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Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Biosynthesis of Nonimmunosuppressive FK506 Analogues with Antifungal Activity Ji Yoon Beom,†,∥ Jin A Jung,†,∥ Kyung-Tae Lee,‡,∥ Areum Hwangbo,‡,∥ Myoung Chong Song,† Yeonseon Lee,‡ Soo Jung Lee,‡ Ji Hoon Oh,§ Sang-Jun Ha,§ Sang-Jip Nam,† Eunji Cheong,*,‡ Yong-Sun Bahn,*,‡ and Yeo Joon Yoon*,† †

Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea § Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

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‡

S Supporting Information *

ABSTRACT: A reduction in the strong immunosuppressive activity of FK506 (1) is essential for developing this compound as an antifungal agent. Seven new FK506 analogues modified at both the FK506-binding protein 12and the calcineurin-binding regions were biosynthesized. 9DeoxoFK520 (7) exhibited a >900-fold reduction in the in vitro immunosuppressive activity but maintained significant antifungal activity, indicating that the C-9 and C-21 positions are critical for separation of immunosuppressive and antifungal activities. 7 exhibited robust synergistic antifungal activity with fluconazole. FK506 (1) is a 23-membered macrolide produced by several Streptomyces species and is used as an immunosuppressive drug to prevent the rejection of transplanted organs. FK506 has also exhibited antifungal, neuroprotective, and neuroregenerative activities. In humans, FK506 binds to FK506binding protein (FKBP) 12, and the resulting FKBP12−FK506 complex interacts with a Ca2+-calmodulin-dependent phosphatase, calcineurin (CaN). Inactivation of CaN by forming the FKBP12−FK506−CaN ternary complex prevents the activation of nuclear factor of activated T cells (NF-AT), inhibiting the production of interleukin-2 and subsequent T-cell proliferation. This CaN signaling pathway also plays a critical role in the growth and pathogenesis of major fungal pathogens such as Cryptococcus neoformans, Candida albicans, and Aspergillus f umigatus. Therefore, the synthesis of FK506 analogues that can discriminate human FKBP12/CaN from its fungal counterparts may separate antifungal activity from the immunosuppressive activity, thereby allowing the development of a novel antifungal agent.

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unique extender unit allylmalonyl-CoA/ACP is synthesized from trans-2-pentenyl-ACP, which is generated by β-ketoacyl synthase TcsB and acyl transferase (AT) TcsA in conjunction with the fatty acid synthase pathway, through reductive carboxylation with the crotonyl-CoA carboxylase/reductase homologue TcsC and C-36−C-37 double-bond formation by TcsD dehydrogenase. 7 The linear polyketide chain is condensed with pipecolate derived from lysine by NRPS FkbP and then cyclized for generation of the macrolide ring.8 This ring is further modified in a parallel manner by the two substrate-promiscuous post-PKS tailoring enzymes (C-9 oxidation by FkbD and C-31-O-methylation by FkbM).9 Interestingly, the AT domain of module 4 (AT4) has broad substrate specificity toward various extender units, thus generating a series of analogues with different C-21 side chains: FK506, FK520, and FK523 possess allyl, ethyl, and methyl side chains at C-21, respectively. In addition, the prolyl

he crystal structure of the mammalian ternary complex of FKBP12−FK506−CaN is the only structure that has been described,1−4 and the limited structural information about the fungal FKBP12−FK506−CaN complex hampers the rational design of antifungal FK506 analogues with reduced immunosuppressive activity. Nevertheless, substantial efforts have been made to modify FK506; for example, a nonimmunosuppressive analogue (L-685,818, a C18-hydroxyFK520) with reduced antifungal activity was reported.5 The structural complexity of FK506 often renders specific chemical modification impractical. A combinatorial biosynthetic approach involving the manipulation of the biosynthetic genes represents an alternative strategy for generating diverse analogues. FK506 is biosynthesized by a hybrid type I modular polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system from a 4,5-dihydroxycyclohex-1-enecarboxylic acid as a starter unit.6 This unit is elongated by 10 condensation steps with two malonyl-CoA units, two methoxymalonyl-acyl carrier proteins (ACPs), five methylmalonyl-CoA units, and an allylmalonyl-CoA/ACP.7 The © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 15, 2019

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DOI: 10.1021/acs.jnatprod.9b00144 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Schematic of the FK506 biosynthetic gene cluster as well as biosynthesis of FK506 and its analogues. Black and white circles depict domains that are predicted to be inactive from the FK506 structure and nonfunctional domains containing mutations in the active sites, respectively. CAS, CoA synthetase; KS, ketoacyl synthase; AT, acyl transferase; DH, dehydratase; ER, enoyl reductase; KR, keto reductase; ACP, acyl carrier protein.

