin (3, Figure 3), or heparin-like compounds, can inhibit HIV through electrostatic interactions with basic amino-acid residues of the transcriptional activator Tat protein [53].

Sulfated Polysaccharides
Sulfated polysaccharides (SP) are the most studied class of antiviral polysaccharides that are structural components of the alga cell wall where they play both the storage and structural role. They are an important source of galactans, commercially known as agar and carrageenan in red alga (Rhodophyta), fucans (fucoidan, sargassan, ascophyllan, and glucuronoxylofucan) in brown alga (Phaeophyta), and ulvans-sulfated heteropolysaccharides that contain galactose, xylose, arabinose, mannose, glucuronic acid, or glucose [35][36][37]. Many studies indicate that, in marine algae, sulfated polysaccharides facilitate water and ion retention in extracellular matrices, which is an important mechanism for coping with desiccation and osmotic stress in a highly salted environment [38][39][40]. The antiviral activity of this group of compounds is mainly connected to the degree of sulfation, constituent sugars, molecular weight, conformation, and dynamic stereochemistry [41,42]. The effect of counter cation should also be considered as an important factor in observed biological activity.
The antagonizing effect of the negatively charged sulfated polysaccharides on the HIV-1 entry into cells may be due to 1) their binding onto the positively charged V3 domain of gp120, thereby preventing the virus attachment to the cell surface [43][44][45] or 2) the masking of the docking sites of gp120 for sCD4 on the surface of T lymphocytes, thereby disrupting the CD4-gp120 interaction [46][47][48] and subsequently inhibiting the expression of the viral antigen and the activity of the viral reverse transcriptase [49,50].

Heparan Sulfate
Heparinoid polysaccharides can interact with the positive-charge regions of cell-surface glycoproteins, leading to a shielding effect on these regions, which prevents the binding of viruses to the cell surface [51]. The sulfated polysaccharides content in marine mollusks is high in comparison with the bovine mucosal heparin (73.5%) and the porcine mucosal heparin (72.8%) [52]. The acidic sulfate groups on heparin (3, Figure 3), or heparin-like compounds, can inhibit HIV through electrostatic interactions with basic amino-acid residues of the transcriptional activator Tat protein [53].
Molecules 2019, 24, x FOR PEER REVIEW 6 of 40 applied immediately upon the HIV-1 infection of cells, indicating that they should be used as a potential treatment in the early stages of HIV infection, probably at the entry stage [31].

Sulfated Polysaccharides
Sulfated polysaccharides (SP) are the most studied class of antiviral polysaccharides that are structural components of the alga cell wall where they play both the storage and structural role. They are an important source of galactans, commercially known as agar and carrageenan in red alga (Rhodophyta), fucans (fucoidan, sargassan, ascophyllan, and glucuronoxylofucan) in brown alga (Phaeophyta), and ulvans-sulfated heteropolysaccharides that contain galactose, xylose, arabinose, mannose, glucuronic acid, or glucose [35][36][37]. Many studies indicate that, in marine algae, sulfated polysaccharides facilitate water and ion retention in extracellular matrices, which is an important mechanism for coping with desiccation and osmotic stress in a highly salted environment [38][39][40]. The antiviral activity of this group of compounds is mainly connected to the degree of sulfation, constituent sugars, molecular weight, conformation, and dynamic stereochemistry [41,42] . The effect of counter cation should also be considered as an important factor in observed biological activity.
The antagonizing effect of the negatively charged sulfated polysaccharides on the HIV-1 entry into cells may be due to 1) their binding onto the positively charged V3 domain of gp120, thereby preventing the virus attachment to the cell surface [43][44][45] or 2) the masking of the docking sites of gp120 for sCD4 on the surface of T lymphocytes, thereby disrupting the CD4-gp120 interaction [46][47][48] and subsequently inhibiting the expression of the viral antigen and the activity of the viral reverse transcriptase [49,50].

Heparan Sulfate
Heparinoid polysaccharides can interact with the positive-charge regions of cell-surface glycoproteins, leading to a shielding effect on these regions, which prevents the binding of viruses to the cell surface [51]. The sulfated polysaccharides content in marine mollusks is high in comparison with the bovine mucosal heparin (73.5%) and the porcine mucosal heparin (72.8%) [52]. The acidic sulfate groups on heparin (3, Figure 3), or heparin-like compounds, can inhibit HIV through electrostatic interactions with basic amino-acid residues of the transcriptional activator Tat protein [53].   [54]. More importantly, these polysaccharides are selective inhibitors of various enveloped viruses, including HIV [54][55][56]. FCSP acts during the early phase of infection by blocking the virus attachment and entry into the host cells, but may also inhibit subsequent replication stages in vitro [57].

Fucoidans
Three fucoidans extracted from three brown seaweeds (Sargassum mcclurei, Sargassum polycystum, Turbinara ornata) inhibit the early stages of HIV-1 entry into target cells, with IC 50 ranging from 0.33 to 0.7 µM. Neither the sulfate content nor the position of sulfate groups are related to the anti-HIV activity of fucoidans, suggesting the involvement of other structural parameters such as the molecular weight, the type of glycosidic linkage, or even a unique fucoidan sequence [56]. Although the presence of sulfo-groups seems to be necessary for anti-HIV activity [58], these data do not support random sulfation as the main antiviral factor. Sulfated fucan polysaccharides, ascophyllan (4, Figure 4), and two fucoidans (S and A) (5 and 6, Table 2), derived from different sources, significantly inhibit (IC 50 1.3; 0.3; 0.6 µg/mL) the early step of HIV-1 (R9 and JR-Fl) infection. They also inhibit the VSV-G-pseudotype HIV-1 infection in HeLa cells [59].

Fucose Containing SP
So far, the main anti-infectious activities documented for the fucose-containing SP are those against viruses [54]. More importantly, these polysaccharides are selective inhibitors of various enveloped viruses, including HIV [54][55][56]. FCSP acts during the early phase of infection by blocking the virus attachment and entry into the host cells, but may also inhibit subsequent replication stages in vitro [57].

