Microbial Sterolomics as a Chemical Biology Tool

Metabolomics has become a powerful tool in chemical biology. Profiling the human sterolome has resulted in the discovery of noncanonical sterols, including oxysterols and meiosis-activating sterols. They are important to immune responses and development, and have been reviewed extensively. The triterpenoid metabolite fusidic acid has developed clinical relevance, and many steroidal metabolites from microbial sources possess varying bioactivities. Beyond the prospect of pharmacognostical agents, the profiling of minor metabolites can provide insight into an organism’s biosynthesis and phylogeny, as well as inform drug discovery about infectious diseases. This review aims to highlight recent discoveries from detailed sterolomic profiling in microorganisms and their phylogenic and pharmacological implications.


Introduction
Sterols, like cholesterol 1, ergosterol 2, and sitosterol 3, as well as secondary metabolites, are amphipathic lipids that contain a 1,2-cyclopentanoperhydrophenanthrene ring nucleus ( Figure 1). Sterols are ubiquitous molecules found in all eukaryotic life, serving a multitude of crucial biological functions [1]. Some prokaryotes synthesize sterols as well, and some prokaryotes contain enzymes with incomplete ∆ 5 sterol biosynthesis [1][2][3][4][5]. While sterol biosynthesis may predate eukaryotes [6], it is often hypothesized that aside from the protomitochondrial lineage, most bacteria have gained these genes via lateral gene transfer [3,4]. The end product of ∆ 5 sterols such as cholesterol 1 and ergosterol 2 ( Figure 1a) contribute to cell membrane fluidity in their bulk insert role in mammals and fungi, respectively [1,7]. Steroidal secondary metabolites of the steroid hormone and bile acid classes serve well-known important roles in inflammation, sex characteristics, and lipid absorption [7].
Novel metabolites isolated from microbial sources are conversely often found to exhibit biological activity. Famously, fusidic acid 7 (Figure 1c), originally isolated from fungal Fusidium spp., is a tetracyclic triterpene antibacterial and has been used in the clinic for decades [27][28][29]. Fusidic acid inhibits growth by restricting protein synthesis via elongation factor G in Gram-positive bacteria, including Streptococcus spp., Clostridium spp., and penicillin-resistant strains of Staphylococcus spp. [28,29]. Structural analogues of fusidic acid, have shown varying antimicrobial, as well as anticholesterolemic and antineoplastic, characteristics [29]. Isolated from a variety of fungi and sponges, as well as vascular plants, ergosterol peroxide 8 possesses broad bioactivity, including antitumor, immunomodulatory, inhibitory hemolytic, anti-inflammatory, antioxidant, and antimicrobial properties. Several other endoperoxides of other phytosterols and of cholestenols have been reported to have similar properties, as well [30][31][32][33][34]. Squalamine 9 is a non-microbially derived natural steroidal, which has demonstrated antimicrobial and antiangiogenic properties and has led to interest in synthetic analogues for structure-activity improvement [35].
The algal pathway was further corroborated by characterization of recombinant C. reinhardtii 24-SMT, found to catalyze the methylation of C24 by introduction of C28 and the methylation of C28 with C29. C. reinhardtii 24-SMT favored cycloartenol as a substrate, and a bifurcation of products to cyclolaudenol 28 and 24(28)-methylenecycloartanol 29 was found in ratios comparable to in vivo ratios of ergosterol and 7-dehydroporiferasterol [50]. A switch to Δ 25 (27) -olefin "algal" products of fungal or plant 24-SMT has been noted upon mutagenesis or incubation with electronically modified substrates [49,51]. In addition, obtusifoliol was found to be a substrate for the second methyltransfer of C. reinhardtii 24-SMT, 24β-methyl-Δ 25 (27) -sterols were not substrates, and incubation with [methyl- In algae, 24-methyl and 24-ethyl sterols arise from a bifurcation of products of biomethylation by sterol methyltransferase (SMT); In higher plants, they arise from alternate pathways from the intermediate 24 (28)-methylene lophenol 30, which can be methylated again or metabolized to campesterol 36. Red methyl groups from SMT co-substrate S-adenosyl methionine (AdoMet) are annotated to show hypothetical labeling patterns of ∆ 5 sterols as discussed in [45,50]. An additional 15 algal sterols were reported in [45]. Truncated fungal phytosterol biosynthesis from protosterol lanosterol 12 is illustrated in Figure 2.