Here, we combinatorially biosynthesized seven new FK506 analogues containing structural modifications in the CaNbinding as well as FKBP-binding regions and evaluated their in vitro immunosuppressive and antifungal activities against three major human fungal pathogens. One of the new derivatives exhibited significantly reduced immunosuppressive activity but improved antifungal activity compared with that of 5 as well as robust synergistic antifungal activity with fluconazole. Importantly, the structure−activity relationship of FK506 analogues for immunosuppressive and antifungal activities could be deduced, elucidating the target moieties for finetuning of the immunosuppressive and antifungal activities.

analogue can also be biosynthesized, owing to the relaxed substrate specificity of FkbP (Figure 1).10 Recently, we reported the in vivo and in vitro antifungal activity of four FK506 analogues containing modifications in the FKBP12-binding region (approximately, one side of the cyclohexane, the pipecolate and pyranose, and the tricarbonyl region)4 generated by modification of post-PKS tailoring steps: 9-deoxo-prolylFK506 (2), 9-deoxoFK506 (3), 31-O-demethylFK506 (4), and 9-deoxo-31-O-demethylFK506 (5).11 Among them, 5 showed 560-fold reduced immunosuppressive activity, but retained significant in vivo and in vitro antifungal activity against C. neoformans, C. albicans, and A. f umigatus, although it was less effective than FK506. This suggests that the C-9 and C-31 positions could be potential sites for controlling the binding affinity of FK506 to human and fungal FKBP12s.11 In addition to the FKBP-binding region, the CaNbinding region of FK506 (roughly, C-13−C-24 region)4 is important for modulating the immunosuppressive and antifungal activities.

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RESULTS AND DISCUSSION Construction of Mutant Strains and Characterization of FK506 Analogues. By exploiting the substrate flexibilities of theFK506 biosynthetic enzymes, we constructed seven mutant strains of FK506-producing Streptomyces sp. KCTC 11604BP where in-frame deletions of f kbD, f kbM, tcsB, and tcsD were combined (Table S1). These yielded several new B



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analogues containing variations at the pipecolate moiety, C-9, C-21, and C-31 positions (Figure 2). The previously

Figure 2. Structures of FK506 and its analogues. a,b,c,dKnown compounds described in refs 10, 9, 12, and 13, respectively.

constructed f kbD deletion mutant (ΔfkbD strain) produced 3, a known intermediate, and 2, a known shunt metabolite containing a proline rather than the pipecolate ring (Figure 3a; Figures S1 and S2).9,10 Additional deletion of tcsD, which encodes the enzyme responsible for the terminal double-bond formation of the allylmalonyl moiety, in the f kbD deletion mutant (ΔtcsD−ΔfkbD strain) generated a new analogue, 9deoxo-36,37-dihydroFK506 (6) (Figure 3b; Table S2; Figures S3 and S4). 6 was purified as a white, amorphous powder through extensive chromatographic separation. Analysis of the 1 H, 13C, and gHSQC NMR spectra in CDCl3 (Figure S4a,b,d) of 6 revealed a ketone carbon (δC 215.4), two carbonyl carbons (δC 174.0; δC 169.4), two olefinic methine signals (δH 5.05/δC 122.2; δH 4.97/δC 128.7), two olefinic nonprotonated carbons (δC 140.8; δC 132.4), a hemiketal carbon (δC 98.5), seven oxymethine signals (δH 5.19/δC 76.5; δH 3.97/δC 69.6; δH 3.84/δC 70.6; δH 3.53/δC 77.1; δH 3.40/δC 74.3; δH 3.39/δC 73.6; δH 3.00/δC 84.1), three methoxy signals (δH 3.39/δC 56.6; δH 3.36/δC 57.6; δH 3.35/δC 56.1), two singlet methyl signals (δH 1.66/δC 13.9; δH 1.63/δC 15.5), three doublet methyl signals (δH 0.93/δC 16.9; δH 0.90/δC 10.1; δH 0.76/δC 18.8), and a triplet methyl signal (δH 0.88/δC 13.9) displaying typical features of an FK506 analogue lacking an exomethylene. Further interpretation of gCOSY and gHMBC data (Figure S4c,e) showed that 6 possesses a pipecolate moiety (δH 4.85/δC 52.6; δH 1.70 and 2.24/δC 26.6; δH 1.31 and 1.72/δC 20.7; δH1.51 and 1.68/δC 24.4; δH 3.19 and 3.69/

Figure 3. UPLC-qTOF-HR-MS analysis of FK506 analogues. (a) Chromatogram selected for m/z = 798.4763 and 812.4919 corresponding to 9-deoxo-prolylFK506 (2) and 9-deoxoFK506 (3), respectively, of culture extracts obtained from the ΔfkbD strain. (b) Chromatogram selected for m/z = 814.5076 corresponding to 9deoxo-36,37-dihydroFK506 (6) of culture extracts obtained from the ΔtcsD−ΔfkbD strain. (c) Chromatogram selected for m/z = 800.4919 and 786.4763 corresponding to 9-deoxoFK520 (7) and 9deoxoFK523 (8), respectively, of culture extracts obtained from the ΔtcsB−ΔfkbD strain. (d) Chromatogram selected for m/z = 812.4555 and 798.4399 corresponding to 31-O-demethylFK506 (4) and 31-O-demethyl-prolylFK506 (●), respectively, of culture extracts obtained from the ΔfkbM strain. (e) Chromatogram selected for m/z = 814.4712 corresponding to 31-O-demethyl-36,37-dihydroFK506 (9) of culture extracts obtained from the ΔtcsD−ΔfkbM strain. (f) Chromatogram selected for m/z = 800.4555 and 786.4399 corresponding to 31-O-demethylFK520 (10) and 31-O-demethylFK523 (■), respectively, of culture extracts obtained from the ΔtcsB−ΔfkbM strain. (g) Chromatogram selected for m/z = 798.4963 and 784.4606 corresponding to 9-deoxo-31-O-demethylFK506 (5) and 9-deoxo-31-O-demethyl-prolylFK506 (11), respectively, of culture extracts obtained from the ΔfkbDM strain. (h) Chromatogram selected for m/z = 800.4919 corresponding to 9deoxo-31-O-demethyl-36,37-dihydroFK506 (12) obtained from the ΔtcsD−ΔfkbDM strain. (i) Chromatogram selected for m/z = 786.4763 and 772.4606 corresponding to 9-deoxo-31-O-demethylFK520 (13) and 9-deoxo-31-O-demethylFK523 (14), respectively, of culture extracts obtained from the ΔtcsB−ΔfkbDM strain. C