Fucoidans
Three fucoidans extracted from three brown seaweeds (Sargassum mcclurei, Sargassum polycystum, Turbinara ornata) inhibit the early stages of HIV-1 entry into target cells, with IC50 ranging from 0.33 to 0.7 µM. Neither the sulfate content nor the position of sulfate groups are related to the anti-HIV activity of fucoidans, suggesting the involvement of other structural parameters such as the molecular weight, the type of glycosidic linkage, or even a unique fucoidan sequence [56]. Although the presence of sulfo-groups seems to be necessary for anti-HIV activity [58], these data do not support random sulfation as the main antiviral factor. Sulfated fucan polysaccharides, ascophyllan (4, Figure 4), and two fucoidans (S and A) (5 and 6, Table 2), derived from different sources, significantly inhibit (IC50 1.3; 0.3; 0.6 µg/mL) the early step of HIV-1 (R9 and JR-Fl) infection. They also inhibit the VSV-G-pseudotype HIV-1 infection in HeLa cells [59].  Chondroitin sulfate with fucosylated branches (FuCS) (7, Figure 5) has also attracted attention as an HIV antiviral compound. Depolymerized fucosylated CS, extracted from the sea cucumber, has shown in vitro activity against a range of viral strains, including the resistant ones [60]. FuCS is effective in blocking the laboratory strain HIV-1IIIB entry and replication by inhibiting the p24 antigen production (4.26 and 0.73 µg/mL, respectively) and the infection of the clinic isolate HIV-1KM018 and HIV-1TC-2 (23.75 and 31.86 µg/mL, respectively) as well as suppressing the HIV-1 drugresistant virus. Additionally, FuCS is also effective in T-20-resistant strains (EC50 values ranging from 0.76 to 1.13 µg/mL). The depolymerized fragments seem to maintain a similar anti-HIV action at the early stages of infection, apparently through interaction with an HIV envelope glycoprotein gp120. The sulfated fucose branches appear necessary for antiviral activity, which is also affected by  Chondroitin sulfate with fucosylated branches (FuCS) (7, Figure 5) has also attracted attention as an HIV antiviral compound. Depolymerized fucosylated CS, extracted from the sea cucumber, has shown in vitro activity against a range of viral strains, including the resistant ones [60]. FuCS is effective in blocking the laboratory strain HIV-1IIIB entry and replication by inhibiting the p24 antigen production (4.26 and 0.73 µg/mL, respectively) and the infection of the clinic isolate HIV-1KM018 and HIV-1TC-2 (23.75 and 31.86 µg/mL, respectively) as well as suppressing the HIV-1 drug-resistant virus. Additionally, FuCS is also effective in T-20-resistant strains (EC50 values ranging from 0.76 to 1.13 µg/mL). The depolymerized fragments seem to maintain a similar anti-HIV action at the early stages of infection, apparently through interaction with an HIV envelope glycoprotein gp120. The sulfated fucose branches appear necessary for antiviral activity, which is also affected by molecular weight and carboxylation [61]. While the in vitro results of the fucosylated CS against HIV are promising, it is questionable whether the antiviral activity would be maintained in vivo. Other polyanionic HIV entry inhibitors, which advanced into clinical trials, failed to prove effective against the heterosexual HIV-1 transmission. This was related to factors not considered in previous development stages, such as the presence of seminal plasma and the concentration and retention of polyanionic inhibitors [62].
Molecules 2019, 24, x FOR PEER REVIEW 8 of 40 molecular weight and carboxylation [61]. While the in vitro results of the fucosylated CS against HIV are promising, it is questionable whether the antiviral activity would be maintained in vivo. Other polyanionic HIV entry inhibitors, which advanced into clinical trials, failed to prove effective against the heterosexual HIV-1 transmission. This was related to factors not considered in previous development stages, such as the presence of seminal plasma and the concentration and retention of polyanionic inhibitors [62]. The complex chemical architecture and the sulfate patterning of marine polysaccharides depends on numerous factors (species, tidal cycles, environmental variations (e.g., salinity), harvesting season, plant age, geographical location etc.) [39,[63][64][65][66][67][68][69], making isolation, purification, and comprehensive chemical characterization a highly challenging task [70]. The development of many polysaccharides into clinical application is hindered by the still limited view of their sophisticated and diverse nature. Despite having good antiviral effects, the use of carbohydrate drugs is still in its infancy, and intensive structure-activity and in vivo studies are needed in the future.
A relatively new strategy in inducing immunity and developing an HIV vaccine is to use carbohydrates. The major difficulty of such an approach lies in mimicking the specific glycan protective epitope. Gp120 of HIV is a highly glycosylated envelope surface glycoprotein responsible for the receptor and co-receptor binding, which, together with gp41, comprises the heterodimeric envelope trimer spikes of HIV. N-linked glycans, mainly mannose and complex-type, cover much of the gp120 surface-accessible face of the HIV envelope spike forming the glycan shield. Inadequate mimicry of the glycan shield, tolerance mechanisms, and/or the inability to induce a domainexchange are reflecting difficulties in creating the proper specificity of Abs [71]. Most of the vaccines for HIV-1 in preclinical trials are based on a Manα1-2Man oligomannosyl epitope (various conjugates, engineered yeast strains, and modified glycoproteins) [72][73][74][75][76][77][78][79]. Better specificity could potentially be gained using carbohydrates of marine origin.

Lectins
Lectins are a group of proteins that specifically, but reversibly, bind glycosylated molecules on the cell surface. Precisely, this group of molecules can affect cell-cell interactions, protect cells from pathogens, influence cell adhesion, and affect the intracellular glycoprotein translocation [80]. Recently, lectins have become promising agents for antiretroviral therapy, and different researches have confirmed their anti-HIV properties. Their antiretroviral activity is manifested through an alteration of the interaction between HIV gp120 or gp41 and the corresponding receptors [81], which, in the end, inhibit the HIV cell function, HIV infectivity, and the formation of the syncytium, multinucleated cells [82][83][84].
Several published review papers describe the previously found marine lectins with antiretroviral action [85,86].  The complex chemical architecture and the sulfate patterning of marine polysaccharides depends on numerous factors (species, tidal cycles, environmental variations (e.g., salinity), harvesting season, plant age, geographical location etc.) [39,[63][64][65][66][67][68][69], making isolation, purification, and comprehensive chemical characterization a highly challenging task [70]. The development of many polysaccharides into clinical application is hindered by the still limited view of their sophisticated and diverse nature. Despite having good antiviral effects, the use of carbohydrate drugs is still in its infancy, and intensive structure-activity and in vivo studies are needed in the future.
A relatively new strategy in inducing immunity and developing an HIV vaccine is to use carbohydrates. The major difficulty of such an approach lies in mimicking the specific glycan protective epitope. Gp120 of HIV is a highly glycosylated envelope surface glycoprotein responsible for the receptor and co-receptor binding, which, together with gp41, comprises the heterodimeric envelope trimer spikes of HIV. N-linked glycans, mainly mannose and complex-type, cover much of the gp120 surface-accessible face of the HIV envelope spike forming the glycan shield. Inadequate mimicry of the glycan shield, tolerance mechanisms, and/or the inability to induce a domain-exchange are reflecting difficulties in creating the proper specificity of Abs [71]. Most of the vaccines for HIV-1 in preclinical trials are based on a Manα1-2Man oligomannosyl epitope (various conjugates, engineered yeast strains, and modified glycoproteins) [72][73][74][75][76][77][78][79]. Better specificity could potentially be gained using carbohydrates of marine origin.

