Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 3.0 https://creativecommons.org/licenses/by/3.0/ Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins © The Royal Society of Chemistry 2022 Published version Heinilä L. M., P.; Jokela, J.; Ahmed M., N.; Wahlsten, M.; Kumar, S.; Hrouzek, P.; Permi, P.; Koistinen, H.; Fewer D., P.; Sivonen, K. Heinilä L. M., P., Jokela, J., Ahmed M., N., Wahlsten, M., Kumar, S., Hrouzek, P., Permi, P., Koistinen, H., Fewer D., P., & Sivonen, K. (2022). Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins. Organic and Biomolecular Chemistry, 20(13), 2681-2692. https://doi.org/10.1039/d1ob02454j 2022 Organic & Biomolecular Chemistry PAPER Cite this: DOI: 10.1039/d1ob02454j Received 21st December 2021, Accepted 3rd March 2022 DOI: 10.1039/d1ob02454j rsc.li/obc Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins† L. M. P. Heinilä, a J. Jokela, a M. N. Ahmed, a,b M. Wahlsten, a S. Kumar, c P. Hrouzek, c P. Permi, d,e H. Koistinen, b D. P. Fewer * a and K. Sivonen * a Low-molecular weight natural products display vast structural diversity and have played a key role in the development of novel therapeutics. Here we report the discovery of novel members of the aeruginosin family of natural products, which we named varlaxins. The chemical structures of varlaxins 1046A and 1022A were determined using a combination of mass spectrometry, analysis of oneand two-dimensional NMR spectra, and HPLC analysis of Marfey’s derivatives. These analyses revealed that varlaxins 1046A and 1022A are composed of the following moieties: 2-O-methylglyceric acid 3-O-sulfate, isoleucine, 2-carboxy-6-hydroxyoctahydroindole (Choi), and a terminal arginine derivative. Varlaxins 1046A and 1022A differ in the cyclization of this arginine moiety. Interestingly, an unusual α-D-glucopyranose moiety derivatized with two 4-hydroxyphenylacetic acid residues was bound to Choi, a structure not previously reported for other members of the aeruginosin family. We sequenced the complete genome of Nostoc sp. UHCC 0870 and identified the putative 36 kb varlaxin biosynthetic gene cluster. Bioinformatics analysis confirmed that varlaxins belong to the aeruginosin family of natural products. Varlaxins 1046A and 1022A strongly inhibited the three human trypsin isoenzymes with IC 50 of 0.62–3.6 nM and 97–230 nM, respectively, including a prometastatic trypsin-3, which is a therapeutically relevant target in several types of cancer. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and provide evidence that the aeruginosin family is a source of strong inhibitors of human serine proteases. Introduction Advanced solid cancers are often difficult to treat because of the ability of tumour cells to escape from the primary tumour and form distant metastases. 1 Proteases play a critical role in tumour progression, especially in the process of metastatic dissemination of cancer cells and the promotion of tumour growth. 2,3 Trypsin is one of the best-characterized proteolytic enzymes. 4,5 The PRSS1,PRSS2 and PRSS3 genes encode trypsinogen-1, -2 and -3 trypsin precursors, respectively, which are especially abundant in pancreatic secretions. 5 These precursors can be proteolytically activated (e.g., by enteropeptidase) to trypsin-1, -2, and -3 (also known as cationic and anionic trypsin, and mesotrypsin, respectively). Trypsin-3 is an enigmatic minor component of pancreatic secretions with a proposed role of modulating overall trypsin activity, as it efficiently degrades proteinaceous trypsin inhibitors. 6,7 Elevated levels of trypsin-3 are associated with several human diseases. Trypsin-3 has also been identified as a potential therapeutic target for the reduction of tumour growth and metastasis in several cancers, such as prostate, breast and pancreatic cancer. 8–12 However, the high sequence identity between trypsins extends to the active sites of the enzymes and makes selective targeting of trypsin-3 challenging. 7,13–16 Nevertheless, human trypsin isoenzymes have slight differences in substrate specificity, which suggests that the development of selective trypsin inhibitors is possible. 17 Aeruginosins are a group of tetrapeptides produced by cyanobacteria. 18 They often possess potent inhibitory activity on trypsin-type serine proteases and, thus, represent potential lead molecules for pharmaceutical applications. 18 This class of non-ribosomal peptides was originally discovered in Microcystis aeruginosa. 18,19 Aeruginosins have also been identified in other cyanobacterial genera, such as Nodularia, †Electronic supplementary information (ESI) available. See DOI: 10.1039/ d1ob02454j a Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland. E-mail: david.fe[email protected], [email protected] b Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, Helsinki, Finland c Laboratory of Algal Biotechnology-Centre Algatech, Institute of Microbiology of the Czech Academy of Sciences, Třeboň, Czech Republic d Department of Chemistry, University of Jyväskylä, Jyväskylä, Finland e Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland This journal is © The Royal Society of Chemistry 2022 Org. Biomol. Chem. Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal Planktothrix, and Nostoc. Aeruginosin subgroups include pseudoaeruginosins, 20 nostosins, 21 suomilide, 22,23 and banyasides. 24 They have a complex chemical structure and typically contain the non-proteogenic 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety and the C-terminal arginine derivatives argininal, argininol, agmatine, 1-amidino-2-ethoxy-3-aminopiperidine, and more rarely a 1-amino-2-(N-amidino-Δ 3 -pyrrolinyl)-ethyl (Aaep) moiety (Fig. S1–S3†). Aeruginosins are sometimes decorated with sugars, which themselves may be further derivatized with fatty acids. 22,24,25 Aeruginosins are the products of a highly variable non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. 23,26–31 The diversity of the aeruginosin family is quite broad and over 130 variants have been described to date (34, Table S1†). However, the chemical diversity of the aeruginosin family of natural products is underestimated given the known genetic diversity of this family. 28,33,34 Members of the aeruginosin natural product family are often strong serine protease inhibitors. 18 It is noteworthy that all the previously reported aeruginosin groups contain wellknown trypsin inhibitors. 18,20–24 The most potent microbial trypsin inhibitor reported from this family of natural products to date is chlorodysinosin A with an IC 50 of 37 nM (Fig. S3 and Table S2†). Most reported biochemical assays employ nonhuman trypsin, such as porcine and bovine trypsin. However, the primary structures of porcine and bovine trypsins differ significantly from those of human trypsins. 35 Thus far, there has been only one study that examined aeruginosins (suomilide) as human trypsin inhibitors. 23 Notably, suomilide was also found to inhibit invasion of trypsin-3 expressing prostate cancer cells. 23 Here we report the discovery of two new members of the aeruginosin family of natural products, varlaxins 1022A and 1046A, that inhibit human trypsin isoenzymes in sub-nanomolar concentrations. Experimental Discovery of varlaxins Varlaxins were discovered serendipitously from Nostoc sp. UHCC 0870 during routine screening of strains of the UHCC culture collection for the production of secondary metabolites. Varlaxins were subsequently identified from Nostoc spp. UHCC 0840, UHCC 0817, UHCC 0758 and UHCC 0757 (Table 1). Nostoc spp. UHCC 0840 and 0870 were collected 2014 from Varlaxudden, Porvoo from the brackish Gulf of Finland waterfront (N 60°12.225′, E 025°37.95′) and isolated into pure culture. Nostoc sp. UHCC 0817 was collected in 2012 from Kobben, Hanko, Finland from the rock surface of Gulf of Finland waterfront (N 59°49.92′, E 023°5.20′). Nostoc sp. UHCC 0758 was collected in 2003 from a mixture of gastropods collected from marina of Jurmo island, Finland (N 59°49.62′,E 021°35.05′). Nostoc sp. UHCC 0757 was collected from an undescribed location in Finland. Strains were grown for 3 weeks at ambient temperature (20–22 °C) under continuous photon irradiance of 15 μmol photons per m 2 per s in 40 mL of saline Z8 medium 36 lacking a source of combined nitrogen. Biomass was collected by centrifugation at 7000gfor 7 min. The pellets were freeze-dried with a BETA 2–8 LSC plus freeze dryer with a LYO CUBE 4–8 chamber (Christ). Freeze-dried pellets were extracted with 1 mL of MeOH in 2 mL plastic tubes containing 0.5 mm glass beads by shaking with a FastPrep cell disrupter (Thermo Electron Corporation, Qbiogene, Inc.) for 30 s at a speed of 6.5 m s −1 . The extract was centrifuged at 10 000gfor 5 min, and the supernatant was analysed with a high-resolution UPLC-QTOF (Acquity I-Class UPLC-Synapt G2-Si, Waters Corp., Milford, MA) system. A 1 μl aliquot was injected into a Kinetex C8 column (50 × 2 mm, 1.7 μm, Phenomenex Inc.), which was eluted at 40 °C with a flow rate of 0.3 mL min −1 from 5% of solvent B (acetonitrile or 1 : 1 acetonitrile/isopropanol with formic acid added to 0.1%) in 0.1% formic acid (solvent A) to 95% of B in 5 min, held for 2 min, then back to 5% of B in 0.5 min, and finally kept for 2.5 min before the next run. In MS/MS mode, the column was eluted from 5% of solvent B (1 : 1 acetonitrile/isopropanol with formic acid added to 0.1%) in 0.1% formic acid (solvent A) to 60% of B in 10 min, then in 0.01 min to 100% of B, held for 3.99 min, then back to 5% of B in 0.5 min, and finally kept for 5.5 min before the next run. QTOF was calibrated using sodium formate and Ultramark® 1621, which yielded a calibrated mass range from m/z91 to 1921. Leucine Enkephalin was used at 10 s intervals as a lock mass