In C. reinhardtii, the biochemical pathway from the "plant" protosterol cycloartenol to the "fungal" ∆ 5 end product was investigated by sterolomic experiments of C. reinhardtii cultures. Sterol profiling of wild-type, mutant, and inhibitor-treated cultures revealed an additional 21 sterols beyond cycloartenol, ergosterol, and 7-dehydroporiferasterol 33 [45] (Figure 3). C. reinhardtii cultures that were not treated with a 24-SMT inhibitor contained only cycloartenol and 24-alkylsterols, indicating that bioalkylation and introduction of C28 by algal 24-SMT occurs upon cycloartenol itself early in the pathway. Further, 24-methylated cycloartenols were 24β-methylcycloart-25 (27)-enol (cyclolaudenol) 28 and 24(28)-methylenecycloartanol 29, signifying a bifurcation of methylated products of algal 24-SMT [45]. The presence of C29 (i.e., a 24-ethyl group) on a 4α,14α-dimethyl sterol 33 led to the identification of obtusifoliol 31 as the substrate for the second biomethylation reaction of the algal sterol side chain, different from the substrate preference in higher plants ( Figure 3). Furthermore, the alkylation product in plants has a 24-ethylene substituent, whereas the product in C. reinhardtii was found to bear a 24β-ethyl group with desaturation at C25 [45].
The algal pathway was further corroborated by characterization of recombinant C. reinhardtii 24-SMT, found to catalyze the methylation of C24 by introduction of C28 and the methylation of C28 with C29. C. reinhardtii 24-SMT favored cycloartenol as a substrate, and a bifurcation of products to cyclolaudenol 28 and 24(28)-methylenecycloartanol 29 was found in ratios comparable to in vivo ratios of ergosterol and 7-dehydroporiferasterol [50]. A switch to ∆ 25(27) -olefin "algal" products of fungal or plant 24-SMT has been noted upon mutagenesis or incubation with electronically modified substrates [49,51]. In addition, obtusifoliol was found to be a substrate for the second methyltransfer of C. reinhardtii 24-SMT, 24β-methyl-∆ 25(27) -sterols were not substrates, and incubation with [methyl-2 H 3 ]S-adenosyl methionine ( 2 H 3 -AdoMet) produced labeled products with three and five deuterium atoms [50].

Trophic and Limnological Sterols
In the cross-class algal study [53], the researchers presented these profiles, along with their quantification, as references to the algal sterolome. As prey, ∆ 7 and ∆ 7,22 sterols are often nutritionally inadequate to invertebrate consumers [53,56,57]. Many invertebrates are auxotrophic for sterols and rely on diet to fulfill their sterol needs for cell membrane and hormonal requirements. Several of these specimens contain alternate enzymes, which dealkylate side chains of phytosterols, yet they lack the enzymes to desaturate C5-C6 or reduce C7-C8 ( Figure 5) [57,58]. It has been proposed that these quantitated algal sterolome references can be used for studies involving the nutritional content of aquatic microorganisms for aquatic invertebrates [53]. Another study monitored the sterol profiles of an algal diet and the amphipod consumer Gammarus roeselii. Prey alga N. limnetica, rich in cholesterol, and alga S. obliquus, lacking cholesterol but rich in ∆ 7 sterols (See Table 1), were fed to G. roeselii. The sterol profile of S. obliquus-fed G. roeselii decreased in cholesterol, and increased in the ∆ 7 metabolite lathosterol 69, detectable when the diet was 50% S. obliquus ( Figure 5) [56].
