Identification of Antimycobacterial Natural Products from a Library of Marine Invertebrate Extracts

Tuberculosis (TB) remains a public health crisis, requiring the urgent identification of new anti-mycobacterial drugs. We screened several organic and aqueous marine invertebrate extracts for their in vitro inhibitory activity against the causative organism, Mycobacterium tuberculosis. Here, we report the results obtained for 54 marine invertebrate extracts. The chemical components of two of the extracts were dereplicated, using 1H NMR and HR-LCMS with GNPS molecular networking, and these extracts were further subjected to an activity-guided isolation process to purify the bioactive components. Hyrtios reticulatus yielded heteronemin 1 and Jaspis splendens was found to produce the bengamide class of compounds, of which bengamides P 2 and Q 3 were isolated, while a new derivative, bengamide S 5, was putatively identified and its structure predicted, based on the similarity of its MS/MS fragmentation pattern to those of other bengamides. The isolated bioactive metabolites and semi-pure fractions exhibited M. tuberculosis growth inhibitory activity, in the range <0.24 to 62.50 µg/mL. This study establishes the bengamides as potent antitubercular compounds, with the first report of whole-cell antitubercular activity of bengamides P 2 and Q 3.


Introduction
Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death, globally. Despite the availability of an effective anti-TB chemotherapy and a neonatal vaccine, an estimated 10 million people contracted TB and 1.4 million people died of the disease, globally, in 2019 [1]. The current standard combination therapy carries a high pill burden (up to four separate anti-TB drugs are used), long duration (minimum six months) of treatment and side effects or toxicities, owing to concomitant use of mixtures of TB drugs and non-TB drugs (for example, anti-HIV drugs) [1,2]. Furthermore, delayed diagnosis, inappropriate treatment and non-adherence has led to the emergence of multidrugresistant (MDR) and extensively drug-resistant (XDR) Mtb strains [1,3]. Treatment of these resistant strains requires the use of non-frontline drugs, often over prolonged periods and with associated risks, including severe side effects and high therapeutic cost [3][4][5]. There is, therefore, an urgent need to discover and develop new potent anti-TB drugs with reduced side effects and effectiveness over shorter periods.
Nature remains our primary source of medication, with the marine environment relatively less explored [6]. The ocean occupies over 70% of the Earth's surface and is uniquely rich in biodiversity. For their survival and existence, marine organisms, especially invertebrates, biosynthesize and secrete secondary metabolites to ward off competitors, predators and parasites [7]. These secondary metabolites have unique structural diversity, with a high degree of stereochemical complexity, and have shown a range of bioactivities, including anticancer, antioxidant, antifungal, antiviral, antibacterial, anti-TB, anti-inflammatory, antimalarial, analgesic and antinematodal properties [8,9]. Representative examples of marine-derived drugs include ziconotide, a peptide analgesic, derived from the marine cone snail Conus magus, and ecteinascidin-743, an alkaloid used in cancer treatment, derived from the sea squirt, Ecteinascidia turbinate [10,11]. Marine-natural products have promise in providing drug leads for the TB drug discovery pipeline and many anti-mycobacterial compounds, including the manzamines, halicyclamines, cyclostellettamines, batzelladines and callyaerins have been isolated from marine organisms [12].
Natural product drug discovery starts with the screening of natural product extracts as the preliminary step to finding new lead compounds. As part of our continuous search for new anti-TB agents from marine invertebrates, we screened several marine-natural product extracts, obtained from the United States of America's National Cancer Institute (NCI) [13]. The whole-cell bioassay approach was employed, in preference to target-based and genetics-driven approaches, given the unknown bioactive compositions of the extracts and the well-described difficulties in ensuring whole-cell activity against Mtb [14]. Herein, we report the initial screening of 984 marine invertebrate extracts and the antimycobacterial activity results of 54 active extracts, with the detection and isolation of the active components of two of the extracts. The extracts of the sponges, Hyrtios reticulatus and Jaspis splendens, were among the most active (Table 1) and were subjected to activity-guided fractionation to isolate the active ingredients. Anti-mycobacterial activity in the extract from Hyrtios reticulatus was found to be associated with heteronemin 1, while, as expected [15], the extract from Jaspis splendens produced the bengamide class of compounds (see Figure 1), with antitubercular activity shown, for the first time, to be associated with bengamides P and Q (Figure 1, compounds 2 and 3), as well as other inseparable mixtures of bengamides. While other marine-derived and synthetically modified bengamides have previously been shown to be potent antitubercular agents and investigated as drug leads [16,17], to the best of our knowledge, this is the first report of antitubercular activity for bengamides P 2 and Q 3.

