Discovery of Druggability-Improved Analogues by Investigation of the

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Discovery of Druggability-Improved Analogues by Investigation of the LL-D49194α1 Biosynthetic Pathway Lei Dong,†,§ Yi Shen,‡ Xian-Feng Hou,‡ Wen-Jun Li,*,†,§ and Gong-Li Tang*,‡ †

Org. Lett. Downloaded from pubs.acs.org by EAST CAROLINA UNIV on 03/11/19. For personal use only.

State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China ‡ State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences (CAS), CAS, Shanghai 200032, China § Southern Laboratory of Ocean Science and Engineering (Guangdong, Zhuhai), Zhuhai 519000, China S Supporting Information *

ABSTRACT: The biosynthetic gene cluster of antitumor antibiotic LL-D49194α1 (LLD) was identified and comparatively analyzed with that of trioxacarcins. The tailoring genes encoding glycosyltransferase, methyltransferase and cytochrome P450 were systematically deleted, which led to the discovery of eight compounds from the mutants. Preliminary pharmaceutical evaluation revealed two intermediates exhibiting higher cytotoxicity, stability and solubility. These results highlighted the modification pathway for LLD biosynthesis, and provided highly potent, structurally simplified “unnatural” natural products with improved druggability.

LL-D49194α1 (LLD, 1) and LL-D49194β2 (2) are naturally occurring cytotoxic antibiotics isolated from Streptomyces vinaceusdrappus.1 Structurally, they share a common highly oxygenated, polycyclic rigid skeleton with another natural product, trioxacarcin A (TXN-A), but differ in glycosylation and methylation modifications at C13-OH and C16-OH (Figure 1). The core structure is featured by a modified

Unfortunately, the unexpected cardiotoxicity hindered further clinical investigations.6 Due to the promising potency in anticancer drug development and the complex chemical structure, efforts to obtain analogues by chemical synthesis have continued.7−10 Although much progress has been achieved, processing such a complex architecture is still a challenging task. Recently, biosynthetic studies of TXN-A not only set up the primary biosynthetic pathway but also generated a series of analogues with high antitumor activity.11,12 Herein, the biosynthetic gene cluster (BGC) of LLD was identified and compared with that of TXNA. By inactivation of the tailoring steps, we obtained eight analogues including intermediates and shunt products, which revealed a proposed biosynthetic pathway. Importantly, two structurally simpler analogues exhibit higher activity and improved druggability, which might serve as potent anticancer agents for further drug development. To locate the entire BGC of LLD, the genome of S. vinaceusdrappus NRRL 15735 was sequenced and a BGC encoding type II polyketide synthase (PKS) with complex tailoring enzymes was subsequently identified (lld, GenBank: MK501817). As expected, most of the genes in this cluster are homologous to the corresponding genes in the txn cluster, except for the additional genes encoding one transporter (LldRr1), one dioxygenase (LldO1), one glycosyltransferase (GT, LldB4), and one methyltransferase (MT, LldM4) (Figure 2A, Table S3). Beyond these, the lld cluster also encodes all the

Figure 1. Structures of LL-D49194α1 (LLD, 1), LL-D49194β2 (2) and trioxacarcin A (TXN-A). The sugar moieties are boxed and defined as S1−S4.

anthraquinone fused with a polyoxygenated 2,7-dioxabicyclo[2,2,1]heptane, which bears a fused spiroepoxide as a pharmacophore for alkylating DNA.2−4 Significantly, these compounds display remarkable antitumor activity and impressive preclinical manifestation,5 which spurred a phase I clinical study of LLD in 15 patients during the early 1990s.6 © XXXX American Chemical Society

Received: February 18, 2019

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DOI: 10.1021/acs.orglett.9b00610 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 2. LLD biosynthesis. (A) Comparation of the biosynthetic gene clusters of LLD and TXN-A. (B) Proposed biosynthetic pathway.

