Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013–0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1–A4 (5–8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1–S3 (9–11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1–AS3 (13–15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3–3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1–L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure–activity relationship (SAR) study on the surugamides 3–15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.


Introduction
Helminth infections in commercial livestock are of major socio-economic importance worldwide [1].First discovered from soil-dwelling Streptomyces in the 1980s, the macrolactone ivermectin and related analogs have emerged as the gold standard for the treatment of helminth infections [2].In addition to safeguarding livestock, anthelmintics such as macrolactones are important for the health and welfare of companion animals (e.g., dogs and cats).With respect to companion animals, Dirofilaria immitis (also known as heartworm) is a major parasitic threat that causes chronic damage to the heart, lungs, and arteries that can result in complications leading to fatal outcomes in dogs, or severe respiratory distress and even sudden death in cats [3].D. immitis is a filarial nematode with a complex lifecycle spanning mosquito vectors (microfilariae-L3 larvae) and mammalian hosts (L3 and L4 larvae, and adult).Adult D. immitis reside in the pulmonary arteries and in the right ventricle of the host, releasing microfilariae (mf) into the bloodstream.If ingested by mosquito vectors of the genera Culex, Aedes, or Anopheles, the mf develop from L1 larvae to infective L3 larvae [3].While anthelmintic macrolactones are the mainstay for preventive control of heartworm infections, the incidence and severity of drug-resistant helminth infections have compromised the efficacy of most marketed classes of commercial anthelmintics (whether used to treat commercial livestock or companion animals).Hence, there is an urgent need to identify new chemical classes that anticipate the spread of resistance and facilitate enhanced user compliance in order to discover, develop, and bring new and improved anthelmintics to the market.
As noted in a recent 2023 review [4], fungi and bacteria can be a prolific source of natural products exhibiting anthelmintic properties, several classes of which have been developed into commercial products.Inspired by these successes, we screened extracts of microbes isolated from diverse substrates sampled across Australia, against both the heartworm D. immitis and livestock gastrointestinal parasite Haemonchus contortus.This screen identified numerous extracts that exhibited potent in vitro inhibition of D. immitis mf motility, including Streptomyces sp.CMB-MRB032 recovered from nematode-infected sheep feces from a farm near Bathurst, VIC, Australia.
Although a preliminary bioassay-guided fractionation supported by a Global Natural Products Social (GNPS) analysis [5] attributed the anthelmintic activity in an ISP2 extract of CMB-MRB032 to the well-known antimycin class of cytotoxin [6], it was also noted that several active fractions incorporated surugamides, a relatively rare class of cyclic octapeptide [7,8].With the cytotoxic properties of antimycins making them ill suited for development as anthelmintics [9], our attention was drawn to the possible anthelmintic potential of the surugamides.In an effort to supplement the low level of surugamide production exhibited by CMB-MRB032, we expanded our GNPS molecular network search to include a more expansive and diverse inhouse microbe library, leading to the detection of multiple additional isolates capable of producing surugamides.Of note, Streptomyces sp.CMB-M0112 isolated from a marine sediment collected near Shorncliffe, Qld., Australia, was a particularly promising surugamide producer.Herein, we report on the isolation, structure characterization, and anthelmintic potential of known and new natural surugamides from Streptomyces spp.CMB-MRB032 and CMB-M0112, as well as an array of semi-synthetic and synthetic surugamides.
Mar. Drugs 2024, 22, x 3 nor 4 exhibited activity against either D. immitis or H. contortus (EC50 > 25 µg/m turned our attention to investigating minor surugamide co-metabolites (includin alternate producers).As noted above, a GNPS molecular network analysis of a library of 1712 m extracts identified 15 additional surugamide producers (Figure S5), all of which ha screened and shown to inhibit D. immitis mf motility (Table S1).Comparative UPL analysis permitted the exclusion of replicates, prioritizing the available unique mide producers such as Streptomyces spp.CMB-MRB032, CMB-M0112, S4S-001 S4S-00007B10, CMB-CS051, and ACM-4361 (Table 1), with a comparison of 16S rR quences confirming taxonomic diversity (Table S2 and Figure S6).While a comp analysis of GNPS molecular networks confirmed that all produced surugamides A some surugamides G-H), as well as new analogs (Table 1 and Figure 2), a single traction of the UPLC-QTOF data proved more effective at detecting antimycins were shown to be present in selected extracts (Table 1 and Figure S78).
The absolute configurations of 5-8 were confirmed by Marfey's analyses (Figure S9).A-C Resonances with the same superscript within a column are overlapping, and assignments can be interchanged.
All natural and synthetic surugamides were also tested and found to be inactive (IC 50 > 30 µM) against Gram-positive bacterium Staphylococcus aureus ATCC 25923 and fungus Candida albicans ATCC 10231 (Figure S83).The selective nature of the surugamide SAR against D. immitis supports the proposition that this class of natural products is not mere cell wall disrupters or cytotoxins, but likely elicit a motility inhibitory effect on D. immitis mf by binding with and modifying the function of a specific molecular target(s)/pathway.3.4 >25 >30 nd = not determined.Highlights: beige: EC 50 < 5 µg/mL in all three assays; green: EC 50 < 5 µg/mL against D. immitis.