Lectins
Lectins are a group of proteins that specifically, but reversibly, bind glycosylated molecules on the cell surface. Precisely, this group of molecules can affect cell-cell interactions, protect cells from pathogens, influence cell adhesion, and affect the intracellular glycoprotein translocation [80]. Recently, lectins have become promising agents for antiretroviral therapy, and different researches have confirmed their anti-HIV properties. Their antiretroviral activity is manifested through an alteration of the interaction between HIV gp120 or gp41 and the corresponding receptors [81], which, in the end, inhibit the HIV cell function, HIV infectivity, and the formation of the syncytium, multi-nucleated cells [82][83][84].
However, in the last few years, there has not been as much research focused on anti-HIV lectins from marine sources. Only Hirayama et al. (2016) reported about the new high-mannose specific lectin and its recombinants that possess anti-HIV activity [87]. In their research performed on the red alga Kappaphycus alvarezii, authors confirmed KAA-1 and KAA-2, two KAA mannose-binding lectin isomers, as potent anti-HIV agents. The anti-HIV role of action of these two compounds includes a strong binding to the virus envelope glycoprotein gp120 and, consequently, the inhibition of HIV entry into the host cells. These KAA recombinants, as well as the native one, inhibited the HIV-1 entry at IC 50 s (neutralization assay in Jurkat cells) of 7.3-12.9 nM. Authors concluded in the end that KAAs, besides their strong inhibitory effect on HIV entry into the cells, have a potential as agents in treatments against other viruses possessing high mannose glycans on their envelope as well.

Peptides
It has been shown that the majority of marine peptides have strong anti-HIV activity. They are usually isolated from marine organisms through the process of enzymatic hydrolysis [88]. The most common source of such constituents is marine sponges that are known for their unique metabolome [89] and are a source of more than 36% of all marine bioactive compounds [90]. Their bioactive peptides can be found in cyclic or linear forms and contain unusual amino acids that form unique structures rarely found in other species. Antiretroviral activity of such structures works on several different levels: blocking of virus entry, inhibition of the cytopathic viral activity, neutralization of viral particles, or inhibition of viral fusion and entry [89,91].
Recently, Shin et al. discovered two new depsipeptides from marine sponges Stelletta sp., stellettapeptin A (8, Figure 6), and stellettapeptin B (9, Figure 6), with the inhibition of the cytopathic effect of HIV-1 infection [92]. Confirming the mentioned theory about the unique metabolome of marine sponges, the authors revealed that these two compounds have previously undescribed nonproteinogenic amino-acid parts on peptides that are rarely found in nature. Namely, stellettapeptin A and stellettapeptin B have an unexpected polyketide subunit, 3-hydroxy-6,8-dimethylnon-4-enoic acid, 3-OHGln, and 3-OHAsn residues. Their high potency is witnessed through low EC 50   However, in the last few years, there has not been as much research focused on anti-HIV lectins from marine sources. Only Hirayama et al. (2016) reported about the new high-mannose specific lectin and its recombinants that possess anti-HIV activity [87]. In their research performed on the red alga Kappaphycus alvarezii, authors confirmed KAA-1 and KAA-2, two KAA mannose-binding lectin isomers, as potent anti-HIV agents. The anti-HIV role of action of these two compounds includes a strong binding to the virus envelope glycoprotein gp120 and, consequently, the inhibition of HIV entry into the host cells. These KAA recombinants, as well as the native one, inhibited the HIV-1 entry at IC50s (neutralization assay in Jurkat cells) of 7.3-12.9 nM. Authors concluded in the end that KAAs, besides their strong inhibitory effect on HIV entry into the cells, have a potential as agents in treatments against other viruses possessing high mannose glycans on their envelope as well.

Peptides
It has been shown that the majority of marine peptides have strong anti-HIV activity. They are usually isolated from marine organisms through the process of enzymatic hydrolysis [88]. The most common source of such constituents is marine sponges that are known for their unique metabolome [89] and are a source of more than 36% of all marine bioactive compounds [90]. Their bioactive peptides can be found in cyclic or linear forms and contain unusual amino acids that form unique structures rarely found in other species. Antiretroviral activity of such structures works on several different levels: blocking of virus entry, inhibition of the cytopathic viral activity, neutralization of viral particles, or inhibition of viral fusion and entry [89,91].
Recently, Shin et al. discovered two new depsipeptides from marine sponges Stelletta sp., stellettapeptin A (8, Figure 6), and stellettapeptin B (9, Figure 6), with the inhibition of the cytopathic effect of HIV-1 infection [92]. Confirming the mentioned theory about the unique metabolome of marine sponges, the authors revealed that these two compounds have previously undescribed nonproteinogenic amino-acid parts on peptides that are rarely found in nature. Namely, stellettapeptin A and stellettapeptin B have an unexpected polyketide subunit, 3-hydroxy-6,8dimethylnon-4-enoic acid, 3-OHGln, and 3-OHAsn residues. Their high potency is witnessed through low EC50 values (inhibition of the cytotoxic effect upon HIV infection)-values of 23 nM for stellettapeptin A and 27 nM for stellettapeptin B. Furthermore, newly discovered anti-HIV constituents derived from marine sponges Verongula rigida and Aiolochoria crassa with amino-acid structure were published by Gomez-Archila et al. (2014) [93]. In their paper, they evaluated and confirmed the anti-HIV effect of 11 bromotyrosine derivatives (Table 3), whereby aeroplysinin-1 (10), 19-deoxyfistularin 3 (15), purealidin B (16), fistularin 3 (17) and 3-bromo-5-hydroxy-O-methyltyrosine (18, Figure 7) were the most potent in their anti-HIV activity. Aeroplysinin 1 (15) and purealidin B (16), compounds found in V. rigida species inhibited the Furthermore, newly discovered anti-HIV constituents derived from marine sponges Verongula rigida and Aiolochoria crassa with amino-acid structure were published by Gomez-Archila et al. (2014) [93]. In their paper, they evaluated and confirmed the anti-HIV effect of 11 bromotyrosine derivatives (Table 3), whereby aeroplysinin-1 (10), 19-deoxyfistularin 3 (15), purealidin B (16), fistularin 3 (17) and 3-bromo-5-hydroxy-O-methyltyrosine (18, Figure 7) were the most potent in their anti-HIV activity. Aeroplysinin 1 (15) and purealidin B (16), compounds found in V. rigida species inhibited the HIV-1 replication in a dose-dependent manner by more than 50%. Specifically, for aeroplysinin 1, HIV-a replication was inhibited by 74% at a concentration of 20 µM, whereas purealidin was less potent with inhibitory power of 57% at a concentration of 80 µM. These two compounds had been previously isolated; however, their anti-HIV activity was proven in this research. The same was with 3-bromo-5-hydroxy-O-methyltyrosine (18) that has a relatively high percentage of inhibition of HIV activity (47%) in a dose-dependent manner. However, the exact mechanism of action remains unclear. In the same study, additional tests with these compounds on the HIV RT inhibition (qPCR of the early and late transcripts), nuclear import (qPCR analysis of 2-LTR transcript), and HIV entry inhibition (viral infectivity assay) were performed. The results showed that aeroplysinin-1 (10), 19-deoxyfistularin 3 (15), purealidin B (16), fistularin 3 (17), and 3-bromo-5-hydroxy-O-methyltyrosine (18) influenced the nuclear import of the HIV virus with around or more than 50% of inhibition: aeroplysinin-1 (10) showed 67% of inhibition at 10 µM, 19-deoxyfistularin 3 62% inhibition at 20 µM, purealidin B 66% of inhibition at 20 µM, fistularin 3 47% of inhibition at 10 µM, and 3-bromo-5-hydroxy-O-methyltyrosine 73% of inhibition at 80 µM. Viral RT inhibition was not high for all compounds, whereby the highest results were around 50% of inhibition. For example, purealidin B had 58% of inhibition at 20 µM in the qPCR analysis of early transcripts. As for the HIV entry inhibition, all compounds were active in a dose-depended manner, with the highest results of inhibition obtained for 3,5-dibromo-N,N,N,O-tetramethyltyraminium (13), from 14% to 30%. Finally, the authors stressed the structural similarity of these compounds with the HIV integrase and protease inhibitors, suggesting that these compounds can have a broader mode of antiviral action. HIV-1 replication in a dose-dependent manner by more than 50%. Specifically, for aeroplysinin 1, HIV-a replication was inhibited by 74% at a concentration of 20 µM, whereas purealidin was less potent with inhibitory power of 57% at a concentration of 80 µM. These two compounds had been previously isolated; however, their anti-HIV activity was proven in this research. The same was with 3-bromo-5-hydroxy-O-methyltyrosine (18) that has a relatively high percentage of inhibition of HIV activity (47%) in a dose-dependent manner. However, the exact mechanism of action remains unclear.
Marine sponges are not the sole source of bioactive proteins. For example, Jang et al. reported about a new small hydroxyproline-rich peptide from Alaska Pollack collagen (APHCP, 21, Figure 8) that exhibits a unique antiviral activity [94]. This peptide is a Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly peptide, and the authors showed that the most important part of a peptide for anti-HIV activity is the hydroxyl group at hydroxyproline, whereas a peptide with only prolines does not exhibit antiviral activity. Its anti-HIV 1 mode of action is manifested through the inhibition of the induced syncytia formation by the interference of an HIV fusion, inhibition of cell lysis, RT activity, and the production of the p24 antigen. It was shown that APHCP can decrease the HIV-1 induced cell lysis at a potency around EC 50 of 459 µM (EC 50 against anti-HIV-1 induced cell lysis-MTT assay). Additionally, through the inhibition of the viral RT at EC 50 at 374 µM, this peptide's crucial role in the inhibition of the conversion of viral RNA to DNA was also confirmed. With EC 50 of 405 µM, this compound effectively suppressed the p24 production in viral cells, as determined by the Western blot analysis.  Figure 8) that exhibits a unique antiviral activity [94]. This peptide is a Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly peptide, and the authors showed that the most important part of a peptide for anti-HIV activity is the hydroxyl group at hydroxyproline, whereas a peptide with only prolines does not exhibit antiviral activity. Its anti-HIV 1 mode of action is manifested through the inhibition of the induced syncytia formation by the interference of an HIV fusion, inhibition of cell lysis, RT activity, and the production of the p24 antigen. It was shown that APHCP can decrease the HIV-1 induced cell lysis at a potency around EC50 of 459 µM (EC50 against anti-HIV-1 induced cell lysis-MTT assay). Additionally, through the inhibition of the viral RT at EC50 at 374 µM, this peptide's crucial role in the inhibition of the conversion of viral RNA to DNA was also confirmed. With EC50 of 405 µM, this compound effectively suppressed the p24 production in viral cells, as determined by the Western blot analysis. Similarly, one new anti-HIV peptide was isolated from Spirulina maxima (SM-peptide) [95]-the Leu-Asp-Ala-Val-Asn-Arg peptide, and the authors showed its HIV-1 infection inhibition in a human T cell line MT4. The peptide inhibited cell lysis, p24 antigen production, and HIV-1 RT. Specifically, IC50 (obtained by a cell viability assay) against an anti-HIV 1 infection was determined as 0.691 mM, the inhibition of the HIV-1-induced RT activation (RT assay kit) in MT4 cells was at a high 90% at a concentration of 1.093 mM, and the p24 production (p24 antigen production assay) was inhibited at 95% at a concentration of 1.093 mM.