reference compound. Data were collected in positive electrospray ionization Resolution Mode at scan range of m/z50–2000. In MS/MS mode, parameters were same as in MS mode and Trap Collision Energy Ramp proceeded from 40.0 eV to 80.0 eV. Varlaxin purification Nostoc sp. UHCC 0870 was cultivated in a 100 L flat-panel bioreactor in Z8 medium bubbled with air enriched with 1.5% CO 2 at a constant temperature (28 °C) and illumination of 50 μmol photon per m 2 per s to obtain 14 g of lyophilized biomass. Biomass was extracted three times with 50% MeOH (in H 2 O). The extract was then concentrated under vacuum using a rotary evaporator. The resulting extract (2.6 g) was chromatographed over normal-phase silica gel (100–200 mesh) flash column yielding 13 fractions eluted with step gradient hexane : diethyl ether (100 : 0, 25 : 75, 50 : 50 and 0 : 100) followed by chloroform : MeOH (100 : 0, 99 : 1, 98 : 2, 96 : 4, 92 : 8, 84 : 16, 68 : 32, 50 : 50 and 0 : 100). Fraction 10 (188 mg in total) eluted with 84 : 16 (chloroform : MeOH) was fractionated on preparative HPLC (Agilent 1260 Preparative Chromatograph equipped with quaternary pumps and MWD detector) on a Phenomenex Kinetex C18 column (250 × 21.2 mm, 5 μm) using H 2 O (A)/acetonitrile (B) both containing 0.1% formic acid as a mobile phase at a flow rate of 10 mL min −1 . The linear gradient was as follows: A/B 85/15 (0 min), 85/15 (over 2 min), 0/100 (over 40 min), and 0/100 (over 45 min). Fraction 6 (eluted at 32–35 min, 25 mg) containing the desired compounds was further purified by semipreparative HPLC on a Phenomenex Kinetex EVO C18 column Phenomenex Kinetex EVO C18 column (250 × 10 mm, 5 μm, 100 Å) eluted with H 2 O (A)/acetonitrile (B) both containing 0.1% formic acid at a flow rate of Paper Organic & Biomolecular Chemistry Org. Biomol. Chem. This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online Table 1 Varlaxin variants identified from five strains in the UHCC culture collection. Mgs = 2-O-methylglyceric acid 3-O-sulfate, Choi = 2-carboxy-6-octahydroindole, Agma = 4-amidinobutylamide, Aaep = 1-amidino-3-(2-aminoethyl)-3-pyrroline, Hpaa = 2-(4-hydroxyphenyl)acetic acid. Δ=difference in parts per million (ppm) between calculated and experimental [M + Na] + .RI= percentage relative intensities of varlaxin variants in a strain. [M −80 + H] + = desulfated (80 = SO 3 ) protonated ion No Var a t R (min) [M −80 + H] + [M + Na] + Structural units UHCC 0870 UHCC 0840 UHCC 0817 UHCC 0758 UHCC 0757 Core Side group [M + Na] + [M −80 +H] + [M + Na] + [M −80 +H] + [M + Na] + [M −80 +H] + [M + Na] + [M −80 +H] + [M + Na] + [M −80 +H] + Calculated (m/z) b 12 c 3 4 Sugar d Hpaa Δ(ppm) RI (%) Δ(ppm) RI (%) Δ(ppm) RI (%) Δ(ppm) RI (%) Δ(ppm) RI (%) 1 740 1.00 661.3767 763.3154 Mgs Val Choi Agma D-Glc —−2.3 e 0.1 −0.4 0.1 0.9 0.4 2764 1.04 685.3767 787.3154 Mgs Val Choi Aaep D-Glc —−0.9 e 1.5 −0.7 1.8 −0.5 0.5 3754 1.24 675.3923 777.3311 Mgs Ile Choi Agma D-Glc —−3.5 0.05 7.7 0.04 −2.4 0.3 −0.7 2.0 1.6 5.6 4778 1.30 699.3923 801.3311 Mgs Ile Choi Aaep D-Glc —−1.5 0.1 −3.3 0.2 0.1 10.5 0.8 39 2.2 7.9 5 874A 1.56 795.4135 897.3522 Mgs Val Choi Agma D-Glc 1 −1.9 0.02 −1.2 0.1 −2.5 0.02 −3.5 0.02 6 898A 1.61 819.4135 921.3522 Mgs Val Choi Aaep D-Glc 1 −0.7 0.05 −1.3 1.9 −0.7 0.4 −3.3 0.1 7 888A 1.64 809.4291 911.3679 Mgs Ile Choi Agma D-Glc 1 −3.0 0.3 −1.7 0.3 −1.8 0.4 −0.7 0.4 8912A 1.67 833.4291 935.3679 Mgs Ile Choi Aaep D-Glc 1 −1.6 1.1 −6.3 0.2 −0.1 11.9 0.4 12 0.8 1.9 9 874B 1.69 795.4135 897.3522 Mgs Val Choi Agma D-Glc 1 −0.6 0.1 −1.6 0.1 −4.1 0.1 −0.6 0.1 10 898B 1.74 819.4135 921.3522 Mgs Val Choi Aaep D-Glc 1 −4.1 0.1 −1.3 0.1 −0.5 2.9 −0.6 1.1 0.4 0.5 11 888B 1.75 809.4291 911.3679 Mgs Ile Choi Agma D-Glc 1 −2.5 1.0 −3.1 0.6 −0.5 0.6 −0.8 1.5 0.1 2.3 12 912B 1.80 833.4291 935.3679 Mgs Ile Choi Aaep D-Glc 1 −1.3 2.2 −1.9 3.1 0.4 27.8 1.5 32 1.3 11 13 1008A 2.02 929.4502 1031.3890 Mgs Val Choi Agma D-Glc 2 −1.3 0.7 0.3 0.2 0.7 0.1 0.0 0.1 14 1032A 2.06 953.4502 1055.3890 Mgs Val Choi Aaep D-Glc 2 −1.9 2.6 −3.7 1.1 0.7 2.9 −1.4 0.3 0.5 0.4 15 1022A 2.07 943.4659 1045.4047 Mgs Ile Choi Agma D-Glc 2 −2.6 25 0.1 12 0.4 0.5 −0.8 0.2 1.7 7.1 16 1046A 2.10 967.4659 1069.4047 Mgs Ile Choi Aaep D-Glc 2 −0.3 55 −0.8 76 0.7 24.3 0.4 6.4 1.8 35 17 1008B 2.13 929.4502 1031.3890 Mgs Val Choi Agma D-Glc 2 −2.0 0.2 −2.3 0.3 −0.5 0.3 18 1032B 2.14 953.4502 1055.3890 Mgs Val Choi Aaep D-Glc 2 −3.7 0.5 0.9 1.6 1.3 1.7 19 1022B 2.20 943.4659 1045.4047 Mgs Ile Choi Agma D-Glc 2 −3.0 2.8 −0.4 4.2 −0.4 0.3 0.4 0.1 0.6 4.1 20 1046B 2.22 967.4659 1069.4047 Mgs Ile Choi Aaep D-Glc 2 −1.8 8.2 0.8 12.5 0.7 2.4 1.3 20 21 1142 —1063.4870 1165.4258 Mgs Val Choi Agma D-Glc 3 22 1166 —1087.4870 1189.4258 Mgs Val Choi Aaep D-Glc 3 23 1156 2.46 1077.5027 1179.4414 Mgs Ile Choi Agma D-Glc 3 −3.2 0.1 −5.1 0.1 24 1180 2.45 1101.5027 1203.4414 Mgs Ile Choi Aaep D-Glc 3 −4.1 0.2 −3.6 0.4 a Numbering base to exact mass of the neutral molecule. Bold font = product ion spectrum annotated. b M−80 originates from neutral loss of SO 3 from M. c According to aa analysis of 1022A and 1046A aa is Ile. In other variants could also be isobaric Leu. d Analyzed from 1022A/1046A, Hpaa = number of Hpaa (4-hydroxyphenylacetic acid) units. e From [M −80 + H] + . Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2022 Org. Biomol. Chem. Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online 3 mL min −1 using following gradient: A/B 85/15 (0 min), 85/15 (in 1 min), 72/28 (in 8 min), and 72/28 (in 30 min). The compound elution was monitored on the MWD detector set to 220 nm, yielding 0.7 mg of purified varlaxin 1046A and 0.6 mg of purified varlaxin 1022A, eluting at 20 min and 19 min, respectively. The purified varlaxins 1046A and 1022A contained approximately 3–4% of other minor varlaxin variants. Amino acid analysis Solid purified varlaxins 1046A and 1022A and reference amino acids were transferred to 200 µL glass tubes inside a closed 4 mL glass vial containing 500 µL of 6 M HCl and hydrolysed at 110 °C for 21 h. Inner-tube HCl residues were vacuum evaporated and 100 µL of 1% Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine or -L-leucine amide in acetone), 50 µL of H 2 O and 20 µL of 1 M NaHCO 3 were added. The solution was incubated at 37 °C for 1 h and the reaction was stopped by adding 20 µL of 1 M HCl. Commercial reference compounds L-Ile, D-Ile, L-allo-Ile, and D-allo-Ile were obtained from SigmaAldrich (St. Louis, MO). The L-Choi reference amino acid was obtained from Microcystis aeruginosa NIES-298. 19 Cells were grown, freeze dried, and extracted as described above. Solvent was evaporated from the crude extract and dry material was used as L-Choi reference material. Monosaccharide analysis Dand L-Glucose were purchased from Sigma-Aldrich. Hydrolysis of varlaxin 1022A and sample derivatizations were performed as previously described 37 with the following exceptions: approximately 0.4 mg of varlaxin 1022A was hydrolysed, freeze dried instead of drying under N 2 gas stream, and the reagents L-cysteine methyl ester (Sigma-Aldrich) and o-tolyl isothiocyanate (Thermo Fisher GmbH, Germany) instead of phenyl isothiocyanate were added simultaneously, and the reaction mixture was incubated at 60 °C for 1 h and diluted with MeOH before UPLC-QTOF analysis. Samples of 1 µL were analysed with two LC methods. The sample was injected into a Kinetex C8 column (50 or 100 × 2 mm, 1.7 μm, Phenomenex Inc.), which was eluted at 0.3 mL min −1 at 40 °C with 95% H 2 O (+0.1% formic acid, eluent A) and 5% ACN : isopropanol (1 : 1, +0.1% formic acid, eluent B). Eluent B was increased linearly to 100% in 5 minutes, kept at 2 minutes, then back to the initial condition in 0.5 minutes with 2.5 min post run before next injection. Additionally, the sample was eluted with Acquity UPLC® BEH C18 column (100 × 2.1 mm, 1.7 µm Waters Corp., MA) as previously described, but eluent B was increased linearly to 70% in 10 minutes, then to 100% in 0.10 minutes, kept at 3.99 minutes, and changed back to initial condition in 0.5 minutes with 5.5 min post run. QTOF was used in resolution mode with positive electrospray ionization. Leucine enkephalin was used as a lock mass, and sodium formate and Ultramark® 1621 for mass calibration. The retention time of monosaccharide thiocarbamoyl-thiazolidine derivative of D-Glc of varlaxin 1022A was 1.97 min (references D-Glc 1.97 min, L-Glc 1.88 min). NMR and UV spectroscopy Spectra were collected on a Bruker Avance III HD 800 MHz NMR spectrometer equipped with a cryogenically cooled TCI 1 H, 13 C, 15 N triple-resonance probe head. Data were collected at 30 °C in DMSO-d 6 (Table S3†). In addition to a 1 H experiment with the solvent pre-saturation and broadband decoupled 13 C experiment, homonuclear, two-dimensional (2D) TOCSY (TOtal Correlation SpectroscopY), NOESY (Nuclear Overhauser Enhancement SpectroscopY) and DQF-COSY (Double Quantum Filtered COrrelation SpectroscopY) experiments were performed. Heteronuclear 2D 1 H– 13 C HSQC and 1 H– 15 N HSQC (Heteronuclear Single Quantum Coherence) and 2D 1 H– 13 C HMBC (Heteronuclear Multiple Bond Correlation) experiments (for the assignment of 1 H, 13 C and 15 N chemical shifts) were also performed. The 2D TOCSY experiment was acquired with an isotropic mixing time of 60 ms. The mixing time for 2D NOESY spectrum was 300 ms. The 13 C HMBC experiment (for observing long-range 1 H– 13 C connectivity) was measured using n J CH transfer time optimized for 8 Hz couplings. For monosaccharide analysis, varlaxin 1046A was hydrolysed for 1 h at 100 °C in 0.5 mL of 2 M D 2 SO 4 in D 2 O. The proton spectrum was collected, and anomeric signals were compared with data from a previous publication. 