Isotopically labeled sterolomic experiments have been used to explore trophic modifications by the Northern Bay scallop Argopecten irradians irradians. Dietary alga Rhodomonas was supplemented with sterols enriched with 13 C at the C22 position. The 13 C-label was noted on new sterol metabolites, including those newly desaturated with ∆ 7 and those bearing an introduced 4α-methyl group. The mollusk's ability to synthesize cholesterol from food was noted to correlate to ∆ 5 double bonds in the dietary sterols. They were more likely to dealkylate side chains possessing 24-ethyl groups. The only 24-methyl sterols dealkylated by A. irradians contained a ∆ 24(28) olefin (i.e., 24-methylene, rather than 24-methyl) [55]. Isotopically labeled sterolomic experiments have been used to explore trophic modifications by the Northern Bay scallop Argopecten irradians irradians. Dietary alga Rhodomonas was supplemented with sterols enriched with 13 C at the C22 position. The 13 C-label was noted on new sterol metabolites, including those newly desaturated with Δ 7 and those bearing an introduced 4α-methyl group. The mollusk's ability to synthesize cholesterol from food was noted to correlate to Δ 5 double bonds in the dietary sterols. They were more likely to dealkylate side chains possessing 24-ethyl groups. The only 24-methyl sterols dealkylated by A. irradians contained a Δ 24(28) olefin (i.e., 24-methylene, rather than 24-methyl) [55].
A recent study investigated the lipid content of 37 strains within 10 classes of phytoplankton. Four classes, Cryptophyceae, Chlorophyceae, Treouciophyceae, and the diatoms are additionally represented in Table 1; this study additionally included dinoflagellates, euglenoids and the conjugatophyceae. Of the 37 strains, 29 sterols were detected, with notable variability of profile as a function of taxonomic class. The authors suggested Δ 5,22 sterols as a potential biomarker for Chlorophyceae Sphaerocystis sp. and ergosterol as a potential biomarker for Chlamydomonas in habitats lacking other aquatic ergosterol-synthesizing microorganisms [59].
A recent study investigated the lipid content of 37 strains within 10 classes of phytoplankton. Four classes, Cryptophyceae, Chlorophyceae, Treouciophyceae, and the diatoms are additionally represented in Table 1; this study additionally included dinoflagellates, euglenoids and the conjugatophyceae. Of the 37 strains, 29 sterols were detected, with notable variability of profile as a function of taxonomic class. The authors suggested ∆ 5,22 sterols as a potential biomarker for Chlorophyceae Sphaerocystis sp. and ergosterol as a potential biomarker for Chlamydomonas in habitats lacking other aquatic ergosterol-synthesizing microorganisms [59].
Molecules 2018, 23, x FOR PEER REVIEW 9 of 38 (11-13%), analyzed as their TMS derivatives. 4α-Methylgorgosterol is uncommon in dinoflagellates and has potential as a biomarker ( Figure 6) [62]. Lipidomic study of the coral Dendrophyllia cornigera revealed a geographical correlation to diet. D. cornigera analyzed from the Cantabrian Sea in the Northeast Atlantic reflected a productive environment, and the coral contained a high diversity of phytosterols. D. cornigera sampled from the Menorca Channel in the Mediterranean had a lower sterol content per dry weight and had less phytosterols. The Mediterranean coral had a higher relative abundance of occelasterol 70, brassicasterol 42, and cholestanol 81, or cholesterol and ergosterol, depending on the sample. The difference in the geographic profiles was attributed to a diet high in phytoplankton and herbivorous grazers in the Cantabrian coral, and a diet primarily consisting of dinoflagellates in the Mediterranean coral [63]. Specimens of the coral Agaricia spp. taken from shallow waters and deep waters were found to have markedly different sterol profiles from one another. From shallow Caribbean waters, Agaricia contained mostly cholesterol and 24-methylenecholesterol, with lower abundances of other phytosterols. Samples from deep waters contained mostly cholesterol and 24ethylcholesterol. No gorgosterol was detected in either set. The Caribbean coral Montastraea cavernosa contained mostly 24-methylcholesterol, followed by cholesterol and gorgosterol, and variation in subsurface depth did not cause a significant change in sterol content. It was concluded that Agaricia spp. relies primarily on heterotrophy, even at greater depths [64].