Other Reported Surugamides and Their Activities
Putting our contribution in context, surugamides A (3) and B-E (16-19) (Figure 7) were first reported in 2013 [7] from a deep-sea sediment-derived Streptomyces sp.JAMM992 collected by a remote unmanned vehicle at -106 m in Kinko Bay, Japan, at which time they were reported to be inhibitors of bovine cathepsin B, a cysteine protease implicated in invasion of metastatic tumor cells-although potency values were modest to negligible (IC 50 16-36 µM).In 2019 [8], the structure of surugamide A (and by inference surugamide C-E) was revised by total synthesis, replacing the D-Ile 2 with a D-allo-Ile 2 residue.Surugamide A (3) was subsequently reisolated in 2014 [13] from Streptomyces sp.RM-27-46 recovered from the soil of a coal mine fire site and was reported to exhibit moderate antibacterial activity against Staphylococcus aureus (MIC 10 µM).A 2016 [14] account attributed the trivial name surugamide F to a structurally unrelated acyclic decapeptide, while a 2017 [15] report on elicitor screens on Streptomyces albus J1074 described the isolation and structure elucidation of surugamides G-J (20-23) and acyl-surugamide A (24), as well as the biosynthetically related albucyclones A-F (25-30) (Figure 7).As a note of caution, this latter study asserted amino acid stereochemical assignments were identical to those (incorrectly) assigned to surugamides A-E in 2013 [7].As such, it seems likely that 20-22 and 24-28 are incorrectly attributed to a D-Ile 2 rather than D-allo-Ile 2 residue (see 2019 [8] reassignment of 3).Most recently, a 2024 [12] report described the new analog acyl-surugamide A2 (6) from a Mediterranean Sea marine tunicate-derived Streptomyces albidoflavus RKJM-0023.Interestingly, a 2013 [16] report on a cyclic octapeptide champacyclin isolated from a marine sediment-derived Streptomyces champavatti described it as having the same amino acid composition as surugamide A, but with a different sequence (cyclo[-D-alloIle 1 -D-Ala 2 -L-Ile 3 -D-Leu 4 -D-Phe 5 -L-Ile 6 -L-Ile 7 -L-Lys 8 -].Comparison of 1 H NMR data (600 MHz, DMSOd 6 ) and MS/MS fragmentation patterns reported for champacyclin [16], with those obtained for surugamide A isolated from CMB-MRB032 (Figures S84-S86), revealed that champacyclin is identical to surugamide A, and hence its assignment should be revised accordingly.Champacyclin (surugamide A) was reported to inhibit the fire blight pathogen Erwinia amylovora at 25 µM (40% inhibition), although this low level of potency might be better characterized as inactive [16].