Screening of Marine Invertebrate Samples
An initial set of 984 organic and aqueous marine invertebrate extracts were screened for their in vitro inhibitory activity against a fully virulent M. tuberculosis H37Rv fluorescent reporter mutant [16,18,19] and the minimum inhibitory concentrations required to inhibit 90% (MIC 90 ) and 99% (MIC 99 ) of the mycobacterial population were recorded. Of the 984 samples, 54 putative actives were identified and retested to confirm activity. A SciFinder ® (https://scifinder.cas.org/, accessed on 10 February 2021) search of the species of marine invertebrates, from which the 54 extracts were obtained, revealed that this was the first report of antimycobacterial activity in 44% of the species.

Dereplication, Molecular Networking, Isolation and Structure Elucidation
The extracts from the two Mauritian marine sponges Hyrtios reticulatus (SS10, MIC 90 = 2.40 µg/mL) and Jaspis splendens (SS2, MIC 90 = 51.00 µg/mL) were selected for preliminary investigations, in an attempt to isolate and identify the active compounds. Both extracts were available in appreciable amounts and were examples of extracts with good and moderate MIC 50 values, respectively. The actives were isolated using an activity-guided isolation procedure. First, the 1 H NMR spectra and HR-LCMS profiles were obtained for the bioactive extracts, and these were then subjected to bioactivity-guided fractionation, using a normal-phase solid-phase extraction (SPE) procedure, which, in each case, yielded five fractions (A-E), corresponding to polarities of the eluents. The fractions were then screened against the Mtb H37Rv reporter strain.
The HR-LCMS profile of the extract of Hyrtios reticulatus (SS10) showed a major peak, with m/z 511.3026 [M + Na] + , which was identified as the antimycobacterial sesquiterpene, heteronemin 1; this was confirmed by the 1 H NMR spectrum. Fractions A and B of SS10 were the most active but had the same 1 H NMR spectrum and were, therefore, combined and further purified, using normal-phase column chromatography to yield the known compound, heteronemin 1, as the most active ingredient. Heteronemin 1 was isolated as a white amorphous solid, with a molecular formula of C 29 H 44 O 6 , deduced from HRESIMS (observed [M + Na] + = 511.3026; calculated [M + Na] + = 511.3036; ∆ = −1.96 ppm), corresponding to eight degrees of unsaturation ( Figure S1). The structure of compound 1 was fully elucidated by the analysis of HRESIMS, together with 1D and 2D NMR data, and this was congruent with that reported in the literature (Figures S1-S6 and Table S1) [24,25].
The HR-LCMS/MS data of the extract of Jaspis splendens (SS2) was analyzed on the GNPS molecular networking platform [26]. Dereplication of the nodes showed molecular network families, which are representative of the bengamide class of compounds; this was confirmed by the analysis of the 1 H NMR spectrum of SS2 ( Figure 2, Table S4). The bengamides are a class of compounds with a core scaffold, comprising a 2(R)-methoxy-3(R),4(S),5(R)-trihydroxy-8-methylnon-6(E)-enoyl moiety, linked to an aminocaprolactam with its cyclic amide, nitrogen free or methylated [15,16,27]. Approximately 23 bengamides have been isolated, with various structural variations, enabling grouping into structural classes [16,27]. The first type contains a hydroxylysine-derived caprolactam, with the OH at C-13 either unsubstituted (bengamides Y, Z) or acylated by a lipid unit (bengamides A, B, G-J, L-O), or by polyketide esters (bengamides C, D). The second type has the lysinederived caprolactam (which therefore does not have an OH at C-13), but with the 5-OH acylated by the lipid unit (bengamides P-R), or not (bengamides E, E , F, F ) [16,27]. Some bengamides have the same molecular formulae and weight and are only differentiated by characteristic NMR signals. For example, bengamides J and M have the same molecular formula of C 33 H 60 N 2 O 8 and weight 635.4228 [M + Na] + (Figure 2, Table S3) [27].
3(R),4(S),5(R)-trihydroxy-8-methylnon-6(E)-enoyl moiety, linked to an aminocaprolactam with its cyclic amide, nitrogen free or methylated [15,16,27]. Approximately 23 bengamides have been isolated, with various structural variations, enabling grouping into structural classes [16,27]. The first type contains a hydroxylysine-derived caprolactam, with the OH at C-13 either unsubstituted (bengamides Y, Z) or acylated by a lipid unit (bengamides A, B, G-J, L-O), or by polyketide esters (bengamides C, D). The second type has the lysinederived caprolactam (which therefore does not have an OH at C-13), but with the 5-OH acylated by the lipid unit (bengamides P-R), or not (bengamides E, E′, F, F′) [16,27]. Some bengamides have the same molecular formulae and weight and are only differentiated by characteristic NMR signals. For example, bengamides J and M have the same molecular formula of C33H60N2O8 and weight 635.4228 [M + Na] + (Figure 2, Table S3) [27].  Figures  1 and 2). Family F2, which is constituted by four nodes, was dereplicated to be the second type of bengamides with a fatty acid chain, including bengamides P 2 (m/z 591.397 [M + Na] + ), Q 3 (m/z 605.413 [M + Na] + ) and R 4 (m/z 619.428 [M + Na] + ), which was linked to its new analogue 5 (node with m/z 633.445 [M + Na] + ), with a mass difference of 14 Da, signifying methylation. From the biosynthesis of bengamides and a keen examination of isolated bengamide structures, the methylation would either be on the caprolactam ring nitrogen or an elongation of the lipid chain of 4. Evaluation of the fragmentation patterns ( Figure S23) of compounds 4 and 5, showed that 5 is an N-methylated analogue of 4. Compound 5 was therefore assigned the name bengamide S. Fractions B and C of SS2 exhibited the best activities, with potent MIC90 values of <0.24 μg/mL and 0.25 μg/mL, respectively. The HR-LCMS profiles and 1 H NMR spectra of active fractions B and C, revealed that they  (Figures 1 and 2). Family F2, which is constituted by four nodes, was dereplicated to be the second type of bengamides with a fatty acid chain, including bengamides P 2 (m/z 591.397 [M + Na] + ), Q 3 (m/z 605.413 [M + Na] + ) and R 4 (m/z 619.428 [M + Na] + ), which was linked to its new analogue 5 (node with m/z 633.445 [M + Na] + ), with a mass difference of 14 Da, signifying methylation. From the biosynthesis of bengamides and a keen examination of isolated bengamide structures, the methylation would either be on the caprolactam ring nitrogen or an elongation of the lipid chain of 4. Evaluation of the fragmentation patterns ( Figure S23) of compounds 4 and 5, showed that 5 is an N-methylated analogue of 4. Compound 5 was therefore assigned the name bengamide S. Fractions B and C of SS2 exhibited the best activities, with potent MIC 90 values of <0.24 µg/mL and 0.25 µg/mL, respectively. The HR-LCMS profiles and 1 H NMR spectra of active fractions B and C, revealed that they comprised similar mixtures of the bengamides, and these fractions were, therefore, combined and subjected to normal-phase column chromatography. From >150 fractions collected, fractions F94 and F100 were sufficiently homogeneous for their structures to be elucidated as compounds 2 and 3, respectively. Compounds 2 and 3 had 1 H NMR signals characteristic of the second type of bengamides, with a lipid chain located as esters of the C-5 oxygen. The fact that this was attached to C-5 and not C-13 was evident from the downfield shift of the signal at δ H 5.47, for the methine proton H-5 in the 1 H NMR spectrum, compared to the shielded signals for the diastereotopic C-13 methylene protons at δ H 1.88 and 1.43. The structures of compounds 2 and 3 were fully elucidated by analyses of their HR-ESIMS, 1D and 2D NMR data and comparison with the literature, and confirmed to be bengamides P and Q, respectively (Figures S7-S19 and Tables S2 and S3) [27].
The 1 H NMR spectra of active fractions F107, F114 and F130 (Figures S20-S22) showed characteristic signals for bengamides of both classes with a lipid chain. However, the main component of fraction F130 was the first type of bengamide, with a branched lipid chain and characteristic upfield signals in the 1 H NMR spectrum at δ H 0.7-1.7, and a signal for an N-methyl group in the caprolactam ring (δ H 3.13). It is proposed that this major component may be bengamide M, as a corresponding peak of m/z 635.423 [M + Na] + was identified in the HR-LCMS profile of fraction F130.