Trypanosoma brucei
Trypanosomatids are flagellated protozoa, all of which are parasitic. Some examples from this clade are Crithidia fasciculata, solely parasitic to insect hosts, Phytomonas serpens, soley phytopathogenic, and a number of human pathogens, including Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei, which are the etiological agents of the following human diseases: leishmaniasis, Chagas' disease, and human African trypanosomiasis (also known as African sleeping sickness), respectively. Most of the species, C. fasciculata [38], P. serpens [40], T. cruzi [38,44], and Leishmania spp. [39] synthesize ergosterol and other 24β-methyl/24(28)-methylene-sterols (ergostenols) de novo as their Δ 5 end products. In light of this de novo biosynthesis, there has been interest in using ergosterol biosynthesis inhibitors (EBIs) to treat Chagas' disease and leishmaniasis, and some molecules have even progressed to the clinic [25,44]. Trypanosoma brucei, conversely, synthesizes ergostenols during its life cycle in the insect vector (procyclic form (PCF)), but uses largely cholesterol from the host's blood as its Δ 5 bulk sterol in the human host (bloodstream form (BSF)) [41][42][43].
In the fly vector, cholesterol comprises a significant portion of the PCF sterol content. The profile contains sterols endogenous to PCF T. brucei, including prominent cholesta-5,7,24-trienol 82 and ergosta-5,7,25(27)-trienol 85. PCF synthesizes trace ergosterol 2; Ergosta-5,7,24(28)-trienol 85 and Lipidomic study of the coral Dendrophyllia cornigera revealed a geographical correlation to diet. D. cornigera analyzed from the Cantabrian Sea in the Northeast Atlantic reflected a productive environment, and the coral contained a high diversity of phytosterols. D. cornigera sampled from the Menorca Channel in the Mediterranean had a lower sterol content per dry weight and had less phytosterols. The Mediterranean coral had a higher relative abundance of occelasterol 70, brassicasterol 42, and cholestanol 81, or cholesterol and ergosterol, depending on the sample. The difference in the geographic profiles was attributed to a diet high in phytoplankton and herbivorous grazers in the Cantabrian coral, and a diet primarily consisting of dinoflagellates in the Mediterranean coral [63]. Specimens of the coral Agaricia spp. taken from shallow waters and deep waters were found to have markedly different sterol profiles from one another. From shallow Caribbean waters, Agaricia contained mostly cholesterol and 24-methylenecholesterol, with lower abundances of other phytosterols. Samples from deep waters contained mostly cholesterol and 24-ethylcholesterol. No gorgosterol was detected in either set. The Caribbean coral Montastraea cavernosa contained mostly 24-methylcholesterol, followed by cholesterol and gorgosterol, and variation in subsurface depth did not cause a significant change in sterol content. It was concluded that Agaricia spp. relies primarily on heterotrophy, even at greater depths [64].

Trypanosoma brucei
Trypanosomatids are flagellated protozoa, all of which are parasitic. Some examples from this clade are Crithidia fasciculata, solely parasitic to insect hosts, Phytomonas serpens, soley phytopathogenic, and a number of human pathogens, including Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei, which are the etiological agents of the following human diseases: leishmaniasis, Chagas' disease, and human African trypanosomiasis (also known as African sleeping sickness), respectively. Most of the species, C. fasciculata [38], P. serpens [40], T. cruzi [38,44], and Leishmania spp. [39] synthesize ergosterol and other 24β-methyl/24(28)-methylene-sterols (ergostenols) de novo as their ∆ 5 end products. In light of this de novo biosynthesis, there has been interest in using ergosterol biosynthesis inhibitors (EBIs) to treat Chagas' disease and leishmaniasis, and some molecules have even progressed to the clinic [25,44]. Trypanosoma brucei, conversely, synthesizes ergostenols during its life cycle in the insect vector (procyclic form (PCF)), but uses largely cholesterol from the host's blood as its ∆ 5 bulk sterol in the human host (bloodstream form (BSF)) [41][42][43].