series of new compounds (Figure 3 III−V). After large-scale fermentation, these newly generated metabolites were isolated and structurally elucidated by LC-MS, NMR, and comparation with 1 and 2 (Figures S2−S5). The lldB4-deletion mutant (ΔlldB4) produced one sugar (S2 at C16-OH)-containing intermediate 7 (Table S4 and Figures S6−S11) and two sugars (S2 at C16-OH and S1 at C4-OH)-containing compound 8 (Figure 3-IV; Table S5 and Figures S12−S13), which suggested that GT LldB4 may be responsible for loading the third sugar moiety (S3) onto C4′−OH (Figure 2B). Meanwhile, the generation of 7 and 9 (Table S6 and Figures S14−S19) by lldB5-deletion mutant (ΔlldB5) hinted that GT LldB5 should function as transferring sugar S1 on C4-OH (Figures 3-V and 2B). In addition, the lldB3-inactivation mutant (ΔlldB3) produced an intermediate 5 (Figure 3-III; Table S7 and Figures S20−S25) without any glycosylation modification, which not only showed that GT LldB3 likely catalyzes sugar S2 loading at C16-OH but also revealed this step precedes both glycosylation reactions by LldB4 and LldB5 (Figure 2B). Further comparison of 5 with the polycyclic skeleton of LLD clearly showed the differences in the ring C of anthraquinone, lacking C2-OH and

enzymes for the deoxysugar pathway including two-component pyruvate dehydrogenase like enzymes (LldB9/B10), which are requested in TXN-A biosynthesis for attaching a two-carbon side chain to yield γ-branched octose but not needed in LLD. Further analysis showed that both LldB9 and LldB10 are truncated compared with their homologous proteins TxnB3 and TxnB4. All collective information highly matched with the requests of biosynthesis of LLD, which needs more GT and MT for additional sugars (S2 and S3) and methylation at C13-OH but lacks the γ-branched octose (S4) (Figure 1). To verify the located BGC is genetically correlated with LLD biosynthesis, the lldA1 gene, encoding a ketosynthase (KS), which is the key enzyme for type II PKS, was inactivated by gene replacement to yield mutant S. vinaceusdrappus TG5019 (Figure S1). This mutant completely abolished the production of 1 and 2 (Figure 3-II), which unambiguously demonstrated that the lld cluster governs the biosynthesis of LLD. Considering the different glycosylation pattern between LLD and TXN, we next constructed the gene-inactive mutants for each of the three GTs to define their roles (Figure S1). All the mutants did not produce 1 or 2 anymore, but accumulated a B

DOI: 10.1021/acs.orglett.9b00610 Org. Lett. XXXX, XXX, XXX−XXX

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After determining the roles of GTs and MTs, subsequent efforts were directed toward the cytochrome P450 oxidases. The null production of any of the intermediates by lldO6 or lldO7inactivated mutant (ΔlldO6 or ΔlldO7) (Figure 3-XI, XII) indicated that they should be involved in the generation of the polycyclic rigid core. While the ΔlldO2 mutant produced 10, which contains two sugars at C16-OH but lacks tailoring modifications of anthraquinone ring C (Figure 3X; Table S10, Figures S38−S43), the ΔlldO10 mutant produced 4, an earlier intermediate lacking a hydroxy group at C16 and a methyl group at C10-OH (Table S11, Figures S44−S49). Collecting all the genetic evidence, we now could propose an approximate tailoring pathway of LLD as depicted in Figure 2B. Similar to TXN-A biosynthesis, the type II PKS system might generate parimycin (3) first using 2-methylbutyryl-CoA as a starter unit derived from L-isoleucine, and LldC3, the homologue of TxnO9, catalyzing heterocyclization/dehydration for anthraquinopyrone formation.12,13 Then, a series of oxidases, including cytochrome P450s LldO6 and LldO7, and MT LldM4 participate in the generation of intermediate 4, which was methylated and hydroxylated by LldM3 and LldO10 to afford 5. Next, after further methylation and glycosylation by LldM2 and LldB3, followed by LldO2-catalyzed hydroxylation and LldO3governed ketone reduction, intermediate 7 was generated. Subsequently, LldB4 and LldB5 mediated further glycosylation steps to yield final product LLD. With eight compounds in hand, we next evaluated the cytotixic activity by comparing with 1 and 2 (Figure S50), and the results were summarized in Table 1. Surprisingly, the new Table 1. Summary of Cytotoxic Activities against Cancer Cellsa

Figure 3. HPLC (UV at 400 nm) analysis of fermentation broth produced by S. vinaceusdrappus (WT) strain and the mutants. * LLDunrelated peaks.