Alkaloids
Marine organisms are well-established sources of natural alkaloids. Although the term 'alkaloid' seems puzzling and is prone to scientific controversy, alkaloids are generally defined as nitrogencontaining compounds derived from plants and animals. Relatively few alkaloids from marine sources have been found to possess antiretroviral properties and, so far, none have found their clinical use.
Aspernigrin C (22, Figure 9) and malformin C (23, Figure 9) have been isolated from marinederived black aspergili, Aspergillus niger SCSIO Jcw6F30, and their inhibitory activity against the chemokine receptor subtype 5 (CCR5) tropic HIV-1 SF162 has been evaluated. They show potent inhibition of infection with IC50 values 4.7 ± 0.4 µM and 1.4 ± 0.06 µM, which is comparable to the nucleoside reverse transcriptase inhibitor-abacavir (IC50 = 0.8 ± 0.1 µM) and the HIV-1 entry inhibitor ADS-J1 (IC50 = 0.8 ± 0.1 µM). In comparison to other aspernigrins, it has been suggested that the 2methylsuccinic moiety is responsible for the potency of aspernigrin C [96]. Similarly, one new anti-HIV peptide was isolated from Spirulina maxima (SM-peptide) [95]-the Leu-Asp-Ala-Val-Asn-Arg peptide, and the authors showed its HIV-1 infection inhibition in a human T cell line MT4. The peptide inhibited cell lysis, p24 antigen production, and HIV-1 RT. Specifically, IC 50 (obtained by a cell viability assay) against an anti-HIV 1 infection was determined as 0.691 mM, the inhibition of the HIV-1-induced RT activation (RT assay kit) in MT4 cells was at a high 90% at a concentration of 1.093 mM, and the p24 production (p24 antigen production assay) was inhibited at 95% at a concentration of 1.093 mM.

Alkaloids
Marine organisms are well-established sources of natural alkaloids. Although the term 'alkaloid' seems puzzling and is prone to scientific controversy, alkaloids are generally defined as nitrogen-containing compounds derived from plants and animals. Relatively few alkaloids from marine sources have been found to possess antiretroviral properties and, so far, none have found their clinical use.

Diterpenes
Many terpenes from marine natural products demonstrated anti-HIV properties. Mechanisms of action involve blocking of different steps of the HIV-1 replicative cycle as reverse transcriptase inhibitors, protease inhibitors, or entry inhibitors. Among them, diterpenes from marine algae are nowadays in the spotlight due to their promising anti-HIV activities [102]. Dolabellane diterpenes are compounds from the diterpene group that have recently been extensively studied for their anti-  Figure 15), respectively [102]. In particular, the new compounds, dolabelladienols A and B, showed potent anti-HIV-1 activities that can be confirmed with their low IC50 values of 2.9 and 4.1 µM and low cytotoxic activity against MT-2 lymphocyte tumor cells. These promising anti-HIV-1 agents were even more active than previously known 2,6-dolabelladienes series.  Figure 16) and secodolastane diterpenes (39, Figure 16) isolated from the brown alga Canistrocarpus cervicornis for anti-HIV-1 activity [103]. They observed that the marine diterpenes 38-40 inhibit the HIV-1 replication in a dose-dependent manner (EC50 values of 0.35, 3.67, and 0.794 µM) without a cytotoxic effect (CC50 values ranging from 935 to 1910 µM). Additionally, they investigated the virucidal effect of these diterpenes and their potential use as microbicides. Dolastane-diterpenes 38 and 40 showed a potent effect on HIV-1 infectivity, whereas no virucidal effect was observed for secodolastane diterpene 39, demonstrating another mechanism of antiretroviral activity. Therefore, the authors suggested a potential use of marine dolastanes 38 and 40 as microbicides that could directly inhibit virus infectivity and possibly act before the virus penetrates the target cells [103].   Figure 15), respectively [102]. In particular, the new compounds, dolabelladienols A and B, showed potent anti-HIV-1 activities that can be confirmed with their low IC 50 values of 2.9 and 4.1 µM and low cytotoxic activity against MT-2 lymphocyte tumor cells. These promising anti-HIV-1 agents were even more active than previously known 2,6-dolabelladienes series.