General Experimental Procedures
Chiroptical measurements ([α] D ) were obtained on a JASCO P-1010 polarimeter in a 100 × 2 mm cell at specified temperatures.Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Avance 600 MHz spectrometer with a 5 mm PASEL 1 H/D-13 C Z-Gradient probe.In all cases, spectra were acquired at 25 • C in DMSO-d 6 or CDCl 3 with referencing to residual 1 H or 13 C signals (DMSO-d 6 , δ H 2.50 and δ C 39.5; CDCl 3 , δ H 7.16 and δ C 77.14).High-resolution ESIMS spectra were obtained on a Bruker micrOTOF mass spectrometer by direct injection in MeOH at 3 µL/min using sodium formate clusters as an internal calibrant.Liquid chromatography-diode array-mass spectrometry (HPLC-DAD-MS) data were acquired either on an Agilent 1260 series separation module equipped with an Agilent G6125B series LC/MSD mass detector and diode array detector or on Shimadzu LCMS-2020.Semi-preparative HPLCs were performed using Agilent 1100 series HPLC instruments with corresponding detectors, fraction collectors, and software inclusively.UPLC chromatograms were obtained on Agilent 1290 infinity UPLC system equipped with a diode array multiple wavelength detector (Zorbax C 8 RRHD 1.8 µm, 50 × 2.1 mm column, eluting at 0.417 mL/min, 2.50 min gradient elution from 90% H 2 O/MeCN to 100% MeCN with a constant 0.01% TFA modifier).UPLC-QTOF analysis was performed on UPLC-QTOF instrument comprising an Agilent 1290 Infinity II UPLC (Zorbax C 8 RRHD 1.8 µm, 50 × 2.1 mm column, eluting with 0.417 mL/min, 2.50 min gradient elution from 90% H 2 O/MeCN to 100% MeCN with a constant 0.1% formic acid modifier) coupled to an Agilent 6545 QTOF.MS/MS analysis was performed on the same instrument for ions detected in the full scan at an intensity above 1000 counts at 10 scans/s, with an isolation width of 4 ~m/z using a fixed collision energy and a maximum of 3 selected precursors per cycle.Chemicals were purchased from Sigma-Aldrich or Merck unless otherwise specified.Analytical-grade solvents were used for solvent extractions.Chromatography solvents were of HPLC grade supplied by Labscan or Sigma-Aldrich and filtered/degassed through 0.45 µm polytetrafluoroethylene (PTFE) membrane prior to use.Deuterated solvents were purchased from Cambridge Isotopes.Microorganisms were manipulated under sterile conditions using a Laftech class II biological safety cabinet and incubated in either MMM Friocell incubators (Lomb Scientific) or an Innova 42R incubator shaker (John Morris).

Cultivation and Fractionation of CMB-MRB032
Medium-scale cultivation of Streptomyces sp.CMB-MRB032 was carried out on ISP2 agar plates (×80), incubated at 30 • C for 14 d.Following incubation, the agar was harvested, diced (~1.5 cm 2 ) and extracted with EtOAc (3 × 600 mL), and the combined organic phase concentrated in vacuo at 40 • C to yield an extract (590.8 mg), which was further partitioned between hexane (2 × 50 mL), and 90% aqueous MeOH (50 mL) and concentrated in vacuo at 40 • C to yield hexane (62.7 mg), and MeOH (173.1 mg) solubles.The MeOH solubles were fractionated by preparative HPLC chromatography (Phenomenex C 8, 10 µm, 21.2 × 250 mm column, gradient elution at 20 mL/min over 30 min from 90% H 2 O/MeCN to 100% MeCN with constant 0.01% TFA/MeCN modifier) to yield 14 fractions, which were tested for anthelmintic activities.UPLC-QTOF and GNPS analysis of the active fractions 9-13 suggested the presence of surugamides and antimycins.The presence of antimycins in these fractions was confirmed by co-injections with authentic standards of antimycins A4a (1) and A2a (2).A subsequent large-scale cultivation was carried out to isolate and identify the surugamides present in the active fractions.
(iii) Cleavage of linear protected peptide from resin: After swelling for 20 min in dry DCM (2 mL), the resin was mixed with 20% HFIP/DCM (2 mL × 3 × 20 min), and the combined filtrate concentrated in vacuo to give the crude fully protected linear peptide.
(iv) Cyclization of protected peptide: Protected linear peptide 0.5 mg/mL in DMF was stirred vigorously and a mixture of 0.4 M HATU (3 eq.), HOBT (3 eq.) and collidine (3 eq.) in DMF (2 mL) added very slowly over 30 min.After 14 h, and/or after LC-MS analysis confirmed completion, the reaction mixture was concentrated in vacuo, and the residue was subjected to preparative HPLC (Phenomenex C 8 , 10 µm, 9.4 × 250 mm column, gradient elution at 20 mL/min over 20 min from 90% H 2 O/MeCN to 100% MeCN with a constant 0.01% TFA/MeCN modifier).After lyophilization, the protected cyclic peptide was obtained as an amorphous powder.
(vi) Deprotection of tert-butyloxycarbonyl (Boc) from Lys: The peptide was stirred for 3 h in a cleavage solution (1 mL) consisting of dry TFA and DCM (50:50) after which the reaction mixture was concentrated under a stream of nitrogen, and the residue taken up in MeCN/water (5 mL) and freeze-dried to give pure cyclic peptide.