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δC 42.7), a propyl chain (δH 1.42 and 1.65/δC 33.8; δH 1.21/δC 20.4; δH 0.88/δC 13.9), and a cyclohexane moiety (δH 2.35/δC 34.8; δH 0.93 and 2.01/δC 39.8; δH 3.00/δC 84.1; δH 3.39/δC 73.6; δH 1.34 and 1.98/δC 30.6; δH 1.06 and 1.61/δC 31.2). The HMBC correlations from a methylene H-9 (δH 2.49 and 2.66) to a carbonyl carbon C-8 (δC 174.0) and a hemiketal carbon C-10 (δC 98.5) identified the location of a methylene at C-9. The attachment of the pipecolate moiety at C-1 was confirmed by the HMBC correlation from a methine H-2 (δH 4.85) to C-1 (δC 169.4). The COSY correlation from an olefinic proton H-28 (δH 4.97) to H-29 (δH 2.35) clearly indicated the location of the cyclohexane moiety. The pyran was placed between H-9 and H-15 based on the clear HMBC correlations from H-9 (δH 2.49 and 2.66) to C-10 and C-11 (δC 98.5; δC 38.4) and the COSY correlation from H-14 (δH 3.84) to H-15 (δH 3.53). The long-range HMBC correlations from the proton of a methine H-21 (δH 3.21) to the two carbons of an olefin group C-19 and C-20 (δC 140.8; δC 122.2), a ketone C-22 (δC 215.4), and two methylene carbons C-35 and C-36 (δC 33.8; δC 20.4) of propyl revealed that a propyl chain is connected to C-21, instead of a propylene group. Three methoxy groups were positioned at C-13, C-15, and C-31 by the HMBC cross-peaks from H-43 (δH 3.35), H44 (δH 3.36), and H-45 (δH 3.39) to C-13 (δC 74.3), C-15 (δC 77.1), and C-31 (δC 84.1), respectively. Thus, the new analogue was identified as 9-deoxo-36,37-dihydroFK506 (6). Combined deletion of tcsB and fkbD (ΔtcsB−ΔfkbD strain) produced 9-deoxoFK520 (7), which has been previously chemically synthesized,12 and 9-deoxoFK523 (8), a new compound (Figure 3c; Table S2; Figures S5−S8). These compounds resulted from the incorporation of methylmalonyland malonyl-CoA, respectively, by the promiscuous AT4 domain in the absence of allylmalonyl-CoA/ACP, owing to the inactivation of tcsB. Detailed analysis of the 1D and 2D NMR spectra of 8 confirmed the compound to be identical to 6 with the exception of the methyl group replacing a propyl moiety at C-21 (Figure S8). In particular, the HMBC correlations from the proton of methine H-21 (δH 3.21) to the two carbons C-19 and C-20 (δC 140.4; δC 123.5) of an olefin group, a ketone C22 (δC 215.4), and a methyl carbon C-35 (δC 17.1) explained the existence of a methyl group instead of a propyl group at C21. The f kbM deletion mutant (ΔfkbM strain) produced 4, a known compound.8 A trace amount of 31-O-demethylprolylFK506 was detected via UPLC-qTOF-HR-MS analysis (Figure 3d; Figure S9), but was insufficient for NMR analysis. Deletion of either tcsD or tcsB in the f kbM deletion mutant (ΔtcsD−ΔfkbM strain and ΔtcsB−ΔfkbM strain), respectively, produced the new analogue 31-O-demethyl-36,37dihydroFK506 (9) (Figure 3e; Table S2; Figures S10 and S11) and 31-O-demethylFK520 (10) (Figure 3f; Table S2; Figures S12 and S13), which has been reported from the biotransformation of FK506 by Actinoplanes sp. ATCC 53771.13 Again, a trace amount of 31-O-demethylFK523 was produced from the ΔtcsB−ΔfkbM strain, which was sufficient only for UPLC-qTOF-HR-MS detection (Figure 3f; Figure S12). The 1H, 13C, and gHSQC NMR spectra in CDCl3 (Figure S11a,b,d) of 9 indicated two ketone carbons (δC 213.8; δC 196.5), two carbonyl carbons (δC 169.4; δC 165.1), two olefinic methine signals (δH 5.09/δC 130.0; δH 5.03/δC 123.6), two olefinic nonprotonated carbons (δC 138.8; δC 132.6), a hemiketal carbon (δC 97.4), seven oxymethine signals (δH 5.33/δC 77.8; δH 3.92/δC 70.4; δH 3.68/δC 73.2; δH 3.59/δC