Diterpenes
Many terpenes from marine natural products demonstrated anti-HIV properties. Mechanisms of action involve blocking of different steps of the HIV-1 replicative cycle as reverse transcriptase inhibitors, protease inhibitors, or entry inhibitors. Among them, diterpenes from marine algae are nowadays in the spotlight due to their promising anti-HIV activities [102]. Dolabellane diterpenes are compounds from the diterpene group that have recently been extensively studied for their anti-  Figure 15), respectively [102]. In particular, the new compounds, dolabelladienols A and B, showed potent anti-HIV-1 activities that can be confirmed with their low IC50 values of 2.9 and 4.1 µM and low cytotoxic activity against MT-2 lymphocyte tumor cells. These promising anti-HIV-1 agents were even more active than previously known 2,6-dolabelladienes series.  Figure 16) and secodolastane diterpenes (39, Figure 16) isolated from the brown alga Canistrocarpus cervicornis for anti-HIV-1 activity [103]. They observed that the marine diterpenes 38-40 inhibit the HIV-1 replication in a dose-dependent manner (EC50 values of 0.35, 3.67, and 0.794 µM) without a cytotoxic effect (CC50 values ranging from 935 to 1910 µM). Additionally, they investigated the virucidal effect of these diterpenes and their potential use as microbicides. Dolastane-diterpenes 38 and 40 showed a potent effect on HIV-1 infectivity, whereas no virucidal effect was observed for secodolastane diterpene 39, demonstrating another mechanism of antiretroviral activity. Therefore, the authors suggested a potential use of marine dolastanes 38 and 40 as microbicides that could directly inhibit virus infectivity and possibly act before the virus penetrates the target cells [103].  Figure 16) and secodolastane diterpenes (39, Figure 16) isolated from the brown alga Canistrocarpus cervicornis for anti-HIV-1 activity [103]. They observed that the marine diterpenes 38-40 inhibit the HIV-1 replication in a dose-dependent manner (EC 50 values of 0.35, 3.67, and 0.794 µM) without a cytotoxic effect (CC 50 values ranging from 935 to 1910 µM). Additionally, they investigated the virucidal effect of these diterpenes and their potential use as microbicides. Dolastane-diterpenes 38 and 40 showed a potent effect on HIV-1 infectivity, whereas no virucidal effect was observed for secodolastane diterpene 39, demonstrating another mechanism of antiretroviral activity. Therefore, the authors suggested a potential use of marine dolastanes 38 and 40 as microbicides that could directly inhibit virus infectivity and possibly act before the virus penetrates the target cells [103]. Dolabelladienetriol from brown alga Dictyota spp has also been evaluated as a potential microbicide against HIV-1 in tissue explants. Namely, Stephens et al. examined the 8,10,18trihydroxy-2,6-dolabelladiene (41, Figure 17) in pretreated peripheral blood cells (PBMC) and macrophages along with their protective effect in the ex vivo explant model of the uterine cervix [104]. Pre-treatment of peripheral PBMC and macrophages with dolabelladienotriol showed inhibitory effects on HIV-1 replication. Furthermore, in the explant model dolabelladienetriol inhibited viral replication in a dose-dependent manner from 20 to 99% in concentrations of 0.15 and 14.4 µM without a loss in the viability of the tissue. The authors concluded that this compound has great potential as a possible microbicide. The same compound was also theoretically analyzed as an inhibitor of the wild-type and mutants' HIV-1 reverse transcriptase [105]. Firstly, the structureactivity relationship studies revealed that a low dipole moment and high HOMO (highest occupied molecular orbital)-LUMO (lowest unoccupied molecular orbital) gap values are related to the antiviral activity. Secondly, molecular docking studies with RT wild-type and mutants showed a seahorse-like conformation of 8,10,18-trihydroxy-2,6-dolabelladiene, hydrophobic interactions, and hydrogen bonds with important residues of the binding pocket. Finally, the authors suggested a new derivative of the 8,10,18-trihydroxy-2,6-dolabelladiene with an aromatic moiety in the double bond to improve its biological activity. Although dolabellane diterpenes of brown alga Dictyota spp showed a strong anti-HIV-1 activity, this was not confirmed for dolabellane diterpenes isolated from octocorals. Therefore, some chemical transformations have been conducted to improve the anti-HIV-1 potency of the main dolabellane 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene from Caribbean octocoral Eunicea laciniata [106]. Oxygenated dolabellanes derivatives (42-44, Figure 18), obtained by epoxidation, epoxide opening, and allylic oxidation of ketodolbellatriene have shown significantly improved antiviral activities and a low cytotoxicity to MT-2 cells, which makes them promising antiviral compounds. Dolabelladienetriol from brown alga Dictyota spp has also been evaluated as a potential microbicide against HIV-1 in tissue explants.
Namely, Stephens et al. examined the 8,10,18-trihydroxy-2,6-dolabelladiene (41, Figure 17) in pretreated peripheral blood cells (PBMC) and macrophages along with their protective effect in the ex vivo explant model of the uterine cervix [104]. Pre-treatment of peripheral PBMC and macrophages with dolabelladienotriol showed inhibitory effects on HIV-1 replication. Furthermore, in the explant model dolabelladienetriol inhibited viral replication in a dose-dependent manner from 20 to 99% in concentrations of 0.15 and 14.4 µM without a loss in the viability of the tissue. The authors concluded that this compound has great potential as a possible microbicide. The same compound was also theoretically analyzed as an inhibitor of the wild-type and mutants' HIV-1 reverse transcriptase [105]. Firstly, the structure-activity relationship studies revealed that a low dipole moment and high HOMO (highest occupied molecular orbital)-LUMO (lowest unoccupied molecular orbital) gap values are related to the antiviral activity. Secondly, molecular docking studies with RT wild-type and mutants showed a seahorse-like conformation of 8,10,18-trihydroxy-2,6-dolabelladiene, hydrophobic interactions, and hydrogen bonds with important residues of the binding pocket. Finally, the authors suggested a new derivative of the 8,10,18-trihydroxy-2,6-dolabelladiene with an aromatic moiety in the double bond to improve its biological activity. Dolabelladienetriol from brown alga Dictyota spp has also been evaluated as a potential microbicide against HIV-1 in tissue explants. Namely, Stephens et al. examined the 8,10,18trihydroxy-2,6-dolabelladiene (41, Figure 17) in pretreated peripheral blood cells (PBMC) and macrophages along with their protective effect in the ex vivo explant model of the uterine cervix [104]. Pre-treatment of peripheral PBMC and macrophages with dolabelladienotriol showed inhibitory effects on HIV-1 replication. Furthermore, in the explant model dolabelladienetriol inhibited viral replication in a dose-dependent manner from 20 to 99% in concentrations of 0.15 and 14.4 µM without a loss in the viability of the tissue. The authors concluded that this compound has great potential as a possible microbicide. The same compound was also theoretically analyzed as an inhibitor of the wild-type and mutants' HIV-1 reverse transcriptase [105]. Firstly, the structureactivity relationship studies revealed that a low dipole moment and high HOMO (highest occupied molecular orbital)-LUMO (lowest unoccupied molecular orbital) gap values are related to the antiviral activity. Secondly, molecular docking studies with RT wild-type and mutants showed a seahorse-like conformation of 8,10,18-trihydroxy-2,6-dolabelladiene, hydrophobic interactions, and hydrogen bonds with important residues of the binding pocket. Finally, the authors suggested a new derivative of the 8,10,18-trihydroxy-2,6-dolabelladiene with an aromatic moiety in the double bond to improve its biological activity. Although dolabellane diterpenes of brown alga Dictyota spp showed a strong anti-HIV-1 activity, this was not confirmed for dolabellane diterpenes isolated from octocorals. Therefore, some chemical transformations have been conducted to improve the anti-HIV-1 potency of the main dolabellane 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene from Caribbean octocoral Eunicea laciniata [106]. Oxygenated dolabellanes derivatives (42-44, Figure 18), obtained by epoxidation, epoxide opening, and allylic oxidation of ketodolbellatriene have shown significantly improved antiviral activities and a low cytotoxicity to MT-2 cells, which makes them promising antiviral compounds. Although dolabellane diterpenes of brown alga Dictyota spp showed a strong anti-HIV-1 activity, this was not confirmed for dolabellane diterpenes isolated from octocorals. Therefore, some chemical transformations have been conducted to improve the anti-HIV-1 potency of the main dolabellane 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene from Caribbean octocoral Eunicea laciniata [106]. Oxygenated dolabellanes derivatives (42-44, Figure 18), obtained by epoxidation, epoxide opening, and allylic oxidation of ketodolbellatriene have shown significantly improved antiviral activities and a low cytotoxicity to MT-2 cells, which makes them promising antiviral compounds. chemical transformations have been conducted to improve the anti-HIV-1 potency of the main dolabellane 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene from Caribbean octocoral Eunicea laciniata [106]. Oxygenated dolabellanes derivatives (42-44, Figure 18), obtained by epoxidation, epoxide opening, and allylic oxidation of ketodolbellatriene have shown significantly improved antiviral activities and a low cytotoxicity to MT-2 cells, which makes them promising antiviral compounds.