IC50 (nM)

reduction of the ketone group at C4 (Figure 2B). This result is unexpected, which indicated that the C4-ketone reduction into the hydroxy group and hydroxylation reaction at C2 most likely occur after the glycosylation at C16-OH by LldB3 (Figure 2B). Next, the four MTs were individually inactivated to investigate their functions in LLD biosynthesis (Figure S1). Either lldM3 or lldM4-deletion mutant (ΔlldM3 or ΔlldM4) lost the ability to produce 1/2 or any related metabolites (Figure 3VIII, IX), which means both of them are likely involved in the early stage for skeleton formation. We therefore tentatively assigned LldM4 for the methylation at C13-OH and LldM3 for C10-OH based on the comparative analysis with TXN-A biosynthesis (Figure 2B), which only bears a methyl group at C10-OH and a sugar moiety at C13-OH (Figure 1). Similar to ΔlldB3 mutant, the production of intermediate 5 in ΔlldM2 mutant suggested that LldM2 is responsible for methylation of C16-OH, and this step should occur prior to the modification of anthraquinone ring C (Figure 2B). Two new metabolites (11 and 12) were produced by mutant ΔlldM1 (Figures 3-VI and 2B), both of which were subsequently isolated and structurally characterized by NMR and HRMS analysis (Tables S8−S9, Figures S26−37). They contain one or two C3′-demethylsugar moieties at C16-OH, but both lose sugar (S1) at C4-OH. The production of new derivatives 11 and 12 by lldM1-inactivated mutant not only demonstrated that LldM1 catalyzes the Cmethylation in sugar pathway (Figure 2B) but also revealed that GTs LldB3 and LldB4 are more tolerant to sugar donors than LldB5.

compound

HL-60

B16-F10

1 2 4 5 7 8 9 10 11 12

5.45 ± 0.01 11.93 ± 0.01 2.84 ± 0.01 3.93 ± 0.01 >100 57.0 ± 5.0 >100 6.32 ± 0.01 >100 >100

23.88 ± 0.09 10.60 ± 0.03 10.05 ± 0.08 6.35 ± 0.01 >100 >100 >100 8.19 ± 0.02 >100 >100

a HL-60: Human promyelocytic acute leukemia cells. B16−F10: Mouse melanoma cells. Cell Counting Kit-8 method was used to assay the cytotoxicity activity.

compounds 4, 5, and 10 exhibit improved antitumor activity against both human promyelocytic acute leukemia cells HL-60 and mouse melanoma cells B16-F10. These three compounds share the same structure of anthraquinone ring C, which suggested that the modification on this ring has no effect on biological activity. Next, these analogues were estimated for optional drug-like properties by a computational approach on XLOGP3 (Table S12),14 which clearly showed that the simpler compounds 4 and 5 exhibit better performance than 1 in drug discovery based on ‘the rule of 5’ proposed by Lipinski.15 Given the fact that this family of antibiotics displays a potent antiproliferative effect depending on the expected spiroepoxide group, which covalently alkylates DNA with guanine (G) at the N7 position,2−4 DNA modification reactions were performed. C

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With 1 as the control, both 4 and 5 were found to form an adduct reaction with a self-complementary DNA oligomer containing a G residue as illustrated by LC-MS (Figure 4A and B). The

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00610. Material and methods, supplementary tables and figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gong-Li Tang: 0000-0003-3149-4683 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful for the supporting grants from the NNSFC (21632007 and 21621002), STCSM (16ZR1443800), and the CAS (XDB20000000, 2019257 and K. C. Wong Education Foundation). W.J.L. was also supported by the Guangdong Province Higher Vocational Colleges and Schools Pearl River Scholar Funded Scheme (2014).