In BSF T. brucei, however, the sterol content is overwhelmingly cholesterol, as well as dietary phytosterols, like sitosterol 3 and campesterol 36, present in the hosts' blood [41][42][43]. Single trace 13 C-labeled sterol was found in BSF cultures fed [1-13 C]glucose [42]. Upon removal of the main sterol component cholesterol, detailed targeted sterolomics of BSF T. brucei cells revealed minor components of the sterol profile. Due to the S-cis double bond configuration in the B ring of ergosterol and compounds 81-87, UV absorbances of 282 nm can be monitored for the presence of endogenous ∆ 5,7 sterols, absent in serum. Endogenous cholesta-5,7,24-trienol and ergostenols were found at trace amounts, while they were undetectable when the presence of cholesterol was predominant. The ergosterol requirements for BSF was estimated to be 0.01 fg/cell, compared to the PCF requirement of 6 fg/cell [41]. Consequently, treatment with the EBIs itraconazole 22 and 25-azalanosterol 25 resulted in parasite death and an increased survival rate of infected mice. Correspondingly, the effects of EBIs on cultures were reversed upon supplementation of ergosterol [41]. 24

Acanthamoeba spp.
Ergosterol is a significant Δ 5 bulk sterol in amoebae, as is 7-dehydroporiferasterol. Sterols are synthesized de novo in amoebae via a biosynthetic pathway involving the protosterol cycloartenol 25, as in green algae and higher plants. Amoebae also synthesize 19(10→6)-abeo-sterols containing aromatic B rings called the amebasterols [22,36]. Amebasterol-1 94, amebasterol-2 95, and amebasterol-4 98 have been described [22]; trace amebasterols-3 96, -5 99, and -6 97 have been identified as of late ( Figure 9). These compounds can be selectively monitored at UV absorbances of 270 nm [36]. The sterol profile of was found to be variable as a function of growth and encystment phases. Analysis of the Acanthamoeba castellanii sterolome throughout the first week and one month after inoculation revealed a variable composition with changes to cell morphology and viability. At the beginning of the excystment-trophozoite-encystment cycle, in early log phase of growth, an accumulation of protosterol cycloartenol 27 and 24-methylenated cycloartanol 29 was noted. As the

Acanthamoeba spp.
Ergosterol is a significant Δ 5 bulk sterol in amoebae, as is 7-dehydroporiferasterol. Sterols are synthesized de novo in amoebae via a biosynthetic pathway involving the protosterol cycloartenol 25, as in green algae and higher plants. Amoebae also synthesize 19(10→6)-abeo-sterols containing aromatic B rings called the amebasterols [22,36]. Amebasterol-1 94, amebasterol-2 95, and amebasterol-4 98 have been described [22]; trace amebasterols-3 96, -5 99, and -6 97 have been identified as of late (Figure 9). These compounds can be selectively monitored at UV absorbances of 270 nm [36]. The sterol profile of was found to be variable as a function of growth and encystment phases. Analysis of the Acanthamoeba castellanii sterolome throughout the first week and one month after inoculation revealed a variable composition with changes to cell morphology and viability. At the beginning of the excystment-trophozoite-encystment cycle, in early log phase of growth, an accumulation of protosterol cycloartenol 27 and 24-methylenated cycloartanol 29 was noted. As the The sterol profile of was found to be variable as a function of growth and encystment phases. Analysis of the Acanthamoeba castellanii sterolome throughout the first week and one month after inoculation revealed a variable composition with changes to cell morphology and viability. At the beginning of the excystment-trophozoite-encystment cycle, in early log phase of growth, an accumulation of protosterol cycloartenol 27 and 24-methylenated cycloartanol 29 was noted. As the cells replicated, trophozoites contained mostly the ∆ 5,7 products ergosterol and 7-dehydroporiferasterol, whereas, in the stationary growth phase, with a mixture of trophozoites and cysts, sterols shifted to the ∆ 5 products brassicasterol and poriferasterol. Supplementation of trophozoite cultures with cholesterol had only a minor stimulation effect on their growth. After one-month incubation, dead cells were mostly comprised of amebasterols, amebasterol-1 94 and amebasterol-2 95 (Figure 9). The shift from ∆ 5,7 products in non-viable encysted cells to the amebasterols was attributed to turnover from stress and a sterol composition associated with altered membrane fluidity affording lysis ( Figure 10) [36].