Antibacterial Assay
LB agar plates inoculated with Staphylococcus aureus ATCC 25923 were incubated at 37 • C for 24 h, after which several colonies were transferred to fresh sterile LB broth, which was incubated at 37 • C for 24 h, and following measurement of optical density, the cell density adjusted to 5 × 10 5 CFU/mL.Analytes (3-8, 13-15, and controls) were dissolved in DMSO and diluted with H 2 O to afford stock solutions (600 µM, 20% DMSO), which were serially diluted with 20% DMSO to yield analyte concentrations ranging from 600 to 0.2 µM.An aliquot (10 µL) of each analyte dilution was transferred to a 96-well microtiter plate along with freshly prepared bacterium broth (190 µL) to final concentrations of 30-0.01 µM in 1% DMSO.The resulting assay plates were incubated at 37 • C for 18 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).The positive control was rifampicin and ampicillin (10 µM in 1% DMSO), and negative control was 1% DMSO in culture broth, together with extracts prepared from LB broth medium without bacterial inoculation.Each analysis was repeated two times and the data represented graphically, and IC 50 and MIC values were calculated using GraphPad Prism version 10.0.1 (Figure S83).

Antifungal Assay
SD agar plates inoculated with Candida albicans ATCC 10231 were incubated at 27 • C for 48 h, after which several colonies were transferred to fresh sterile SD broth (4 mL), which was incubated at 27 • C for 48 and, following measurement of optical density, the cell density adjusted to 5 × 10 5 CFU/mL.An aliquot (10 µL) of analytes (3-8, 13-15) prepared as above for antibacterial assays was transferred to a 96-well microtiter plate, and freshly prepared fungal broth (190 µL) was added to each well to give final concentrations of 30-0.01 µM in 1% DMSO.The resulting assay plates were incubated at 27 • C for 48 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).The positive control was amphotericin (10 µM in 1% DMSO) and negative control was 1% DMSO, together with extracts prepared from SD broth without fungal inoculation.Each analysis was repeated two times and the data represented graphically, and IC 50 and MIC values calculated using GraphPad Prism version 10.0.1 (Figure S83).

Cytotoxic Assay
Aliquots (3000 cells/well in 190 µL of Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum) of human colorectal (SW620) carcinoma cells were transferred to 96-well microtiter plates and incubated at 37 • C in 5% CO 2 for 3 days.An aliquot (10 µL) of analytes (3-8, 13-15), as prepared above for antibacterial assays, was transferred to a 96-well microtiter plate and incubated again for 24 h, after which an aliquot (10 µL) of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate-buffered saline (5 mg/mL) was added to each well, which were again incubated for 4 h.The media were then carefully removed (pipette) and the residue dissolved in DMSO (100 µL) by shaking at 50 rpm for 2 min.Finally, the absorbance of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).The positive control was sodium dodecyl sulfate (SDS) and negative control was 1% DMSO.Each analysis was repeated two times and the data represented graphically, and IC 50 and MIC values were calculated using GraphPad Prism version 10.0.1 (Figure S83).

Anthelmintic Assays
Inhibition of motility of D. immitis microfilariae.Approximately two hundred and fifty Dirofilaria immitis microfilariae suspended in a total volume of 100 µL RPMI 1640 media (Hyclone) was added to wells of a microtiter plate containing various concentrations of test compounds formulated in 100% DMSO.Plates were incubated for ~72 h at 37 • C and 5% CO 2 .Parasite motility quantitative descriptors were calculated for each well after imaging in a camera-based system.The properties of each test compound at a given dose were expressed as percentage motility inhibition after normalization with the average motility of positive (1.0 µM Gramicidin) and negative (DMSO) controls on each plate.Dose-response assays were conducted to determine EC 50 values.
H. contortus L1-L3 larvae development assay (LDA).Approximately 20 L1 stage Haemonchus contortus were delivered to wells of a microtiter plate containing nutrient medium and various concentrations of test compounds dissolved in 100% DMSO.Plates were incubated for four days at 27 • C and 85% relative humidity.The resulting worms (L3s) were imaged in a camera-based system and quantitative motility descriptors were calculated.The properties of each test compound at a given dose were expressed as percentage inhibition after normalization of the motility descriptor values with the average motility of positive (1.0 µM Ivermectin) and negative controls (DMSO only), respectively.Dose-response assays were conducted to determine an EC 50 value.

