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Priming of Azabicycle Biosynthesis in the Azinomycin Class of Antitumor Agents Shogo Mori, Keshav K. Nepal, Gilbert T. Kelly, Vasudha Sharma, Dinesh Simkhada, Vishruth Gowda, Dioscar Delgado, and Coran M. H. Watanabe Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01108 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on January 30, 2017
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Priming of Azabicycle Biosynthesis in the Azinomycin Class of Anti-tumor Agents Shogo Mori,‡ Keshav K. Nepal,‡ Gilbert T. Kelly,‡ Vasudha Sharma,‡ Dinesh Simkhada, Vishruth Gowda, Dioscar Delgado, Coran M. H. Watanabe* Department of Chemistry, Texas A&M University, College Station, TX 77843, United States Supporting Information Placeholder
ABSTRACT: The biosynthesis of the azabicyclic ring system of the azinomycin family of anti-tumor agents represents the “crown jewel” of the pathway and is a complex process involving at least 14 enzymatic steps. This study reports on the first biosynthetic step, the inroads, to the construction of the novel aziridino [1,2-a]pyrrolidine, azabicyclic core, enabling us to advance a new mechanism for azabicycle formation.
The azinomycins (Figure 1) are produced by the soil dwelling microorganism Streptomyces sahachiroi and display potent anti-cancer activities in cell culture and against murine transplantable tumors.(1-3) An early phase clinical investigation with azinomycin B 2 has also shown favorable results against 36 cases of malignant neoplasms.(4) Covalent linkages made between the two electrophilic carbons (C10 and C21) imbedded within the epoxide and aziridino[1,2a]pyrrolidine motifs of the azinomycins and the N7 positions of suitably disposed purine bases of DNA(57) are central to the biological actions of the molecule.
mechanistic underpinnings of the enzymology. The formation of the aziridino[1,2-a]pyrollidine ring, poses a fascinating biosynthetic question. While the biosynthetic operon has been sequenced and one can speculate on gene function, elucidation of the chemical logic of the pathway cannot be made until the biosynthetic origin of the azabicycle has been determined and individual genes are functionally characterized. Here we have identified the key precursor to the azabicyclic core and established AziC2 as mediating the first step in the formation of the aziridino pyrrolidine moiety. Ornithine 3 was initially envisioned to serve as a direct precursor to the azabicyclic ring system (Scheme 1). Scheme 1. Plausible biosynthetic route forming the azabicycle from ornithine O
A
N
21
20
O
O
O H N O O
O
O N H
O
O 3'
1'
4'
8'
N
O HO
Azinomycin A, 1
O 18
O H N 7 17
O O
13
5'
7'
O HO
6 N 3
OP
ATP OH
O
O [H] H 2N
OH
H NH 2
5
OH
O -OOC
2 O
OH
9
N
H
10
NH 2
O
11
O
10
[H]
COO-
OH
Azinomycin B, 2
N H
NH 2
COO-
11
[H]
N H
12
ATP
O
Figure 1. Chemical structures of the azinomycins Given the cancer relevance of these compounds, considerable effort has been expended towards total synthesis and SAR studies with azinomycin and its derivatives.(1) From a biosynthetic perspective, the dense functionalization of the natural product has spurred considerable interest to unveil the complex
NH
OH
4
NH 2
H
8
6
7
OH
O
NH
N
8
3 O
OH
OH [O]
OP
N
N
8
N H
14
13
B
O H 3N
O O
H 3N N
+
8
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15
OH
[O] N
HO
HO
16
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Radiolabeling studies with S. sahachiroi cell-free extracts had shown that 14C-ornithine labels azinomycin albeit the position(s) of label was not determined.(8) The terminal carboxylate of ornithine 3 could be reduced to the alcohol while transamination of the γ-amino group of 3 gives the aldehyde to facilitate ring closure (Scheme 1A, pathway marked in blue), resulting in 5. Phosphorylation would enable the generation of the azabicyclic ring system 7 with liberation of phosphate. Synthetic studies have shown that the 1-azabicyclo[3.1.0]-hexane ring system can be achieved in this type of fashion, i.e. initial formation of the 5-membered ring system which sets the stage for ring closure, formation of the aziridine ring system by SN2 attack and loss of a mesolate group.