Fungal Sterol Profiles in Drug-Treated Cultures
EBIs are a staple of antimycotic drug discovery [23][24][25]. A general hypothetical biosynthetic pathway, as well as popular block points for EBIs, are presented in Figure 2. Sterolomics can be used to confirm the inhibition of ergosterol biosynthesis upon treatment with new small molecules with antifungal properties.
The noted pairs of cycloartenol and 24(28)-methylenecycloartanol (24-H/24-Me), and pairs of ergosterol/poriferasterol, brassicasterol/poriferasterol, and amebasterol-1/amebasterol-2 (each 24-Me/24-Et) in the various portions of the Acanthamoebic life cycle [36], along with product outcomes being largely determined by biomethylation patterns of A. castellanii SMTs [36,72], underscores the crucial nature of SMT function in the pathogen. Subtle alterations in substrate selectivity were noted to have a profound impact on the balance of 24-methyl and 24-ethyl sterols [36]. After treatment with the 24-SMT inhibitor 24(R,S),25-epiminolatnosterol 26 and the azole 14-SDM inhibitor voriconazole 20 (See Figure 2 for structures), and small increase in amounts of cycloartenol and obtusifoliol were noted [72,73]. Upon treatment with EBIs, trohpozoites were stimulated to encyst, while excystment was insensitive to treatment. The correlation between stage-specific sterol compositions and the physiological effects of EBIs provide insight on opportunities for therapeutics ( Figure 10). It is imagined that EBIs targeting the enzyme that reduces the ∆ 7 olefin of ergosterol/7-dehydroproferasterol to brassicasterol/poriferasterol could be used to modulate Acanthamoeba growth phases and prevent recurrence of the disease [36].
Azole inhibitors of 14-SDM have been reported to restrict Acanthamoeba growth in the nanomolar to micromolar range [36,37,[73][74][75][76], and inhibitors of 24-SMT have been reported with nanomolar activity against Acanthamoeba cultures [36,72]. Treatments of 14-SDM-and 24-SMT-inhibitors in combination led to complete eradication of the amoeba parasite at concentrations as low as their respective IC 50 s [36].

Fungal Sterol Profiles in Drug-Treated Cultures
EBIs are a staple of antimycotic drug discovery [23][24][25]. A general hypothetical biosynthetic pathway, as well as popular block points for EBIs, are presented in Figure 2. Sterolomics can be used to confirm the inhibition of ergosterol biosynthesis upon treatment with new small molecules with antifungal properties.
Series of amidoesters substituted with imidazolylmethyl groups were reported to have bioactivity against opportunistic fungal pathogens Candida albicans, Candida tropicalis, Cryptococcus neoformans, and Aspergillus fumigatus [77,78]. Some of these compounds, including 100 [77] and 101 [78] (Figure 11a) displayed better antifungal properties than fluconazole 21 (cf. Figure 2). The sterols of C. albicans administered with these compounds were analyzed to confirm a mechanism of disrupting ergosterol biosynthesis. Ergosterol normally comprises of the vast majority of the sterol profile in C. albicans (>98%), and treatment with 100 [77] or 101 [78] reduced ergosterol in a dose dependent manner. Dose-dependent increases in lanosterol 12 were noted, as well as increases in 14α-methylsterol by-products eburicol 102 and obtusifoliol 31 (Figure 11a). The increase in lanosterol (substrate for C. albicans 14-SDM), the increase in 14-methylsterols, and a commensurate decrease in ergosterol itself, suggested 14-SDM as a target for these molecules [77,78].