Conclusions
This study demonstrates the value of screening microbial extracts to detect natural products exhibiting anthelmintic properties, and the use of GNPS molecular networking as a means to rapidly dereplicate new from known, and rare from common, and detect new producers of prioritized microbial natural products.The study also discloses for the first time that members of the surugamide family of Streptomyces cyclic octapeptides are non-cytotoxic, selective inhibitors of the motility of D. immitis microfilaria.
Furthermore, the detection of six taxonomically and chemically unique surugamideproducing Australian Streptomyces spp, from both marine and terrestrial sources, demonstrated that despite limited reports on the structural diversity of this compound class in the scientific literature, the biosynthetic genes needed to produce surugamides are relatively widespread.Of particular note was the co-occurrence with the surugamides of the highly cytotoxic and anthelmintic antimycins.This latter observation warrants comment as it highlights a limitation of GNPS molecular networking as a "first-line" tool to prioritize/deprioritize extracts-where the detection of a known nuisance class (e.g., antimycins) in an anthelmintic extract may be seen as sufficient to deprioritize and abandon further investigation.While such an approach is not without merit, on this occasion, it would have excluded investigation of the anthelmintic properties of the non-cytotoxic surugamides.As noted in this study, such exclusions can be avoided by reacquiring bioassay and GNPS analyses on fractionated extracts and/or extracts obtained from cultivation profiling on multiple media (MATRIX).
Finally, a D. immitis mf SAR assessment of the natural, synthetic, and semi-synthetic surugamides assembled during this study highlights the importance of the core octapeptide sequence and selective acylation of the Lys 3 -ε-NH 2 residue.That the acyl-surugamides A3 (7) and AS3 (15) were the sole surugamide anthelmintic actives, but were neither cytotoxic, antibacterial, or antifungal, suggests a molecular target-oriented mode-of-action (i.e., discrete binding interaction).Given the rise in anthelmintic resistance, future investigations into the mode of action of the acyl-surugamides would appear warranted, as such knowledge may reveal a promising new target that could inform the development of future anthelmintics.

Figure 2 .
Figure 2. The surugamide molecular family from a GNPS molecular network of surugamide producers cultivated on ISP2 agar, highlighting surugamides A-E, and G-H, and new analogs.

Figure 2 .
Figure 2. The surugamide molecular family from a GNPS molecular network of surugamide producers cultivated on ISP2 agar, highlighting surugamides A-E, and G-H, and new analogs.

Figure 2 .
Figure 2. The surugamide molecular family from a GNPS molecular network of surugamide producers cultivated on ISP2 agar, highlighting surugamides A-E, and G-H, and new analogs.

Author Contributions:
Conceptualization, R.J.C.; experimental-microbial cultivation and isolation, K.S. and T.W.; experimental-peptide synthesis, W.M.H., Y.Z. and T.W.; experimental-anthelmintic assays, Y.M. and D.F.B.; experimental-antibacterial and antifungal assays, T.W.; experimental-cytotoxicity assays, S.J.; NMR data analysis, T.W. and A.A.S.; genomic data analysis, Z.G.K.; writing-original draft preparation, T.W. and A.A.S.; writing-review and editing, A.A.S. and R.J.C.; funding acquisition, Z.G.K. and R.J.C.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by the Australian Research Council (LP190100083 and LE210100148), Boehringer Ingelheim Animal Health and the UQ Institute for Molecular Bioscience.T.W., K.S. and S.J. were supported by UQ International Postgraduate Scholarships, and Z.G.K. through an Australian Research Council Future Fellowship (FT230100468).

Table 1 .
Microbial surugamide producers, and chemical and biological analysis of ISP2 ext

Table 1 .
Microbial surugamide producers, and chemical and biological analysis of ISP2 extracts.
A-C Resonances with the same superscript within a column are overlapping, and assignments can be interchanged.

Table 5 .
Effect of 1-15 on inhibition of motility of D. immitis mf and H. contortus L3 larvae.