(9) Subsequent oxidation would give 8. Alternatively, one might envision a mechanism by which ornithine 3 is first converted to proline 12 (Scheme 1A, pathway marked in red), a well established metabolic process, to generate azabicycle 8. Exogenous feeding of dually labeled [1,2-13C] ornithine 3, however, was suggestive of incorporation at C-6 and C-7 of azinomycin (ca. 2-4%). Isolated yields were exceedingly low based upon multiple feeding attempts. Likewise, supplying either [1-13C] or [213 C]glycine 15 to S. sahachiroi cultures only resulted in scattering of label and no site specific incorporation into the azabicycle. These results were further supported by the failure of [1-13C] proline 12 to be incorporated into the natural product. These data make the notion of preforming the azabicyle ring prior to coupling to the amino acid backbone of glycine (Scheme 1B) a highly unlikely event. Likewise, while L-lysine 17 could be envisaged as a potential precursor (Scheme 2), where oxidation facilitates formation of the 5-membered ring, exogeneous feeding of [1-13C]lysine 17 to S. sahachiroi cultures failed to give convincing incorporation of Llysine. This corroborates our earlier findings with cell-free extracts.(8) Moreover, S-adenosylmethionine, by way of (13C-methyl)-methionine, fails to give sitespecific incorporation into the azinomycins.(10) Scheme 2. Plausible biosynthetic route forming the azabicycle from lysine O H 2N
O OH
H 2N
O OH
[O]
H 2N
O
17
NH 2
NH
18
NH 2
O OH
19
H 2N
OH
[O] SAM
HO
HO
N
16
Given the site-specific labeling results with labeled ornithine (albeit with very low isolated yields), in this study we turned to the possibility of glutamic acid 9 (a precursor of ornithine) serving as the fundamental building block to the azabicycle. Feeding of [1-13C]
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glutamic acid 9 to whole cell cultures was shown to site-specifically incorporate at C-6 of the azinomycins (up to 5%) with favorable yields. It is thus tempting to speculate that ornithine 3 inhibits biosynthetic enzymes of the pathway and any incorporation seen is due to that metabolized to glutamic acid 9. Bioinformatic analysis of the azinomycin gene cluster(1, 11) reveals the presence of a series of three genes, aziC2 a putative N-acetyltransferase,(12) aziC3 a putative N-acetyl glutamate kinase,(13) and aziC4 a putative N-acetyl-γ-glutamyl-phosphate reductase.(11) These transformations display homology to those observed in bacterial arginine biosynthesis and is consistent with glutamic acid 9 serving as the key precursor, with N-acetylation playing a protective function. Deprotection and protection steps are frequented in organic synthesis schemes and more specifically utilized in peptide syntheses.(14, 15) To evaluate its putative biochemical function as a “protection step,” aziC2 was successfully cloned and overexpressed in E. coli. The purified protein was successfully reconstituted in vitro when AziC2 was incubated in the presence of glutamic acid 9 and acetyl-CoA. The formation of N-acetylglutamic acid 20 was confirmed by HPLC and ESI/MS/MS. (Figure 2). Figure 2. HPLC analysis (λ 320 nm): Reconstitution of AziC2 activity A] AziC2 incubated with glutamic acid and acetylCoA, B] HPLC standards of glutamic acid and N-acetylglutamic acid
AziC2 displays homology to N-acetyltransferases such as L-2-aminoadipate N-acetyltransferase as well as the lysine biosynthetic gene LysX.(12) The protein displays 46% identity to L-2-aminoadipate Nacetyltransferase from Bellilinea caldifistulae and 45% identity to LysX from Chloroflexux aggregans. LysX exhibits homology to the ATP-dependent carboxylate-amine/thiol ligase superfamily, which is known to activate the carboxyl group of the substrate by phosphorylation and to catalyze the condensation of the carboxyl group to the amino group, forming a C-N bond. As phosphorylation typically involves the use of ATP, we also evaluated the ability of AziC2 to effect N-acetylation by incubating the protein in the presence of glutamic acid 9, acetyl-
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CoA and ATP. Interestingly, ATP inhibited the reaction and N-acetylglutamic acid was not produced.