Phlorotannins and Xanthones
Phlorotannins are tannin derivatives made from several phloroglucinol units linked to each other in different ways. Phlorotannins contain phenyl linkage (fucols), ether linkage (fuhalols and phlorethols), phenyl and ether linkage (fucophloroethols), and dibenzodioxin linkage (eckols) [86,107]. So far, a series of phlorotannins have been identified with potent anti-HIV activity. For example, 8,8 -bieckol and 6,6 -bieckol from marine brown alga Ecklonia cava has shown an enhanced HIV-1 inhibitory effect [112,113]. Karadeniz et al. reported that 8,4 -dieckol (45, Figure 19) is another phlorotannin derivative isolated from the same brown alga that could be used as a drug candidate for the development of new generation anti-HIV therapeutic agents [107]. The compound showed HIV-1 inhibitory activity at noncytotoxic concentrations. More precisely, the results indicated that 8,4 -dieckol inhibited the cytopathic effects of HIV-1, including HIV-1 induced syncytia formation, lytic effects, and viral p24 antigen production. Furthermore, 8,4 -dieckol inhibited an HIV-1 entry and RT enzyme with the inhibition ratio of 91% at a concentration of 50 µM.

Phlorotannins and Xanthones
Phlorotannins are tannin derivatives made from several phloroglucinol units linked to each other in different ways. Phlorotannins contain phenyl linkage (fucols), ether linkage (fuhalols and phlorethols), phenyl and ether linkage (fucophloroethols), and dibenzodioxin linkage (eckols) [86,107]. So far, a series of phlorotannins have been identified with potent anti-HIV activity. For example, 8,8′-bieckol and 6,6′-bieckol from marine brown alga Ecklonia cava has shown an enhanced HIV-1 inhibitory effect [112,113]. Karadeniz et al. reported that 8,4′′′-dieckol (45, Figure 19) is another phlorotannin derivative isolated from the same brown alga that could be used as a drug candidate for the development of new generation anti-HIV therapeutic agents [107]. The compound showed HIV-1 inhibitory activity at noncytotoxic concentrations. More precisely, the results indicated that 8,4′′′-dieckol inhibited the cytopathic effects of HIV-1, including HIV-1 induced syncytia formation, lytic effects, and viral p24 antigen production. Furthermore, 8,4′′′-dieckol inhibited an HIV-1 entry and RT enzyme with the inhibition ratio of 91% at a concentration of 50 µM. Recently, for the first time, xanthone dimer was identified as a potential anti-HIV-1 agent [108]. Xanthones are secondary metabolites from higher plant families, fungi, and lichen [114,115]. Although structurally related to flavonoids, xanthones are not as frequently encountered in nature [9]. Penicillixanthone A (PXA) (46, Figure 20), a natural xanthone dimer, has been isolated from the jellyfish-derived fungus Aspergillus fumigates with fourteen other natural products [108]. However, only penicillixanthone A showed inhibitory activities in an HIV infection. Marine-derived PXA displayed potent anti-HIV-1 activity against CCR5-tropic HIV-1 SF162 and CXCR4-tropic HIV-1 NL4-3, with IC50 of 0.36 and 0.26 µM, respectively. A molecular docking study confirmed that PXA might bind to either CCR5 or CXCR4 to prevent HIV entry into target cells. Therefore, PXA, as a CCR5/CXCR4 dual-coreceptor antagonist, may be seen as a new potential lead product type for the development of anti-HIV therapeutics. Recently, for the first time, xanthone dimer was identified as a potential anti-HIV-1 agent [108]. Xanthones are secondary metabolites from higher plant families, fungi, and lichen [114,115]. Although structurally related to flavonoids, xanthones are not as frequently encountered in nature [9]. Penicillixanthone A (PXA) (46, Figure 20), a natural xanthone dimer, has been isolated from the jellyfish-derived fungus Aspergillus fumigates with fourteen other natural products [108]. However, only penicillixanthone A showed inhibitory activities in an HIV infection. Marine-derived PXA displayed potent anti-HIV-1 activity against CCR5-tropic HIV-1 SF162 and CXCR4-tropic HIV-1 NL4-3, with IC 50 of 0.36 and 0.26 µM, respectively. A molecular docking study confirmed that PXA might bind to either CCR5 or CXCR4 to prevent HIV entry into target cells. Therefore, PXA, as a CCR5/CXCR4 dual-coreceptor antagonist, may be seen as a new potential lead product type for the development of anti-HIV therapeutics.