Antimycobacterial Activity of Isolated Compounds and Semi-Pure Fractions
Heteronemin 1 exhibited an MIC 90 of 8.23 µg/mL against Mtb. Shown in Table 2 are the antimycobacterial activity results of isolated pure compounds and the most active semipure fractions obtained from purification of fractions B and C of SS2. Fraction F114 showed the greatest activity, with MIC 90 < 0.24 µg/mL. Although bengamides have been isolated from the genus Jaspis, this is the first report of the detection and isolation of bengamides from Jaspis splendens, which is known to be a prolific producer of jasplakinolides [16]. The bengamides are potent antitumor agents, which target methionine aminopeptidase (MetAP) [15,16]. Synthetic MetAP inhibitors, based on the bengamide structural scaffold, have been described with activity against the Mtb MetAPs (the bacillus encodes two isoforms, MetAP1a and MetAP1c); however, the antimycobacterial potential (whole-cell or target-based) of natural bengamides is less explored [28,29]. There is a report of antimycobacterial activity of bengamide B 9, which exhibited strong in vitro activity against whole-cell Mtb and the purified Mtb MetAP1C protein and was also non-toxic against human cell lines [17]. To our knowledge, ours is the first report of antimycobacterial activity for the second type of bengamides with lipid chains, namely bengamides P 2 and Q 3. This suggests that the bengamides are potential antitubercular leads and that all known natural analogs could be screened against Mtb, to further aid in SAR and QSAR design and studies. There also appears to be value in further exploration of the Mtb MetAPs as potential drug targets.