azinomycin production (Figure 3). Complementation of the mutant strain with N-acetylglutamic acid 20
ArgA (N-acetylglutamate synthase) which ‘primes’ glutamic acid for the arginine biosynthetic pathway by acetylating the amino group is well characterized.(16) The N-terminal domain is responsible for its high substrate specificity towards L-glutamic acid.(16, 17) An ArgA ortholog that lacks the N-terminal domain has been shown to have high Km values for glutamic acid indicating decreased specificity.(18) Truncated ArgA might also utilize a protein partner to enhance specificity for glutamic acid.(16) AziC2 also lacks the N-terminal domain that enhances specificity for Lglutamic acid. To evaluate the specificity of AziC2 for glutamic acid, the kinetics of the N-acetylation reaction were measured with a DTNB (Ellman’s reagent, 5,5’-dithiobis-2-nitrobenzoic acid) spectrophotometric assay (see supporting information for details). An assay of this type has been utilized to evaluate the TE domain of the erythromycin and azinomycin polyketide synthases.(19, 20) Substrates L-glutamic acid 9, L-lysine 17 and D-α-amino adipic acid were evaluated in the presence of acetyl-CoA and AziC2. DTNB allows for the detection of released free thiol by monitoring the progress of the reaction spectrophotometrically at 412 nm.(21) The reaction is rapid and stoichiometric resulting in cleavage of the DTNB disulfide bond. The reaction was carried out in the absence of any thiol reducing agents to decrease background absorbance. The kcat/Km values were similar for all three substrates with a slight enhancement when L-glutamic acid 9 was provided as a substrate (14 + 3 µM-1s-1). L-lysine 17 and D-α amino adipic acid gave kcat/Km values of 11 + 2.5 µM-1s-1 and 6.6 + 1.9 µM-1s-1, respectively. These findings are consistent with the notion that enzymes involved in primary metabolite biosynthesis tend to be much more specific in comparison to those involved in secondary metabolite biosynthesis.(21) Moreover, as has been suggested from ArgA in Mycobacterium tuberculosis, another protein could partner with AziC2 to make it more specific towards L-Glutamic acid.
Figure 3. HPLC analysis of metabolites from ΔaziC2 mutant in comparison to the S. sahachiroi wild-type strain (λ 254 nm): A] Extract of the ΔaziC2 mutant B] Extract of the wild-type strain
As N-acetylation can not only serve as a protection step in biosynthetic reaction schemes, but also serve to inactivate natural products as an antibiotic resistance mechanism by microorganisms,(22, 23) we additionally sought to inactivate the aziC2 gene by genetic knockout to evaluate its affect on azinomycin production. The resulting S. sahachiroi/ΔaziC2 strain was confirmed by RT-PCR and Southern hybridization of thiostrepton resistant and apramycin sensitive colonies (See supporting information for details). As anticipated, evaluation of the metabolites generated from the ΔaziC2 disruptant strain revealed the loss of
∆
∆
∆: Naphthoate
•◊
●: Azinomycin A
◊: Azinomycin B