Bioactive Steroidal Metabolites
Endogenous oxysterols play essential roles in human biology, including signaling, development, and immunology [8][9][10][11]. Similarly, several oxysterols isolated from microbial sources have been reported to exhibit therapeutic properties. Many of these compounds from microbes are oxyphytosterols, i.e., unlike human endogenous sterols, they possess alkyl groups at C24 and therefore do not occur in human biology. Bioactivities include those against cancer cell lines, as well as ligands for nuclear receptors, antioxidants, anti-inflammatory agents, and inhibitors of amyloid-β (Aβ) aggregation.
Minor steroidal metabolites often possess bioactivity against other microbes, like fusidic acid, as discussed above. Study of these natural products can further lead to semi-synthetic analogues for structure-activity relationship studies and improvement of antimicrobial agents. For instance, squalamine 9 (Figure 1c), isolated from dogfish shark, is a steroid with polyamine substitution. The cationic polyamine moiety and its polyvalence have been attributed to much of its antimicrobial and anticancer properties [35], and, as a result, a class of synthetic and semi-synthetic analogues, collectively termed cationic steroid antibiotics, have been developed [35,83,84]. For the purposes of this review, only isolated compounds are discussed, though these compounds can inform synthetic and semisynthetic analogues for increased bioactivity. Likewise, steroidal metabolites with a compromised cyclopentanoperhydrophenanthrene nucleus are omitted here.

Peroxides
Michosterol A 107 ( Figure 13) is a newly described polyoxygenated sterol with a C20 hydroperoxyl group and a C25 acetoxyl group, isolated by the ethyl acetate extract of the soft coral Lobophytum michaelae. Michosterol A demonstrated moderate cytotoxic effects against A549 cells, with an IC50 of 14.9 µg/mL, and was not cytotoxic (IC50s > 20 µg/mL) to DLD-1 and LNCap cell lines. Its anti-inflammatory activity was examined by assaying against superoxide formation in human neutrophils and against elastase release. Michosterol A had IC50s of 7.1 µM and 4.5 µM for superoxide anion generation and elastase release, respectively. A second hydroperoxyl polyoxygenated sterol (C15 hydroperoxyl, and Δ 17 (20) ), named michosterol B 108 ( Figure 13) was discovered in this extract. Michosterol B did not display cytotoxicity against the cell lines tested, but inhibited superoxide anion generation 14.7% and elastase release 31.8% each at 10 µM michosterol B [85].

Bioactive Steroidal Metabolites
Endogenous oxysterols play essential roles in human biology, including signaling, development, and immunology [8][9][10][11]. Similarly, several oxysterols isolated from microbial sources have been reported to exhibit therapeutic properties. Many of these compounds from microbes are oxyphytosterols, i.e., unlike human endogenous sterols, they possess alkyl groups at C24 and therefore do not occur in human biology. Bioactivities include those against cancer cell lines, as well as ligands for nuclear receptors, antioxidants, anti-inflammatory agents, and inhibitors of amyloid-β (Aβ) aggregation.
Minor steroidal metabolites often possess bioactivity against other microbes, like fusidic acid, as discussed above. Study of these natural products can further lead to semi-synthetic analogues for structure-activity relationship studies and improvement of antimicrobial agents. For instance, squalamine 9 (Figure 1c), isolated from dogfish shark, is a steroid with polyamine substitution. The cationic polyamine moiety and its polyvalence have been attributed to much of its antimicrobial and anticancer properties [35], and, as a result, a class of synthetic and semi-synthetic analogues, collectively termed cationic steroid antibiotics, have been developed [35,83,84]. For the purposes of this review, only isolated compounds are discussed, though these compounds can inform synthetic and semisynthetic analogues for increased bioactivity. Likewise, steroidal metabolites with a compromised cyclopentanoperhydrophenanthrene nucleus are omitted here.