75.8; δH 3.39/δC 75.2; δH 3.39/δC 74.0; δH 3.36/δC 75.6), two methoxy signals (δH 3.30/δC 57.3; δH 3.40/δC 56.6), two singlet methyl signals (δH 1.62/δC 14.4; δH 1.59/δC 16.2), three doublet methyl signals (δH 1.00/δC 16.5; δH 0.93/δC 20.8; δH 0.87/δC 9.8), and a triplet methyl signal (δH 0.90/δC 14.4), displaying features typical of an FK506 analogue. Further interpretation of gCOSY and gHMBC data (Figure S11c,e) showed that 9 possesses a pipecolate moiety, a propyl chain, and a cyclohexane moiety. The HMBC spectrum gave correlations from H-43 (δH 3.40) and H-44 (δH 3.30) to C-13 (δC 74.0) and C-15 (δC 75.2), respectively, indicating the two methoxy groups were positioned at C-13 and C-15. 9 was confirmed as 31-O-demethyl-36,37-dihydroFK506 bearing a propyl group at C-35−C-37 and a hydroxy group instead of a methoxy group at C-31. Deletion of both f kbD and f kbM (ΔfkbDM strain) produced the known biosynthetic intermediate 5 and 9-deoxo-31-Odemethyl-prolylFK506 (11) (Figure 3g; Table S2; Figures S14−S16). 1H, 13C, and gHSQC NMR spectroscopic data in CDCl3 of 11 (Figure S16a,b,d) indicated a ketone carbon (δC 214.0), two carbonyl carbons (δC 173.0; δC 170.1), exomethylene signals (δH 5.00 and 5.03/δC 116.8), three olefinic methine signals (δH 5.72/δC 135.8; δH 5.01/δC 129.8; δH 4.99/ δC 122.0), two olefinic nonprotonated carbons (δC 141.2; δC 132.6), a hemiketal carbon (δC 98.7), seven oxymethine signals (δH 5.18/δC 78.3; δH 4.04/δC 69.2; δH 3.85/δC 71.1; δH 3.54/ δC 77.4; δH 3.44/δC 75.3; δH 3.43/δC 74.7; δH 3.35/δC 75.8), two methoxy signals (δH 3.38/δC 56.5; δH 3.37/δC 58.0), two singlet methyl signals (δH 1.67/δC 15.9; δH 1.65/δC 14.4), and three doublet methyl signals (δH 0.96/δC 17.2; δH 0.90/δC 10.1; δH 0.77/δC 19.1), illustrating typical features of an FK506 analogue. Further interpretation of gCOSY and gHMBC data (Figure S16c,e) showed that 11 possesses a proline moiety (δH 4.36/δC 59.1; δH 1.97 and 2.19/δC 29.4; δH 1.96 and 1.98/δC 24.9; δH 3.55 and 3.64/δC 47.6), a propylene chain (δH 2.25 and 2.45/δC 35.8; δH 5.72/δC 135.8; δH 5.00 and 5.03/δC 116.8), and a cyclohexane moiety (δH 2.35/δC 35.2; δH 1.14 and 1.90/δC 39.4; δH 3.35/δC 75.8; δH 3.44/δC 75.3; δH 1.36 and 1.98/δC 32.3; δH 1.06 and 1.61/δC 31.2). The long-range HMBC correlations from a methylene signal at H-9 (δH 2.56 and 2.63) to a carbonyl group at C-8 (δC 173.0) and a hemiketal carbon at C-10 (δC 98.7) allowed the establishment of the methylene at C-9. The HMBC correlations from the proton of a methine H-21 (δH 3.38) to the two carbons of an olefin group C-19 and C-20 (δC 141.2; δH 4.99/δC 122.0), a ketone (δC 214.0), and a methylene carbon (δC 35.8) and an olefinic methine carbon (δC 135.8) of propylene indicated a propylene chain on C-21. The proton singlets H-43 (δH 3.38) and H-44 (δH 3.37) of two methoxy groups showed HMBC signals connecting them to C-13 (δC 74.7) and C-15 (δC 77.4), respectively. 11 was confirmed as an FK506 analogue with a methylene at C-9 and a hydroxy group at C-31. Introduction of a tcsD deletion to the f kbD−f kbM double mutant (ΔtcsD−ΔfkbDM strain) yielded 9-deoxo-31-Odemethyl-36,37-dihydroFK506 (12), a new compound (Figure 3h; Figure S17). Detailed analysis of 1D and 2D NMR spectra including COSY, HSQC, and HMBC (Table S2; Figure S18) disclosed that the signals of 12 were also very similar to those of 6, and the structure of compound 12 was established as a congener of FK506 bearing a propyl group at C-35−C-37 and a hydroxy group instead of a methoxy group on C-31. The long-range HMBC correlations from the two methoxy groups H-43 (δH 3.35) and H-44 (δH 3.35) to C-13 (δC 74.7) and CD