38 UV spectra of varlaxins 1022A and 1046A, and a reference compound 4-hydroxyphenylacetic acid were measured in MeOH with a UV-1800 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). Trypsin inhibition The inhibition of human trypsins was determined as described previously. 23 The recombinant trypsinogens-1, -2 and -3 were produced in Escherichia coli and activated to trypsin-1, -2, and -3 as described previously. 39 Enzyme inhibitory activity of varlaxins was determined in 96-well plates by adding 10 μL of varlaxins diluted in ultra-purified H 2 O (containing up to 0.027% DMSO), 15 μL of 0.1% bovine serum albumin in 50 mM Tris-buffered saline (BSA/TBS) and 25 μLof each trypsin (1.8 nM). Ultra-purified H 2 O (10 µL) was used as a control. DMSO at the concentrations used did not have any effect on trypsin activity (data not shown). The varlaxins were pre-incubated with trypsins at room temperature for 15 min before addition of 50 μL of 0.2 mM chromogenic substrate S-2222 (ChromogeniX) in ultra-purified H 2 O. The tested varlaxin 1046A and 1022A concentrations, after the addition of substrate, ranged from 0.01 nM, 1076 nM to and from 0.73 nM to 7339 nM to 0.73 nM, respectively. The change of absorbance at 405 nm was followed for 15 min (VICTOR X4 Multiple plate reader, PerkinElmer). The experiment was performed three times, each with 2 replicates (n= 3). The absorbance change was determined at the phase of the substrate reaction during which the absorbance of the control reaction increased linearly over time. The absolute IC 50 values were estimated using Quest Graph™(https://www.aatbio.com/tools/ic50-calculator). The porcine trypsin assay was performed as previously described. 22 Paper Organic & Biomolecular Chemistry Org. Biomol. Chem. This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online Genome sequencing and gene cluster analysis The cyanobacterial strain Nostoc sp. UHCC 0870 was grown for 8daysat20–21 °C under photon irradiance of 15 μmol m −2 s −1 in 150 mL of Z8 culture medium in a 500 mL Erlenmeyer flask. Genomic DNA was isolated using phenol–chloroform extraction as previously described. 40 The quality and concentration of isolated DNA samples were assessed using an Agilent TapeStation (Agilent Technologies) and a NanoDrop 1000 spectrophotometer (Thermo Scientific), respectively. Whole-genome sequencing was performed using a combination of PacBio RSII and Illumina MiSeq. Nextera adapters were removed from Illumina data by cutadapt v1.14. PacBio data were assembled with HGAP3. Illumina data were assembled with SPAdes v3.11.1 with the “careful”option. Gap4 was used to combine assemblies and check sequence circularity. Bwa 0.7.12-r1039 was used to map Illumina reads against the combined assembly. Homopolymer errors were corrected using Pilon v1.16. The complete sequence was annotated using Prokka v1.13 and PANNZER2. All sequencing, assembly, and annotation were performed at the DNA Sequencing and Genomics laboratory, Institute of Biotechnology, University of Helsinki, Finland. The complete genome sequence of Nostoc sp. UHCC 0870 was deposited in the GenBank database (CP091913–CP091924). We analysed the substrate specificity of adenylation domains using NRPSpredictor2 analysis website (http://nrps.informatik.uni-tuebingen.de/). The condensation domain of VarB was analyzed with Natural Product Domain Seeker (NaPDoS) and compared with other known aeruginosin AerB. Results Analysis of methanol extracts of Nostoc sp. UHCC 0870 using UPLC-QTOF mass spectrometry led to the serendipitous discovery of two novel aeruginosins, which we named varlaxins 1046A and 1022A, after Varlaxudden on the Gulf of Finland from where the strain was isolated. Total ion current chromatogram from the methanol extract showed prominent peaks (retention time 2.07 and 2.10 min) at the peptide region (Fig. S4A†). The upper-range mass spectrum from these peaks contained two pairs of mass peaks, representing sodiated ions. The same compounds in desulfated protonated form showed the presence of a single sulfate group (Fig. S4B†). A broaderrange mass spectrum showed neutral loss of methylglyceric acid (MgA)-Ile and ions matching to one of the common aeruginosin structures (Fig. S4C†). Low-mass nonspecific product ion spectrum (MS E ) from these peaks showed diagnostic ions for aeruginosin substructures as Choi, Aaep, and MgA-Ile (Fig. S4D†). Analysis of product ion spectra (MS 2 ) from the desulfated protonated ions m/z967.46 and 943.46 gave preliminary structures (Fig. 1 and 2) without the exact locations of 4-hydroxyphenylacetic acid (Hpaa) residues or clarifying the nature of the hexose sugar (Fig. S5 and Table S4†). Further analysis of strains from the UHCC culture collection led to identification of 22 varlaxin chemical variants from five strains (Table 1). Mass spectrometric analyses (Fig. S5 and Table S4†) and retention time data showed that the chemical variation originates from the