Penicisteroid A 115 ( Figure 14) is an analogue of anicequol bearing a 7α-hydroxyl rather than a 7-oxo-group. Extracted from Penicillium chrysogenum QEN-24S, an endophytic fungus isolated from a red alga of the genus Laurencia, penicisteroid A exhibited both antimycotic and cytotoxic effects. Against the pathogenic fungi Aspergillus niger and Alternaria brassicae, penicisteroid A gave ZOIs (20 µg) of 18 mm and 9 mm, respectively, compared to 24 mm and 16 mm for control amphotericin B. Penicisteroid A also inhibited HeLa, SW1990, and NCI-H460 cancer cell lines with IC50s of 15 µg/mL, 31 µg/mL, and 40 µg/mL, showing selectivity variable from the anicequol parent compound [91]. Penicisteroid C 116 (Figure 14) also has a C16 acetate, but is less oxygenated than penicisteroid A. It was isolated from a co-cultivation of bacteria Streptomyces piomogenus AS63D and fungus Aspergillus niger using solid-state fermentation on rice medium. Penicisteroid C displayed selective antimicrobial Penicisteroid A 115 ( Figure 14) is an analogue of anicequol bearing a 7α-hydroxyl rather than a 7-oxo-group. Extracted from Penicillium chrysogenum QEN-24S, an endophytic fungus isolated from a red alga of the genus Laurencia, penicisteroid A exhibited both antimycotic and cytotoxic effects. Against the pathogenic fungi Aspergillus niger and Alternaria brassicae, penicisteroid A gave ZOIs (20 µg) of 18 mm and 9 mm, respectively, compared to 24 mm and 16 mm for control amphotericin B. Penicisteroid A also inhibited HeLa, SW1990, and NCI-H460 cancer cell lines with IC 50 s of 15 µg/mL, 31 µg/mL, and 40 µg/mL, showing selectivity variable from the anicequol parent compound [91]. Penicisteroid C 116 (Figure 14) also has a C16 acetate, but is less oxygenated than penicisteroid A. It was isolated from a co-cultivation of bacteria Streptomyces piomogenus AS63D and fungus Aspergillus niger using solid-state fermentation on rice medium. Penicisteroid C displayed selective antimicrobial activity against tested organisms. ZOIs for penicisteroid C were 7 mm, 9 mm, and 10 mm for bacterial cultures Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis, respectively, and were 8 mm and 12 mm for fungal cultures Candida albicans and Saccharomyces cerevisiae, respectively [92].

Cyclopropanes
From the marine sponge Xestospongia testudinaria, oxyphytosterols 129 and 130 (Figure 15), with a side chain cyclized at C26-C27 were recently reported to posess anti-adhesion properties against bacteria Pseudoalteromonas spp. and Polaribacter sp. New compounds 129 and 133, as well as known compounds xestokerol A 130, 7α-hydroxypetrosterol 132, and aragusterol B 143 (Figure 15), had antifouling EC 50 s ranging from 10 to 171 µM. New compound 133 and petrosterol 135 had an EC 50 > 200 µM [98]. Some of these compounds, other known analogues, and seven new analogues have also been extracted from the marine sponge Petrosia (Strongylophora) sp. Compounds 130, 131, 134-141, and 143-147 ( Figure 15) displayed micromolar inhibition across various human cancer cell lines tested, with the ketal 139 showing weaker activity [99]. Representatives from this class of steroids from Xestospongia spp. tested against human cancer cell line K562 yielded IC 50 s for aragusterol J 149 of IC 50

Other Bioactive Steroids
Several sponge sterols, such as solomonsterol A 158 and B 159, theonellasterol 160, conicasterol  161 (Figure 16), and their analogues, can serve as ligands for human nuclear receptors; many of these compounds and their activities have been reviewed [104]. Ganoderic acid A 163 ( Figure 16) and related compounds isolated from the higher fungus Ganoderma sp. possess broad therapeutic properties, including those of anti-tumor and anti-inflammation [105]. Additional recently reported bioactivities of sterols from microorganisms and algae are presented in Table 2 and Figure 16.    Figure 16. Bioactive sterols and steroids. Activities are given in Table 2. Figure 16. Bioactive sterols and steroids. Activities are given in Table 2.