Fish Oil as an Adjuvant to HAART Therapy
HAART therapy can cause severe side effects, e.g., insulin resistance, lipoatrophy, dyslipidemia, and abnormalities of fat distribution. Therefore, finding an adequate diet and supplementation to lower the negative effects of the HAART combination therapy is desirable [116]. Fish oil contains omega-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic (EPA, 20:5n-3) (47, Figure 21) and docosahexaenoic (DHA, 22:6n-3) (48, Figure 21) acids, which may have beneficial effects for HIVinfected patients. It has been shown that the addition of fish oil to the diet of HIV-infected individuals receiving usual antiretroviral therapy can significantly lower serum triglycerides levels [117], which is highly relevant knowing that HIV dyslipidemia is a serious problem related to an increased frequency of cardiovascular disease. Recently, He et al. analyzed the influence of DHA on the locomotor activity in ethanol-treated HIV-1 transgenic rats [109]. The prevalence of alcohol use and alcohol abuse in infected individuals is much higher, and numerous ethanol and HIV-1 viral proteins have synergistic effects on inflammation in the central nervous system [118][119][120]. HIV remains in the body in its latent form after HAART therapy and, as such, can induce neuroinflammation. DHA depletion has been found to be associated with various neurological abnormalities, and its administration can have a neuroprotective effect. DHA taken daily could reverse the effects of the ethanol negative effect on the locomotor activity in the presence of HIV viral proteins. An in vivo study, using real-time quantitative PCR, showed that the addition of DHA can reduce elevated levels of IL-6, IL-18, and increase the expression of NF-κB in the striatum. This proved the potential of this fish oil constituent as an adjuvant in HIV patients' treatment that can help in lowering the interactive effects of ethanol consumption during HIV infection.

Fish Oil as an Adjuvant to HAART Therapy
HAART therapy can cause severe side effects, e.g., insulin resistance, lipoatrophy, dyslipidemia, and abnormalities of fat distribution. Therefore, finding an adequate diet and supplementation to lower the negative effects of the HAART combination therapy is desirable [116]. Fish oil contains omega-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic (EPA, 20:5n-3) (47, Figure 21) and docosahexaenoic (DHA, 22:6n-3) (48, Figure 21) acids, which may have beneficial effects for HIV-infected patients. It has been shown that the addition of fish oil to the diet of HIV-infected individuals receiving usual antiretroviral therapy can significantly lower serum triglycerides levels [117], which is highly relevant knowing that HIV dyslipidemia is a serious problem related to an increased frequency of cardiovascular disease.

Fish Oil as an Adjuvant to HAART Therapy
HAART therapy can cause severe side effects, e.g., insulin resistance, lipoatrophy, dyslipidemia, and abnormalities of fat distribution. Therefore, finding an adequate diet and supplementation to lower the negative effects of the HAART combination therapy is desirable [116]. Fish oil contains omega-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic (EPA, 20:5n-3) (47, Figure 21) and docosahexaenoic (DHA, 22:6n-3) (48, Figure 21) acids, which may have beneficial effects for HIVinfected patients. It has been shown that the addition of fish oil to the diet of HIV-infected individuals receiving usual antiretroviral therapy can significantly lower serum triglycerides levels [117], which is highly relevant knowing that HIV dyslipidemia is a serious problem related to an increased frequency of cardiovascular disease. Recently, He et al. analyzed the influence of DHA on the locomotor activity in ethanol-treated HIV-1 transgenic rats [109]. The prevalence of alcohol use and alcohol abuse in infected individuals is much higher, and numerous ethanol and HIV-1 viral proteins have synergistic effects on inflammation in the central nervous system [118][119][120]. HIV remains in the body in its latent form after HAART therapy and, as such, can induce neuroinflammation. DHA depletion has been found to be associated with various neurological abnormalities, and its administration can have a neuroprotective effect. DHA taken daily could reverse the effects of the ethanol negative effect on the locomotor activity in the presence of HIV viral proteins. An in vivo study, using real-time quantitative PCR, showed that the addition of DHA can reduce elevated levels of IL-6, IL-18, and increase the expression of NF-κB in the striatum. This proved the potential of this fish oil constituent as an adjuvant in HIV patients' treatment that can help in lowering the interactive effects of ethanol consumption during HIV infection.

Others
Resorcyclic acid lactones, namely radicicol (49, Figure 22) pochonin B (50, Figure 22) and C (51, Figure 22) isolated from H. fuscoatra exhibited a 92-98% reactivation efficiency of the latent HIV-1 relative to SAHA (subeoylanilide hydroxamic acid, vorinostat, HDAC inhibitor) and EC50 of 9.1, 39.6 Recently, He et al. analyzed the influence of DHA on the locomotor activity in ethanol-treated HIV-1 transgenic rats [109]. The prevalence of alcohol use and alcohol abuse in infected individuals is much higher, and numerous ethanol and HIV-1 viral proteins have synergistic effects on inflammation in the central nervous system [118][119][120]. HIV remains in the body in its latent form after HAART therapy and, as such, can induce neuroinflammation. DHA depletion has been found to be associated with various neurological abnormalities, and its administration can have a neuroprotective effect. DHA taken daily could reverse the effects of the ethanol negative effect on the locomotor activity in the presence of HIV viral proteins. An in vivo study, using real-time quantitative PCR, showed that the addition of DHA can reduce elevated levels of IL-6, IL-18, and increase the expression of NF-κB in the striatum. This proved the potential of this fish oil constituent as an adjuvant in HIV patients' treatment that can help in lowering the interactive effects of ethanol consumption during HIV infection.

Others
Resorcyclic acid lactones, namely radicicol (49, Figure 22) pochonin B (50, Figure 22) and C (51, Figure 22) isolated from H. fuscoatra exhibited a 92-98% reactivation efficiency of the latent HIV-1 relative to SAHA (subeoylanilide hydroxamic acid, vorinostat, HDAC inhibitor) and EC 50 of 9.1, 39.6 and 6.3 µM [110]. The reactivation strategy is, indeed, a promising strategy to expunge the HIV-1 infection by reactivating latent viral loads, mainly in CD4 + T-cells, which quickly rebound when antiviral treatment is interrupted. It was noted that all active compounds contain Michael acceptor functionality. The PKC-independent mechanism of reactivation of the latent HIV-1 remains to be elucidated.
Molecules 2019, 24, x FOR PEER REVIEW 33 of 40 and 6.3 µM [110]. The reactivation strategy is, indeed, a promising strategy to expunge the HIV-1 infection by reactivating latent viral loads, mainly in CD4 + T-cells, which quickly rebound when antiviral treatment is interrupted. It was noted that all active compounds contain Michael acceptor functionality. The PKC-independent mechanism of reactivation of the latent HIV-1 remains to be elucidated. A team of researchers led by Zhao isolated new isoprenylated cyclohexanols from the spongeassociated fungus Truncatella angustata named truncateols O-V [111]. In vitro testing showed that truncateols O and P (52 and 53, Figure 23), analogues bearing the alkynyl group in the side chain, exhibit a significant inhibition toward the HIV-1 virus with IC50 values of 39.0 µM and 16.1 µM, respectively. These compounds could be considered as new anti-HIV lead compounds due to lower cytotoxicity (CC50 > 100 µM) in comparison with the positive control efavirenz (CC50 = 40.6 µM).