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15 (δC 77.2), respectively, indicated two methoxy groups placed at C-13 and C-15. The tcsB−f kbD−f kbM triple mutant (ΔtcsB−ΔfkbDM strain) produced the new analogues 9-deoxo-31-O-demethyFK520 (13) and 9-deoxo-31-O-demethyFK523 (14) (Figure 3i; Table S2; Figures S19−S22). Compound 13 was elucidated as an FK506 analogue with an ethyl chain replacing a propyl chain at C-21 through comprehensive analysis of 1D and 2D NMR spectra including COSY, HSQC, and HMBC (Figure S20). The HMBC correlations from the proton of a methine H-21 (δH 3.20) to the two carbons of an olefin group C-19 and C-20 (δC 141.3; δC 122.3), the ketone C-22 (δC 215.4), the methylene carbon C-35 (δC 25.1), and the methyl carbon C-36 (δC 12.0) of an ethyl group were observed in 13, indicating an ethyl chain on C-21. The 1H, 13C, and gHSQC NMR spectroscopic data (Table S2; Figure S22a,b,d) of 14 were similar to those of 8 or 13 except for an absence of a methoxy signal at C-31 of 8. In particular, the HMBC correlations from the proton of methine H-21 (δH 3.21) to the two carbons C-19 and C-20 (δC 140.4; δC 123.5) of an olefin group, a ketone (δC 215.4), and a methyl carbon (δC 17.1) explained the existence of a methyl group at C-21. The two methoxy groups H-41 (δH 3.37) and H-42 (δH 3.37) showed HMBC signals connecting them to C-13 (δC 74.6) and C-15 (δC 77.1), respectively. The structure of 14 was confirmed as a new FK523 analogue having a methylene at C-9, a hydroxy group at C-31, and a methyl group at C-35. Taken together, we obtained seven new FK506 analogues (6, 8, 9, and 11−14) by a combined gene deletion approach. Immunosuppressive and Antifungal Activities of FK506 Analogues. To assess the immunosuppressive activity, CD3/CD28-stimulated CD4+ T cell proliferation was analyzed after 72 h of exposure to FK506 and its analogues. When FK506 and its analogues were listed in order of immunosuppressive activity, we found that the 50% inhibitory concentration (IC50) increased as the structural modifications were made at more diverse positions. This was true except for the analogues containing the proline ring or methyl side chain at C21. FK506 was the most potent (0.034 nM), followed by derivatives modified at a single position (3: 19-fold reduction, 4: 9-fold reduction,), two positions (5: 559-fold reduction, 6: 946-fold reduction, 7: 925-fold reduction, 9: 64-fold reduction, 10: 121-fold reduction), and three positions (12: 1699-fold reduction, 13: 9559-fold reduction). In general, the reduction in immunosuppressive activity of compounds with shorter C21 side chains was greater than that of the compounds with longer chains, and the combined modification at the C-9 and C-21 positions is more effective in reducing immunosuppressive activity than the modification at the C-21 and C-31 positions. The level of immunosuppressive activity dropped significantly for compounds containing a proline moiety (2: 9937-fold reduction, 11: 3.3 × 104-fold reduction) as well as compounds with a C-21 methyl side chain (8: 7.4 × 104-fold reduction, 14: 1.3 × 105-fold reduction) (Table 1; Figure S23). Taken together, the immunosuppressive activity of each analogue was significantly lower than that of FK506. The in vitro antifungal activity of the analogues against three major pathogens, C. neoformans H99 strain,14 C. albicans SC5314 strain,15 and A. f umigatus Af293 strain16 (Table 1; Figures S24−S27) was determined. FK506 exhibited the lowest IC50 value (0.001, 0.005, and 0.015 μg/mL) against C. neoformans, C. albicans, and A. f umigatus, respectively. Compared with the previously reported 5 modified at the C-

Table 1. Evaluation of FK506 Analogue Activities IC50 (μg/mL)

compound FK506 (1)c 2c 3c 4c 5c 6 7 8 9 10 11 12 13 14

50% immunosuppression (ng/mL)

Cryptococcus neoformansa

Candida albicansb

Aspergillus f umigatusa

0.027

0.001

0.005

0.015

268.3 0.513 0.246 15.09 25.55 24.98 1985.0 1.723 3.273 886.5 45.87 258.1 3378.0

6.112 0.036 0.005 0.154 0.151 0.355 2.466 0.033 0.027 221.1 8.846 3.297 54.54

322.0 0.963 0.106 2.796 0.817 0.662 6.073 0.059 0.079 3444.0 4.264 2.847 837.1

>50.0 24.19 0.165 29.77 14.07 7.822 230.4 0.415 12.21 >50.0 >50.0 306.8 >50.0

a

Assay performed using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. b2,3Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) reduction assay. cIC50 value of the compounds reported in ref 11.

9 and C-31 positions, the derivatives containing C-9 and C-21 modifications, such as 6 and 7, were generally more effective in retaining the antifungal activity. This was true except for 8 (which has a methyl side chain at C-21), which showed significantly reduced antifungal activity against all three pathogens, and 7 exhibited relatively weaker activity against C. neoformans compared to 5. Notably, compared with 5 and 6, 7 was characterized by a lower IC50 value against C. albicans and A. f umigatus, while the IC50 value for immunosuppressive activity of 7 was approximately 1.7-fold higher than that of 5. On the other hand, 6 exhibited stronger antifungal activity than 7 against C. neoformans. In general, compared with 6 and 7, analogues 9 and 10 modified at the C-21 and C-31 positions exhibited considerably higher antifungal activity, except 10, which exhibited relatively weaker activity against A. f umigatus, but also maintained high immunosuppressive activity. The fungal growth of C. albicans was inhibited by 50% at 0.059 μg/ mL of 9 and 0.079 μg/mL of 10. A similar growth-suppression trend was observed for A. f umigatus (9: 0.415 μg/mL, 10: 12.21 μg/mL). In contrast, 10 presented a lower IC50 value (0.027 μg/mL) than 9 (0.033 μg/mL) against C. neoformans. Modifications at the C-9, C-31, and C-21 positions (compounds 12−14) greatly reduced not only inhibition of T cell proliferation (1.7 × 103- to 1.3 × 105-fold reduction) but also antifungal activity (>500-fold reduction). Particularly, 14, containing a methyl side chain, did not show an inhibitory effect, even at high doses, as observed with 8 (Figures S24− S27). The present observation is supported by the ternary structure of the FKBP12−FK506−CaN complex, whereby the C-21 allyl side chain binds to the hydrophobic groove between the catalytic A subunit and the Ca2+-binding regulatory B subunit of CaN.4,17 The analogues substituted with a proline also exhibited significant reduction in both immunosuppressive and antifungal activities (>9000-fold and >6000-fold, respectively). This is consistent with previous reports that identified the pipecolate of FK506 fitting the hydrophobic pocket of FKBP12.18,19 Taken together, the combination of modification E