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REFERENCES

(1) Maiese, W. M.; Labeda, D. P.; Korshalla, J.; Kuck, N.; Fantini, A. A.; Wildey, M. J.; Thomas, J.; Greenstein, M. J. Antibiot. 1990, 43, 253. (2) Fitzner, A.; Frauendorf, H.; Laatsch, H.; Diederichsen, U. Anal. Bioanal. Chem. 2008, 390, 1139. (3) Pfoh, R.; Laatsch, H.; Sheldrick, G. M. Nucleic Acids Res. 2008, 36, 3508. (4) Š venda, J.; Hill, N.; Myers, A. G. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6709. (5) Underberg, W. J. M.; Hofman, G. A.; Lubbers, S. C. M.; Bekers, O.; Ten Bokkel Huinink, W. W.; Beijnen, J. H. J. Pharm. Biomed. Anal. 1989, 7, 1791. (6) Cassidy, J.; Graham, M. A.; Ten Bokkel Huinink, W.; McDaniel, C.; Setanoians, A.; Rankin, E. M.; Kerr, D. J.; Kaye, S. B. Cancer Chemother. Pharmacol. 1993, 31, 395. (7) Magauer, T.; Smaltz, D. J.; Myers, A. G. Nat. Chem. 2013, 5, 886. (8) Pröpper, K.; Dittrich, B.; Smaltz, D. J.; Magauer, T.; Myers, A. G. Bioorg. Med. Chem. Lett. 2014, 24, 4410. (9) Nicolaou, K. C.; Cai, Q.; Sun, H.; Qin, B.; Zhu, S. J. Am. Chem. Soc. 2016, 138, 3118. (10) Nicolaou, K. C.; Chen, P.; Zhu, S.; Cai, Q.; Erande, R. D.; Li, R.; Sun, H.; Pulukuri, K. K.; Rigol, S.; Aujay, M.; Sandoval, J.; Gavrilyuk, J. J. Am. Chem. Soc. 2017, 139, 15467. (11) Qi, L.-H.; Zhang, M.; Pan, H.-X.; Chen, X.-D.; Tang, G.-L. Youji Huaxue 2014, 34, 1376. (12) Zhang, M.; Hou, X.-F.; Qi, L.-H.; Yin, Y.; Li, Q.; Pan, H.-X.; Chen, X.-Y.; Tang, G.-L. Chem. Sci. 2015, 6, 3440. (13) Hou, X.-F.; Song, Y.-J.; Zhang, M.; Lan, W.; Meng, S.; Wang, C.; Pan, H.-X.; Cao, C.; Tang, G.-L. Angew. Chem., Int. Ed. 2018, 57, 13475. (14) Cheng, T. J.; Zhao, Y.; Li, X.; Lin, F.; Xu, Y.; Zhang, X. L.; Li, Y.; Wang, R. X. J. Chem. Inf. Model. 2007, 47, 2140. (15) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev. 2012, 64, 4. (16) Bingham, T. W.; Hernandez, L. W.; Olson, D. G.; Svec, R. L.; Hergenrother, P. J.; Sarlah, D. J. Am. Chem. Soc. 2019, 141, 657. (17) Venkataramanan, R.; Zang, S.; Gayowski, T.; Singh, N. Antimicrob. Agents Chemother. 2002, 46, 3091.

Figure 4. Comparation of the DNA-modification effects, solubility and stability. (A) HPLC and (B) LC-MS chromatograms of adduct formation reaction of the DNA oligomer d(AATTACGTAATT) (50 μM) and compounds (100 μM) after 12 h at room temperature. DNA oligomer without (I) and with compound 1 (II), 5 (III), or 4 (IV). HRESI negative mode MS spectrum affords the m/z of [M − 3H]3−. (C) Aqueous solubility assessed by miniaturized shake flask approach. (D) Stability was assessed in human liver microsomes at 37 °C for 2 h, and the remaining percentage t1 was compared to initial t0. Error is SD, n ≥ 3.

results indicate that analogue 5 bears a higher alkylation rate and efficiency during the same reaction time. We then assessed the solubility of 1 and its simpler analogues 4 and 5 using the miniaturized shake flask approach.16 Despite clinical trials, it was unexpectedly found that 1 was poorly soluble, whereas the aldehyde hydrate-containing analogue 5 shows improved aqueous solubility (5 times higher than that of 1) (Figures 4C and S51). In addition, to mimic the endoplasmic reticulum activity for these potential anticancer agents, we introduced a 10-donor human liver microsome assay in vitro.17 Both 4 and 5 were more stable in this assay compared to 1 (Figure 4D), which suggested that they may serve as better drug candidates. Taken together, the structurally simpler 4 and 5 display improved antitumor activity, solubility, and stability. Additionally, the aldehyde hydrate group of 5 could be considered as a further chemical modification point for more derivatives. In summary, we have identified and characterized the BGC of LLD, which revealed unexpected tailring orders and obtained eight newly generated analogues including three simpler members with significantly improved druggability. This study not only set the stage for deep exploration of the biosynthetic pathway and enzymatic reactions but also encourages us to further discover and develop new anticancer agents. D

DOI: 10.1021/acs.orglett.9b00610 Org. Lett. XXXX, XXX, XXX−XXX