following three positions: Ile or Val in position 2, Aaep or Agma in position 4, and the number and location of Hpaa residues in the structure. Two variants with identical masses, but with different retention times, were detected from varlaxin chemical variants with one and two Hpaa residues (Table 1). Two variants with three Hpaa residues were detected, but low abundance prevented further characterization of these variants. This variation was not observed when Hpaa was absent from the structure. These observations suggest that variants with identical masses differ with respect Fig. 1 Structures of varlaxins 1046A and 1022A from Nostoc sp. UHCC 0870. Mgs=2-O-methylglyceric acid 3-O-sulfate, Choi = 2-carboxy-6octahydroindole, Agma = 4-amidinobutylamide, Aaep = 1-amidino-3- (2-aminoethyl)-3-pyrroline, Hpaa = 4-hydroxyphenylacetic acid. Fig. 2 Major varlaxin MS 2 fragments from desulfated variants 1046A, 1022A, 912A, 888B, 778, 764, and 754. Fragment numbers are given in Table S3.† Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2022 Org. Biomol. Chem. Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online to the positions of the Hpaa residues in the hexose unit. Therefore, the subunit sequence for the 22 varlaxins can be summarized as Mgs 1 -Ile/Val 2 -(Choi-Glc-(0–3 Hpaa)) 3 -Agma/ Aaep 4 (Table 1). In all strains, Ile and Aaep dominated in positions 2 and 4, respectively, and in three strains variants with two Hpaa residues dominated. The Choi moiety was decorated with a hexose esterified with 0–3 Hpaa residues. UV spectra of varlaxin 1046A and 1022A near their maximum of 278 nm matched closely with spectrum of reference Hpaa, which is consistent with the existence of Hpaa substructure in varlaxins (Fig. S6†). The structures of purified varlaxins 1046A and 1022A were completed and verified with 1D 1 H and 13 C spectra and 2D DQF-COSY, TOCSY, NOESY, 1 H– 1 C HSQC, 1 H– 15 N HSQC, and HMBC NMR spectra (Fig. S7, S8 and Tables S5, S6†). Two sets of δ C/H values were recognized from both 1046A and 1022A data originating from cis/trans Ile-Choi rotamers in a 4 : 6 ratio (Table S5†). DQF-COSY correlations and HMBC/NOESY correlations were used for subunit connections (Fig. 3). HMBC correlations connected the two Hpaa residues to hexose positions 2 and 6 (Fig. 3). The hexose connected to Choi-OH was analysed from the hydrolysis mixture with NMR and LC-MS to be D-glucose (Fig. S8 and S9†). The configuration of the two amino acids in the varlaxin substructure were analysed with LC-MS of the Marfey’s derivatives of purified varlaxin 1046A and 1022A acid hydrolysates. Comparison to commercial amino acid reference Ile isomers and Choi from Microcystis aeruginosa NIES-298 raw extract showed that Ile was in D-configuration and Choi was in the L-configuration in both varlaxins, which is the same configuration as Choi from Microcystis aeruginosa NIES 298 (Fig. S10†). We sequenced the complete 7.17 Mb genome of Nostoc sp. UHCC 0870, which was organized in a 6.42 Mb chromosome, 10 plasmids, and a plasmid prophage. Bioinformatic analysis identified the putative 36 kb varlaxin (var) biosynthetic gene cluster, which contained 22 ORFs of which 10 shared similarity to gene products residing in other aeruginosin biosynthetic gene clusters (Table 2 and Fig. 4). VarB and VarG encode two bimodular NRPS enzymes, which form the varlaxin peptide backbone (Fig. 4). The first module of VarB consists of an O-methylglyceric acid transferase (OMT), a FkbH domain, peptidal carrier protein (PCP) and sulfotransferase (ST) (Fig. 4). The second module consists of condensation (C), adenylation (A), PCP and epimerization (E) domains (Fig. 4). The first module of this NRPS likely incorporate glycerate, which is further methylated and sulfated (Fig. 4), the second module loads allo-isoleucine. A loading module with an identical set of catalytic domains from the suomilide and dysinosin biosynthetic pathways is also proposed to synthesize Mgs. 23,30 The VarB second adenylation domain binding pocket Stachelhaus sequence is DALWMGGVFK, which predicts, with a moderate 80% match, Val in the second position (Table S7†). However, according to chemical analysis by Marfey’s method, this amino acid in varlaxins 1022A and 1046A was D-Ile. Biosynthetic gene clusters for ulleungmycin and desotamide cyclic peptides contain identical adenylation domain binding pocket sequence DALWMGGVFK in genes UlmA 4 and DsaH 4 without following epimerase. 