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Figure 4. Synergistic antifungal activity of FK506 analogues with fluconazole (FCZ) against Cryptococcus neoformans H99 strain. Checkerboard assay for determining fractional inhibitory concentration (FIC) index between FK520, 7, 8, or 13 and fluconazole. C. neoformans cells grown in YPD medium at 30 °C overnight were inoculated in liquid RPMI 1640 medium at a cell density of 0.01 OD600 cells/mL. FK520, 7, 8, and 13 were 2-fold serially diluted from the indicated concentrations.

8, and 13; Figure S28), consistent with a previous study showing that the Δf rr1 mutant conferred FK506 resistance.21 These results indicate that, similar to FK506, the analogues target FKBP12 in C. neoformans. Although the production levels of the new analogues obtained in this study were significantly lower than that of FK506 from the wild-type strain, further efforts including metabolic engineering of the precursor supply and biosynthetic pathway, metabolic model-aided genome editing using the CRISPR/Cas9 system, optimization of culture conditions, and heterologous production of the analogues in high-producing industrial strains will improve the production of valuable analogues.22,23 Finally, application of the simple strategy altering post-PKS tailoring steps toward various polyketide biosynthetic pathways that include multiple post-PKS steps will facilitate the biosynthesis of natural product analogues with therapeutic potentials.

at C-9 and C-21 was found to be more effective at retaining antifungal activity than immunosuppressive activity. This is especially true for 7 (with a hydrogen at C-9 and ethyl side chain at C-21 position), which exhibited significant antifungal activity against each of the tested fungal pathogens, while its IC50 value for immunosuppression was 925-fold lower than that of FK506. The synergistic effects of analogues 7, 8, and 13 with fluconazole, an azole drug widely used as an antifungal agent against C. neoformans H99, were also investigated. A previous study has shown that FK506 has a synergistic effect with azole compounds.20 Compound 7 was selected based on its high potential as a new antifungal agent against a broad spectrum of fungal pathogens. 8 and 13 were chosen because they showed significantly reduced immunosuppressive activities but exhibited a certain degree of specific activities against C. neoformans and C. albicans. To determine the fractional inhibitory concentration (FIC) index, a checkerboard assay was performed to analyze synergism between compounds 7/8/ 13 and fluconazole (Figure 4). FIC index values of 7/8/13 with fluconazole were 0.19, 0.5, and 0.19, respectively, which were comparable to those of FK506 (0.38)11 and FK520 (0.50) with fluconazole, suggesting that synergism exists between 7/8/13 and fluconazole. Notably, in the presence of 2.5 μg/mL fluconazole, the IC50 of 7 could be lowered to 0.031 μg/mL, which was close to the IC50 value of immunosuppression (0.025 μg/mL). Moreover, in the presence of 1.25 μg/mL fluconazole, the IC50 values of 8 and 13 could be lowered to 0.125 and 0.0625 μg/mL, respectively, which were lower than their IC50 values of immunosuppression (1.985 and 0.258 μg/mL, respectively). Collectively, these data further suggest that combination therapy with 7/8/13 and fluconazole could be clinically more useful than monotherapy. To verify whether the antifungal activity of 7/8/13 is still mediated through inhibition of the FKBP12−CaN pathway, the susceptibility of the C. neoformans FKBP12 mutant (the Δf rr1 mutant) to the FK506 analogues was evaluated. The Δf rr1 mutant was completely resistant to FK506 analogues (7,

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were acquired using a Varian INOVA 500 spectrometer (Palo Alto, CA, USA) operating at 500 MHz for 1H and 125 MHz for 13C nuclei. Samples for NMR analysis were prepared by dissolving each compound in 250 μL of CDCl3 and placing the solutions in 5 mm Shigemi Advanced NMR microtubes matched to the solvent. All NMR data processing was done using Mnova software (Mestrelab Research S.L., Santiago de Compostela, Spain). UPLC-qTOF-HR-MS analysis of the FK506 analogues generated from several deletion mutants was performed on a Waters XEVO G2S Q-ToF mass spectrometer coupled with a Waters Acquity UPLC system equipped with an Xbridge C18 column (2.1 × 100 mm, 3.5 μm) consisting of an Acquity I-Class system. A gradient elution using solvent A (20% MeCN(aq)) and solvent B (MeCN) as the mobile phase at a flow rate of 0.43 mL/min at 40 °C was applied. Construction of Mutants and Culture Conditions. The tcsB, tcsD, f kbD, and fkbM genes were inactivated in the FK506-producing strain Streptomyces sp. KCTC 11604BP by in-frame deletion via double-crossover homologous recombination. Details regarding DNA manipulation and construction of plasmids for gene deletion and the resulting mutant strains are described in the Supporting Information F