confirmed its role in azinomycin biosynthesis. Taking into account the mechanistic constraints detailed above, a plausible mechanism is provided in Scheme 3 (it is anticipated that the azinomycin gene locus does encode for all putative genes required for the chemistry put forth). N-acetylation of glutamic acid 9 is invoked as a first step, serving as a protection” step in the biosynthesis of the 1azabicyclo[3.1.0]-hexane amino acid. The protective role prevents cyclization of the amino acid and the formation of the Δ1-pyrrolidine carboxylate, analogous to that observed in bacterial arginine biosynthesis. N-acetylation reflects at least one mechanism by which azabicycle biosynthesis can be primed. An N-acetylglutamate kinase AziC3 would phosphorylate the carboxylate of 21, facilitating reduction to the aldehyde 22, by an N-acetyl-γglutamyl-phosphate reductase (AziC4). These first three steps have now been validated through experimentation,(24) are highlighted in blue in Scheme 3, and demonstrate the current state of knowledge of the pathway. A transketolase would give rise to 24, where a transaminase (aziC1 or C7) could conceivably give the amine diol 25. Dehydration and oxidation would give the diol 28. Sulfation could facilitate aziridine ring formation resulting in 29. Precedence for the sulfotransferase mediated step comes from experiments with rat liver homogenate in which conversion of ephedrine to aziridine is observed in the presence of ATP and sulfate.(25) Subsequent forma tion of the aziridine 31 could either occur spontaneously or be enzyme-catalyzed. While the possibility exists that aziridine formation might not necessitate sulfation of the alcohol for ring closure, the additional ring strain imposed by the formation of the azabicycle may necessitate the involvement of a more
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Scheme 3 Proposed biosynthetic route to the formation of the aziridinopyrrolidine ring system O H 2N
HO
O
H N
OH
AziC2
HO
O
OH
O 2-O
O
3PO
AziC5/C6
H N
OH
O
AziC1 or C7
OH H
HO
O
22
O
HO OH
24
HO
TPP
23 O
H N
O
H N
OH
O
-H2O
O NH 2
HO
H 2N
25
HO
[D]
O HO
NH 2
HO
29
O HO HO
OSO3-
HO
N
33
H N
OH
O HO HO
31
AziC10
H 2N
OH N
HO HO
Corresponding Author Author Contributions
O
H N
OH
AUTHOR INFORMATION *E-mail:
[email protected] N
32
O OH
O HO HO
OSO3-
O
H N
[D]
30
O
O HO
NH 2
AziH3
OH
28
H N
OH
AziH1/2 NH 2
HO
27
O
H N
OH
HO
OH
O HO
[O]
Supporting Information. Additional supplemental material including details on feeding experiments, gene disruptions experiments, and kinetic analyses. The Supporting Information is available free of charge on the ACS Publications website at DOI:
O
H N
OH
NH 2
26
OH
O
O
OH
O
H N
H N
OH
Here, we have provided experimental support for the “priming” of the pathway in the formation of the aziridino [1,2-a]pyrrolidine, azabicyclic ring system; experiments are currently underway to evaluate the remaining biosynthetic steps. ASSOCIATED CONTENT
O
OH
O
O
O
O
H N OH
AziC4
21
20
O
H N
AziC3
O
9
O
H N
OH
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TPP = Thiamin pyrophosphate D = Dehydrogenase
16
facile leaving group. Dehydrogenation would generate the azabicycle, where deacetylation ultimately gives 16.