41,42 This resulted in incorporation and existence of L-allo-Ile in these peptides. Notably, the second domain of VarB contains an epimerase domain. When the substrate of this domain is L-allo-Ile, the C 2 epimerization results in D-Ile, which is found in varlaxins 1022A and 1046A. Based on these three cases, the Stachelhaus sequence DALWMGGVFK is more specific for L-allo-Ile than for Val. The binding pocket of second adenylation domain of AerB from aeruginosins 98-A, -B, and -C is DAFFLGVTFK, of which amino acid prediction is Ile with strong 100% match (Tables S7 and S28†). Thus, the action of these two VarB second domains resulted in the incorporporation of D-Ile or D-allo-Ile in varlaxins or aeruginosins 98-A, -B, and -C, as adenylation domains recognize different amino acids (L-allo-Ile or L-Ile, respectively), and C 2 epimerization in both cases generate the final Ile configurations (Fig. 4 and Table S7†). VarG, consists of C, A, PCP, C, and PCP domains, which load the Choi and arginine to the preceding moiety (Fig. 4). VarI glycosylates the Choi hydroxyl group. VarO is an adenylation (A) domain that could recognize the Hpaa substrate, which would be bound to acyl carrier protein (ACP) VarP. VarQ belongs to the MBOAT enzyme family, which could catalyse the addition of Hpaa to the glucose (Fig. 4). The trypsin inhibitory activity of varlaxins 1046A and 1022A was tested using both porcine and human trypsins. Inhibition of porcine trypsin was very effective with both varlaxin variants, with IC 50 values of 14 nM and 740 nM, respectively (Fig. 5). This makes the varlaxin 1046A the most potent porcine trypsin inhibitor among the reported aeruginosins (Table S2†). As varlaxins are strong porcine trypsin inhibitors, the three human trypsin isoenzymes were also tested for inhibition. Both varlaxin variants inhibited all human trypsin isoenzymes (Fig. 5). Varlaxin 1046A inhibited human trypsin-2 and -3 with an IC 50 values of 0.62 nM and 0.96 nM, respectively (Fig. 5), while trypsin-1 was inhibited at higher concentrations (IC 50 3.6 nM). Fig. 3 DQF-COSY, HMBC, and NOESY correlations of varlaxins 1022A and 1046A. Paper Organic & Biomolecular Chemistry Org. Biomol. Chem. This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online Discussion We report here the identification and structural characterization of novel members of the aeruginosin family of natural products, varlaxin 1046A and 1022A. All identified varlaxin variants contain Choi at the third amino acid, a moiety which is characteristic for most of the aeruginosins, except for subclasses such as pseudoaeruginosins, suomilide, banyasides and nostosins (Table 1 and Table S1†). Thus, we propose that varlaxins are members of the aeruginosin family of natural compounds. The Choi moiety of the varlaxins reported here is decorated with a sugar, a decoration previously identified in about one quarter of the aeruginosins that contain a Choi moiety (Table S1†). The varlaxin α-D-glucopyranose moiety is esterified by 0–3 carboxylic acids (Table 1 and Fig. 1, 4). Approximately half of the other aeruginosin sugar units are decorated with carboxylic or sulfate groups. 32 However, the Hpaa residue of varlaxins is part of the Choi-glucose subunit, making this substructure markedly different and unique among aeruginosins. This unique sugar decoration has been reported in plant sesquiterpene lactones, where glucose is decorated with three Hpaa residues. 43 The N-terminus moiety of varlaxin is Mgs, which is rare among aeruginosins (Table 1 and Table S1†); only suomilide, banyasides, and four dysinosins contain this moiety. 22,24,32 While the second moiety of varlaxins is primarily allo-Ile (identified from variants 1046A and 1022A, but could be isobaric Leu in other variants), Val was also found (Table 1). Both are nonpolar amino acids, which are the typical second amino acids in aeruginosins (Table S1†). The fourth substructure unit of varlaxins is either Aaep or Agma, of which Aaep is rarer than Agma in aeruginosins (Table S1†). In summary, the Mgs and Aaep structure and the Choi-Glu-Hpaa moiety make varlaxins unique amongst the aeruginosin family of natural products and justifies naming these compounds as a separate aeruginosin subclass. The 36 kb varlaxin biosynthetic gene cluster is similar to previously reported aeruginosin biosynthetic gene clusters. 23,26–31 Varlaxin biosynthesis begins with the incorporation of Mgs, catalyzed by a FkbH domain, which tailors O-methylglyceric acid (OMT) and sulfotransferase (ST) Table 2 Predicted varlaxin (var) biosynthetic gene cluster gene products Protein Accession number Predicted function Length (aa) BLASTp top hit Description Scientific name Identity % Accession ORF1 UKO95746 156 Tetratricopeptide repeat protein Anabaena minutissima 96 WP_190695341 ORF2 UKO95747 449 FAD-binding oxidoreductase Anabaena minutissima 94 WP_190696554 VarB UKO95748 NRPS 2853 Amino acid adenylation domaincontaining protein Nostoc sp. FACHB-145 85 WP_190754768 ORF3 UKO95749 274 Hypothetical protein Nostoc sp. KVJ20 86 WP_069074352 ORF4 UKO95750 126 Nuclear transport factor 2 family protein Nostoc sp. 84 WP_199337282 ORF5 UKO95751 Sulfotransferase 192 Adenylyl-sulfate kinase Scytonema sp. NIES-4073 90 WP_096564148 VarC NA 720 Rieske 2Fe-2S domain-containing protein