Article

scatter area) and SSC-A (side scatter area) using the MagniSort Mouse CD4 T cell enrichment kit (eBioscience). To extract the FSCA and SSC-A from lymphocyte populations, spleens were dissected from B6J mice of 6−8 weeks of age. Before red blood cells were lysed using ammonium-chloride-potassium lysing buffer (Gibco), spleens were ground into single cells using a cell strainer. CD4+ T cells were activated by Dynabeads mouse T-activator CD3/CD28 for T-cell expansion (Gibco) and incubated for 72 h in 96-well U-bottom culture plates before surface staining and flow cytometry analysis.27 CD4+ T cells were stained with a CellTrace Violet cell proliferation kit (Molecular Probes) on the day of isolation to monitor generations of proliferating T cells. The surface of cells was dyed with CD4 markers using Brilliant Violet 605 anti-mouse CD4 antibodies (RM4-5 Clone) (Biolegend) and CD44 using the activation marker CD44 FITC (BD Biosciences) after 72 h of incubation. For evaluation of cytotoxicity, the LIVE/DEAD fixable near-IR dead cell stain kit (Molecular Probes) was used. Flow cytometry was performed using a BD FACS CANTO II flow cytometer (BD Biosciences). IC50 curves were generated using the software Prism. Minimum Inhibitory Concentration Assay. All cultures and materials for evaluation of the half-maximal inhibitory concentration (IC50) values and checkerboard assays were prepared by following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, except for assessing the IC50 in C. albicans using an XTT reduction assay in the presence of 100% fetal bovine serum (FBS; see below). C. neoformans H99 strain grown in YPD medium at 30 °C for 20 h was inoculated in RPMI liquid medium (8.4 g/L RPMI1640, 34.5 g/L MOPS, and 20 g/L dextrose, pH 7) at cell densities of 0.01 optical density at 600 nm (OD600) in 96-well microdilution plates.28 For A. f umigatus Af293, 105 conidia/mL were inoculated in the RPMI liquid medium. Then the resuspended cells were added with 2-fold serially diluted FK506 analogues (from 10 μg/ mL), which results in a final 100 μL of suspension per well. The microplates were further incubated at 35 °C (for A. f umigatus) or 37 °C (for C. neoformans) for 48 h, and OD600 for each well was measured with an iMark microplate absorbance reader. Relative growth (% growth) in each well was measured in comparison with the average growth from noninhibitor control wells after subtracting an average RPMI medium only background value. IC50 was calculated as previously described.28,29 XTT Reduction Assay. C. albicans SC5314 strain was cultured in YPD liquid medium at 30 °C for 16 h, washed twice with phosphatebuffered saline (PBS), and resuspended in PBS, and their cell concentration was measured by using automated cell counter TC10 (Bio-Rad). Then cells were inoculated into each well of a 96-well plate at a concentration of 1.25 × 105 cells in 100 μL of 100% FBS containing 2-fold serially diluted FK506 or its analogues from 10 μg/ mL and incubated at 37 °C for 24 h.30,31 To prepare the XTT solution, 500 μg/mL XTT sodium salt (Sigma) dissolved in PBS was added with menadione (final concentration: 10 μM). A 100 μL amount of the XTT solution was added to each well and incubated in the dark at 37 °C for 2 h. Then cell viability was measured using an iMark microplate absorbance reader at OD490 after shaking for 60 s.32 Evaluation of Fractional Inhibitory Concentration Index. To determine interactions between the antifungal agents being tested by this study, the FIC index value was calculated for each strain as previously described.29 An FIC of ≤0.5 indicates synergism between two agents, an FIC of >0.5 and ≤1.0 indicates an additive effect, an FIC of >1.0 and ≤2.0 indicates no interaction, and an FIC of >2.0 indicates antagonism. Growth Inhibition Assays on Solid Medium. To qualitatively evaluate the growth inhibition of A. f umigatus by FK506 analogues, YPD solid medium containing 1 μg/mL of FK506 or its analogues was spotted with 5000 spores/2 μL, incubated at 30 °C for 2 d, and photographed.33