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally. Funding Sources
We are grateful to the National Science Foundation (CHE-1608580) and the Welch Foundation (A-1828). Notes
The authors declare no competing financial interest
REFERENCES (1) Foulke-Abel, J., Agbo, H., Zhang, H., Mori, S., and Watanabe, C. M. H. (2011) Nat. Prod. Rep. 28, 693-704. (2) Hata, T., Koga, F., Sano, Y., Kanamori, K., Matsumae, A., Sugawara, R., Hoshi, T., Shima, T., Ito, S., and Tomizawa, S. (1954) J Antibiot (Tokyo) 7, 107-112. (3) Ishizeki, S., Ohtsuka, M., Irinoda, K., Kukita, K., Nagaoka, K., and Nakashima, T. (1987) J Antibiot (Tokyo) 40, 60-65. (4) Shimada, N., Uekusa, M., Denda, T., Ishii, Y., Iizuka, T., Sato, Y., Hatori, T., Fukui, M., and Sudo, M. (1955) J Antibiot (Tokyo) 8, 67-76. (5) Coleman, R. S., Perez, R. J., Burk, C. H., Navarro, A. (2002) J. Am. Chem. Soc. 124, 13008-13017. (6) Armstrong, R. W., Salvati, M. E., and Nguyen, M. (1992) J. Am. Chem. Soc. 114, 3144-3145. (7) Alcaro, S., Ortuso, F., and Coleman, R. S. (2002) J Med Chem 45, 861-870. (8) Liu, C. K., G. T.; Watanabe, C. M. H. . (2006) Org. Lett. 8, 1065-1068. (9) Hashimoto, M., Matsumoto, M., Yamada, K., and Terashima, S. (2003) Tetrahedron 59, 3089-3097. (10) Kelly, G. T., Sharma, V., and Watanabe, C. M. H. (2008) Bioorg. Chem. 36, 4-15. (11) Zhao, Q., He, Q., Ding, W., Tang, M., Kang, Q., Yu, Y., Deng, W., Zhang, Q., Fang, J., Tang, G., and Liu, W. (2008) Chem. Biol. 15, 693-705. (12) Horie, A., Tomita, T., Saiki, A., Kono, H., Taka, H., Mineki, R., Fujimura, T., Nishiyama, C., Kuzuyama, T., and Nishiyama, M. (2009) Nat. Chem. Biol. 5, 673-679.
(13) Sundaresan, R., Ragunathan, P., Kuramitsu, S., Yokoyama, S., and Kumarevei, T. (2012) Biochem. Biophys. Res. Commun. 420, 692-697. (14) Shendage, D. M., Fröhlich, R., and Haufe, G. (2004) Org. Lett. 6, 3675-3678. (15) Schnölzer, M., Alewood, P., Jones, A., Alewood, D., and Kent, S. B. H. (1992) Int. J. Peptide Protein Res. 40, 180-193. (16) Xu, Y., Labedan, B., and Glansdorff, N. (2007) MMBR 71, 36-47. (17) Powers-Lee, S. G. (1985) [4] N-Acetylglutamate synthase, Methods in Enzymology, 113, 27-35, Academic Press. (18) Errey, J. C., and Blanchard, J. S. (2005) J. Bacteriol. 187, 3039-3044. (19) Mori, S., Simkhada, D., Zhang, H., Erb, M. S., Zhang, Y., Williams, H., Fedoseyenko, D., Russell, W. K., Kim, D., Fleer, N., Ealick, S. E., and Watanabe, C. M. H. (2016) Biochemistry 55, 704-714. (20) Gokhale, R. S., Hunziker, D., Cane, D. E., and Khosla, C. (1999) Chem. Biol. 6, 117-125. (21) Bai, L., Chang, M., Shan, J., Jiang, R., Zhang, Y., Zhang, R., and Li, Y. (2011) Biochemie 93, 1401-1407. (22) Oda, K., Matoba, Y., Noda, M., Kumagai, T., and Sugiyama, M. (2010) J. Biol. Chem. 285, 1446-1456. (23) Sugiyama, M., Paik, S. Y., and Nomi, R. (1985) J. Gen. Microbiol. 131, 1999-2005. (24) Nepal, K. K., Lee, R. P., Rezenom, Y. H., and Watanabe, C. M. H. (2015) Biochem. 54, 4415-4418. (25) Bicker, U., Fischer, W. (1974) Nature 249, 344-345.
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O
O
O H N O O
O
R N H
O
ca. 14 enzymatic steps
N
O H 2N
O HO A, R=H 2 B, R=
H N OH
O
O
OH
OH
aziC2
O HO
H 2N
OH
N HO
O
AcCoA
HO
O
HO
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