Future Directions in the Anti-HIV Marine Drug Development
Marine organisms have been acknowledged as a precious source of bioactive compounds that may provide novel anti-HIV structures or lead structures for structural optimization. A large amount of evidence from scientific research confirmed a high biological potential of these compounds to treat serious diseases, including infective ones. Some of the marine-derived bioactive compounds discovered much earlier have emerged with novel properties and potential applications after a decade or two. Isolation and structural elucidation of compounds from marine organisms is not an easy task and still carries challenges. Identification of all the compounds is a daunting task, especially with regards to complex structural motifs that may be present in a single marine extract. Taxonomic knowledge is still insufficient to enable unambiguous species classification that can result in the false prediction of chemical constituents and hamper structural analysis. Furthermore, a temporal lag between the discovery, chemical characterization, and associated pharmacological activities is quite common, and the majority of marine metabolites are usually tested for anticancer activity, whereas anti-HIV and other possible biological effects are neglected or mostly not performed due to a lack of funding. Targeted assays and in vivo analyses are similarly performed only for some of the potential candidates, while the translation into clinical trials remains very limited. Thus, the financial gap is certainly a relevant factor contributing to the slow drug development process in this area. In particular, the development of anti-HIV compounds, which act by mechanisms that differ from existing antivirals, requires a well-designed and focused approach to studying the mode of action. A team of researchers led by Zhao isolated new isoprenylated cyclohexanols from the sponge-associated fungus Truncatella angustata named truncateols O-V [111]. In vitro testing showed that truncateols O and P (52 and 53, Figure 23), analogues bearing the alkynyl group in the side chain, exhibit a significant inhibition toward the HIV-1 virus with IC 50 values of 39.0 µM and 16.1 µM, respectively. These compounds could be considered as new anti-HIV lead compounds due to lower cytotoxicity (CC 50 > 100 µM) in comparison with the positive control efavirenz (CC 50 = 40.6 µM).
Molecules 2019, 24, x FOR PEER REVIEW 33 of 40 and 6.3 µM [110]. The reactivation strategy is, indeed, a promising strategy to expunge the HIV-1 infection by reactivating latent viral loads, mainly in CD4 + T-cells, which quickly rebound when antiviral treatment is interrupted. It was noted that all active compounds contain Michael acceptor functionality. The PKC-independent mechanism of reactivation of the latent HIV-1 remains to be elucidated. A team of researchers led by Zhao isolated new isoprenylated cyclohexanols from the spongeassociated fungus Truncatella angustata named truncateols O-V [111]. In vitro testing showed that truncateols O and P (52 and 53, Figure 23), analogues bearing the alkynyl group in the side chain, exhibit a significant inhibition toward the HIV-1 virus with IC50 values of 39.0 µM and 16.1 µM, respectively. These compounds could be considered as new anti-HIV lead compounds due to lower cytotoxicity (CC50 > 100 µM) in comparison with the positive control efavirenz (CC50 = 40.6 µM).

Future Directions in the Anti-HIV Marine Drug Development
Marine organisms have been acknowledged as a precious source of bioactive compounds that may provide novel anti-HIV structures or lead structures for structural optimization. A large amount of evidence from scientific research confirmed a high biological potential of these compounds to treat serious diseases, including infective ones. Some of the marine-derived bioactive compounds discovered much earlier have emerged with novel properties and potential applications after a decade or two. Isolation and structural elucidation of compounds from marine organisms is not an easy task and still carries challenges. Identification of all the compounds is a daunting task, especially with regards to complex structural motifs that may be present in a single marine extract. Taxonomic knowledge is still insufficient to enable unambiguous species classification that can result in the false prediction of chemical constituents and hamper structural analysis. Furthermore, a temporal lag between the discovery, chemical characterization, and associated pharmacological activities is quite common, and the majority of marine metabolites are usually tested for anticancer activity, whereas anti-HIV and other possible biological effects are neglected or mostly not performed due to a lack of funding. Targeted assays and in vivo analyses are similarly performed only for some of the potential candidates, while the translation into clinical trials remains very limited. Thus, the financial gap is certainly a relevant factor contributing to the slow drug development process in this area. In particular, the development of anti-HIV compounds, which act by mechanisms that differ from existing antivirals, requires a well-designed and focused approach to studying the mode of action.

Future Directions in the Anti-HIV Marine Drug Development
Marine organisms have been acknowledged as a precious source of bioactive compounds that may provide novel anti-HIV structures or lead structures for structural optimization. A large amount of evidence from scientific research confirmed a high biological potential of these compounds to treat serious diseases, including infective ones. Some of the marine-derived bioactive compounds discovered much earlier have emerged with novel properties and potential applications after a decade or two. Isolation and structural elucidation of compounds from marine organisms is not an easy task and still carries challenges. Identification of all the compounds is a daunting task, especially with regards to complex structural motifs that may be present in a single marine extract. Taxonomic knowledge is still insufficient to enable unambiguous species classification that can result in the false prediction of chemical constituents and hamper structural analysis. Furthermore, a temporal lag between the discovery, chemical characterization, and associated pharmacological activities is quite common, and the majority of marine metabolites are usually tested for anticancer activity, whereas anti-HIV and other possible biological effects are neglected or mostly not performed due to a lack of funding. Targeted assays and in vivo analyses are similarly performed only for some of the potential candidates, while the translation into clinical trials remains very limited. Thus, the financial gap is certainly a relevant factor contributing to the slow drug development process in this area. In particular, the development of anti-HIV compounds, which act by mechanisms that differ from existing antivirals, requires a well-designed and focused approach to studying the mode of action. Libraries should be created for specifically defined crude extracts, their corresponding simplified fractions as well as for pure compounds for a well-balanced natural product discovery program. Additionally, there exist but few publications in which scientists have tried to modify known compounds of marine origin to improve their bioactivity. We are, however, continuously witnessing advancements in the deep-sea exploration technology, sampling strategies, genome sequencing, genome mining, genetic engineering, chemo-enzymatic synthesis, nanoscale NMR structure determination, and development and optimization of suitable fermentation strategies to ensure a continued supply of unique bioactive compounds from the oceans. Therefore, the grounds have been met for a broad, international effort based on scientific collaboration that would rely on well-equipped infrastructure and human resources as a prerequisite for a full advancement in the field and development of new drug candidates for the pharmaceutical market in the future.