Antimycobacterial Activity
The crude extracts, semi-pure and isolated compounds were dissolved in DMSO and their minimum inhibition concentrations (MIC 90 / 99 values) determined against the green fluorescent protein (GFP)-tagged Mtb H37Rv pMSp12:GFP bioreporter using the standard broth microdilution method developed by Collins et al. [18]. Mtb H37Rv was cultured in Middlebrook 7H9 broth medium supplemented with albumin-dextrose complex, Dglucose, and Tween-80 (7H9/ADC/Glu/Tw). The minimum concentration which inhibited the growth of 90% (MIC 90 ) or 99% (MC99) of bacilli for the tested samples was determined at 1 week and 2 weeks post-inoculation using microplate detection of GFP fluorescence intensity and expressed in µg/mL. The fluorescence was measured with excitation at 485 nm and emission at 520 nm. Growth media and 5% dimethyl sulfoxide (DMSO) were used as a negative control, with rifampicin used as a positive control.

Fractionation, Isolation and Purification of Active Compounds
The extracts SS2 and SS10 were subjected to an NCI DIOL SPE fractionation process. The organic extract (150 mg) was weighed and solubilized in 1.8 mL of 1:1 MeOH/CH 2 Cl 2 and sonicated to mix. The mixture was loaded on a 2 g DIOL SPE column and eluted stepwise, with solvent mixtures of increasing polarity (a column volume/solvent mixture of 6 mL): 9:1 hexane/CH 2 Cl 2 (A), 20:1 CH 2 Cl 2 /EtOAc (B), 100% EtOAc (C), 5:1 EtOAc:MeOH (D), 100% MeOH (E). The fractionation process was repeated with 3 × 150 mg of extract and each fraction, from A to E, was combined and dried.
Fractions A and B of SS10 were combined and further purified through a normal-phase column chromatography with an increasing polarity of a mixture of hexane/EtOAc from 1:0 hexane/EtOAc to 0:1 hexane/EtOAC to yield compound 1 (17.01 mg).
Fractions B and C of SS2 were also combined and subjected to a normal-phase column chromatography with an increasing polarity of a mixture of hexane/EtOAc from 1:0 hexane/EtOAc to 0:1 hexane/EtOAc to yield compounds 2 (3.08 mg) and 3 (0.69 mg) and a mixture of bengamides.

Dereplication and Molecular Networking
Raw LC-MS/MS data of sample SS2 was converted to mzXML format using the ProteoWizard tool MSconvert (version 3.0.10051, Vanderbilt University, United States) [30]. The mzXML data was uploaded to the Global Natural Products Social (GNPS) Molecular Networking (MN) webserver3 (http://gnps.ucsd.edu, accessed on 12 August 2020) and analyzed using the MN workflow [23]. The data was filtered by removing all MS/MS fragment ions within +/− 17 Da of the precursor m/z. MS/MS spectra were window filtered by choosing only the top 6 fragment ions in the +/− 50Da window throughout the spectrum. The precursor ion mass tolerance was set to 0.02 Da and a MS/MS fragment ion tolerance of 0.02 Da. A network was then created where edges were filtered to have a cosine score above 0.7 and more than 3 matched peaks. Further, edges between two nodes were kept in the network, only if each of the nodes appeared in each other's respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set to 100, and the lowest scoring edges were removed from molecular families until the molecular family size was below this threshold. The spectra in the network were then searched against GNPS' spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 3 matched peaks. The output of the molecular network was visualized using Cytoscape version 3.7.2 [31] and displayed using the settings "preferred layout" with "directed" style. The nodes (compounds) originating from the solvent control (MeOH) were excluded from the original network to enable visualization of only the compounds in SS2.

Conclusions
Marine-natural products are a reliable source of potent antitubercular leads, as this screening project identified 54 actives in whole-cell growth inhibition assays (MIC 90 range: < 0.244-125 µg/mL). Notably, 44% of the species from which these extracts were obtained are reported here, for the first time, as possessing antimycobacterial activity. A combination of 1 H NMR and HR-LCMS dereplication, along with GNPS molecular networking and a bioactivity-guided isolation techniques was employed to detect and isolate heteronemin and the bengamides P and Q, as the respective active ingredients of the extracts of the Mauritian sponges Hyrtios reticulatus and Jaspis splendens. A new bengamide derivative was detected in the molecular network of SS2. In this study, the bengamides have been identified as potent antimycobacterial compounds and should be explored further. Moreover, the isolation of promising active compounds from crude extracts, with high and moderate activity, provides compelling evidence for the continuous need to explore the rich resource of the NCI's repository of marine invertebrate extracts further, for novel anti-TB leads.