Nostoc sp. B(2019) 86 WP_162397143 VarK UKO95752 351 Type 2 isopentenyl-diphosphate Delta-isomerase Scytonema sp. NIES-4073 92 WP_096564146 VarD UKO95753 Choi formation 198 Bacilysin biosynthesis protein BacABacA Nostoc sp. LEGE 12450 90 WP_193945672 VarE UKO95754 Choi formation 235 Cupin domain-containing protein Scytonema sp. NIES-4073 84 WP_096564145 VarF UKO95755 Choi formation 264 SDR family oxidoreductase Nostoc sp. B(2019) 90 WP_162397147 VarG UKO95756 NRPS 1615 Non-ribosomal peptide synthetase Scytonema sp. NIES-4073 88 WP_096564143 ORF6 UKO95757 267 SDR family oxidoreductase Nostoc sp. ‘Peltigera membranacea cyanobiont’213 93 WP_094328879 VarN1 UKO95758 ABC transporter 596 ABC transporter substrate-binding protein Anabaena sp. 4-3 83 WP_066426904 ORF7 UKO95759 385 Hypothetical protein Anabaena sp. CA = ATCC 33047 78 WP_066382146 ORF8 UKO95760 214 Hypothetical protein Anabaena 84 WP_066382147 VarO UKO95761 NRPS 541 Amino acid adenylation domaincontaining protein Nostoc linckia 82 WP_096542302 VarP UKO95762 ACP 88 Acyl carrier protein Anabaena 80 WP_066382151 VarQ UKO95763 Hpaa transfer 504 MBOAT family protein Anabaena sp. 4-3 89 WP_066426900 VarI UKO95764 Glycosylation 423 Glycosyltransferase family 4 protein Anabaena sp. 4-3 85 WP_066426897 ORF9 UKO95765 457 Major facilitator superfamily transporter Nostoc sp. ‘Peltigera membranacea cyanobiont’N6 87 AVH65385 VarN2 UKO95766 ABC transporter 600 ATP-binding cassette domaincontaining protein Anabaena sp. CA = ATCC 33047 83 WP_066382156 ORF10 UKO95767 294 Hypothetical protein Anabaena minutissima 89 WP_190700187 ORF11 UKO95768 273 3-Deoxy-7-phosphoheptulonate synthase Anabaena minutissima 97 WP_190700184 Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2022 Org. Biomol. Chem. Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online domains (Fig. 5). A similar organization of Mgs-incorporating domains is seen in the biosynthesis of cyanopeptolin 1138, 48 dysinosin 30 and suomilide. 23 Phormidolide and oscillapeptin G both contain similar glyceric acid units that are incorporated by FkbH domains. 49,50 FkbH domains use phosphoglycerate as their substrate. 51 The predicted substrate for sulfotransferase is 3′-phosphoadenylyl sulfate, produced by ORF5 (adenylylsulfate kinase). The varlaxin biosynthetic gene cluster encodes the three Choi-forming enzymes, AerDEF (VarDEF in varlaxin BGC). 27 The varlaxin biosynthetic gene cluster also includes both AerC (VarC) and AerK (VarK), which are predicted to participate in Choi formation. However, Choi-containing aeruginosin NAL2 is produced without either of these enzymes. 29 No study to date has provided a reasonable model for formation of Aaep and this is likely to involve novel biochemistry. However, when comparing aeruginosin biosynthetic gene clusters and their products, AerC (VarC) can be found exclusively in biosynthetic gene clusters that produce aeruginosins with Aaep. 25 AerH has been predicted to act in Aaep formation. 27 However, no AerH homolog was found in the varlaxin biosynthetic gene cluster (Fig. 5). The varlaxin biosynthetic gene clusters encodes three unique proteins, VarO-Q, that are not found in other aeruginosin biosynthetic gene clusters and predicted here to attach Hpaa residues to the glucose (Fig. 5). MBOAT enzymes have also been shown to act on saccharides. 52 The timing of individual biosynthetic steps is unclear but glycosylation of Choi may occur prior to release of the final product because it appears in all of the varlaxins detected. The fact that there are mono-, diand tri-Hpaa-ester derivatives suggest that this step occurs subsequent to release from VarG. Varlaxin 1046A is produced in two-fold higher abundance than varlaxin 1022A, indicating that varlaxin 1046A is the main product of the varlaxin biosynthetic gene cluster (Table 1). The other varlaxin chemical variants are likely to be side products from unspecific substrate specificities of the VarB adenylation domains and incomplete action of the possible tailoring reactions. The aeruginosins have frequently been observed to inhibit serine proteases. 18 Varlaxin 1046A appears to be the most potent cyanobacterial trypsin inhibitor reported and is conFig. 4 The varlaxin (var) biosynthetic gene cluster and putative biosynthetic scheme in Nostoc sp. UHCC 0870. A –Organization of predicted biosynthetic genes. B –Proposed biosynthetic pathway of varlaxin 1046A. FkbH (domain of unknown function), OMT (O-methylglyceric acid transferase); PCP (peptidyl carrier protein); ST (sulfotransferase); C (condensation domain); A (adenylation domain); E (epimerization domain); and MBOAT (membrane bound O-acyl transferase). Paper Organic & Biomolecular Chemistry Org. Biomol. Chem. This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 09 March 2022. Downloaded on 3/17/2022 8:25:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online