(see also Table S1). Spores of gene deletion mutants were generated on an ISP4 agar plate,24 and seed culture was prepared in R2YE broth.25 The seed culture of deletion mutants was prepared in a 250 mL baffled flask containing 50 mL of R2YE, and then 10 mL of vegetative cells grown in the seed culture was inoculated into a 3 L baffled flask containing 1 L of the same medium as described above. The production cultures were grown on an orbital shaker (set at 180 rpm) for 5 d at 28 °C. Extraction and Isolation. The mutant strains (Table S1) were cultured in production media and processed separately. The individual culture broths (25 L) of each strain were extracted twice using EtOAc, and the EtOAc layers of each culture were combined and concentrated in vacuo. Each EtOAc extract was reconstituted in 5 mL of methylene chloride (MC) and loaded onto a SiO2 column that had been previously conditioned with 12 mL of MC to be eluted with a gradient of MC−MeOH (100:0, 100:1, 10:1, 1:1, and only MeOH, 100 mL of each volume), resulting in five fractions. The whole fractions were evaporated to dryness at room temperature by an EYELA rotary vacuum evaporator (Tokyo Rikakikai Co., Tokyo, Japan) and then kept in a freezer. The third and fourth fractions were subjected to prep-HPLC and eluted with isocratic MeCN(aq) (45%, 50%, or 55%) as the mobile phase at a flow rate of 4 mL/min. PrepHPLC was performed with a preparative Phenomenex Luna 5 μm C18(2) 100 Å column (10 × 250 mm, 5 μm) on an ACME 9000 HPLC system (YL Instrument Co., Anyang, Korea) consisting of an SP930D gradient pump coupled with a UV730D UV detector set to 205 nm and a CTS30 column oven set to 40 °C. The injection volume was 100 μL. Finally, 9-deoxo-36,37-dihydroFK506 (6) (9.7 mg), 9-deoxoFK520 (7) (7.8 mg/L), 9-deoxoFK523 (8) (8.3 mg), 31-O-demethyl-36,37-dihydroFK506 (9) (20.6 mg), 31-O-demethylFK520 (10) (11.2 mg/L), 9-deoxo-31-O-demethyl-prolylFK506 (11) (11.0 mg), 9-deoxo-31-O-demethyl-36,37-dihydroFK506 (12) (12.1 mg), 9-deoxo-31-O-demethylFK520 (13) (8.7 mg), and 9deoxo-31-O-demethylFK523 (14) (8.0 mg) were isolated as pure compounds. Approximately 125 mg of FK506 could be isolated from a 25 L culture of the wild-type Streptomyces sp. KCTC 11604BP strain. 9-Deoxo-36,37-dihydroFK506 (6): amorphous, white powder; 1H and 13C NMR data, see Table S2 and Figure S4; (+)-HRESIMS m/z 814.5086 [M + Na]+ (calcd for C44H73NNaO11+, 814.5076), see Figure S3. 9-DeoxoFK523 (8): amorphous, white powder; 1H and 13C NMR data, see Table S2 and Figure S8; (+)-HRESIMS m/z 786.4771 [M + Na]+ (calcd for C42H69NNaO11+, 786.4763), see Figure S7. 31-O-Demethyl-36,37-dihydroFK506 (9): amorphous, white powder; 1H and 13C NMR data, see Table S2 and Figure S11; (+)-HRESIMS m/z 814.4708 [M + Na] + (calcd for C43H69NNaO12+, 814.4712), see Figure S10. 9-Deoxo-31-O-demethyl-prolylFK506 (11): amorphous, white powder; 1H and 13C NMR data, see Table S2 and Figure S16; (+)-HRESIMS m/z 784.4623 [M + Na]+ (calcd for C42H67NNaO11+, 784.4606), see Figure S15. 9-Deoxo-31-O-demethyl-36,37-dihydroFK506 (12): amorphous, white powder; 1H and 13C NMR data, see Table S2 and Figure S18; (+)-HRESIMS m/z 800.4928 [M + Na]+ (calcd for C43H71NNaO11+, 800.4919), see Figure S17. 9-Deoxo-31-O-demethylFK520 (13): amorphous, white powder; 1 H and 13C NMR data, see Table S2 and Figure S20; (+)-HRESIMS m/z 786.4766 [M + Na]+ (calcd for C42H69NNaO11+, 786.4763), see Figure S19. 9-Deoxo-31-O-demethylFK523 (14): amorphous, white powder; 1 H and 13C NMR data, see Table S2 and Figure S22; (+)-HRESIMS m/z 772.4601 [M + Na]+ (calcd for C41H67NNaO11+, 772.4606), see Figure S21. Evaluation of the Immunosuppressive Activity by FK506 Analogues. The immunosuppressive activity of FK506 analogues was assessed by measuring their effect on the viability and proliferation of CD4+ helper T cells, which are essential for adaptive immunity by the release of cytokines activating other immune cells.26 The CD4+ helper T cells were isolated form the FSC-A (forward G



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00144. Experimental details; tables for bacterial strains and plasmids used in this study; and qTOF-HR-MS and 1D and 2D NMR spectra of 2−14 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +82-2-2123-5885. Fax: +82-2-2123-8284. E-mail: [email protected]. *Tel: +82-2-2123-5558. Fax: +82-2-362-7265. E-mail: [email protected]. *Tel: +82-2-3277-4446. Fax: +82-2-3277-3419. E-mail: [email protected] ORCID

Kyung-Tae Lee: 0000-0002-0068-6220 Myoung Chong Song: 0000-0002-3658-025X Sang-Jip Nam: 0000-0002-0944-6565 Yeo Joon Yoon: 0000-0002-3637-3103 Author Contributions ∥

J.Y.B., J.A.J., K.-T.L., and A.H. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) grants funded by the Ministry of Science and ICT (MSIT) (2019R1A2B5B03069338; 2016R1E1A1A01943365; 2018R1A5A1025077; 2017R1A2B3011098; 2017M3C7A1023471), the Bio & Medical Technology Development Program of the NRF funded by the MSIT (2018M3A9F3079662), a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI18C1664), and the Strategic Initiative for Microbiomes in Agriculture and Food funded by Ministry of Agriculture, Food and Rural Affairs (918012-4), Republic of Korea. J.Y.B. was the recipient of the Ewha Womans University scholarship of 2015. K.-T.L. was also supported by the Yonsei University Research Fund (Yonsei Frontier Lab Young Researcher Supporting Program) of 2018.

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