Mutasynthesis of fluorinated pactamycin analogues and their antimalarial activity

Article abstract of DOI:10.1021/ol4004614

A mutasynthetic strategy has been used to generate fluorinated TM-025 and TM-026, two biosynthetically engineered pactamycin analogues produced by Streptomyces pactum ATCC 27456. The fluorinated compounds maintain excellent activity and selectivity toward chloroquine-sensitive and multidrug-resistant strains of malarial parasites as the parent compounds. The results also provide insights into the biosynthesis of 3-aminobenzoic acid in S. pactum.

Full text of DOI:10.1021/ol4004614

ORGANIC
LETTERS
2013
Vol. 15, No. 7
1678–1681
Mutasynthesis of Fluorinated Pactamycin
Analogues and Their Antimalarial Activity
Khaled H. Almabruk,†,§ Wanli Lu,†,§ Yuexin Li,‡ Mostafa Abugreen,† Jane X. Kelly,‡
and Taifo Mahmud*,†
Department of Pharmaceutical Sciences, Oregon State University, Corvallis,
Oregon 97331-3507, United States, and Veterans Affairs Medical Center, Portland,
Oregon 97239, United States
taifo.mahmud@oregonstate.edu
Received February 19, 2013
ABSTRACT
A mutasynthetic strategy has been used to generate fluorinated TM-025 and TM-026, two biosynthetically engineered pactamycin analogues produced by
Streptomyces pactum ATCC 27456. The fluorinated compounds maintain excellent activity and selectivity toward chloroquine-sensitive and multidrug-
resistant strains of malarial parasites as the parent compounds. The results also provide insights into the biosynthesis of 3-aminobenzoic acid in S. pactum.
Pactamycin, a bacterial-derived natural product discov-
ered by the Upjohn Company in the early 1960s, has been
shown to have broad-spectrum growth inhibitory activity
against bacteria,1 mammalian cells,2 viruses,3 and protozoa.4
This broad-spectrum activity is primarily due to its strong
binding to a conserved region within the ribosome of most
organisms, leading to nonselective inhibition of protein
synthesis.5,6 Consequently, its wide-ranging cytotoxicity,
coupled with stability issues and difficulties to generate
analogues of pactamycin through organic synthesis, have
hampered its further development. As one of the most
complex aminocyclitol natural products, pactamycin had
presented great synthetic challenges; although, through
seminal work of Hanessian and co-workers, more recently,
this densely functionalized aminocyclitol antibiotic has
finally surrendered to total synthesis.7,8 In addition, a number
of synthetic methodologies to access the aminocyclopentitol
moiety of pactamycin have also been reported.9,10 However,
long synthetic routes and low overall yields remain major
limitations of the synthetic approach to generate pactamycin
analogues for clinical uses.
Recently, using biosynthetic engineering technology,
we were able to generate a number of mutant strains of
Streptomyces pactum ATCC 27456 that produce novel
analogues of pactamycin with improved chemical and
biological properties.11,12 One of the mutants, in which
† Oregon State University.
‡ Veterans Affairs Medical Center.
(7) Hanessian, S.; Vakiti, R. R.; Dorich, S.; Banerjee, S.; Lecomte, F.;
DelValle, J. R.; Zhang, J.; Deschenes-Simard, B. Angew. Chem., Int. Ed.
2011, 50, 3497–500.
(8) Hanessian, S.; Vakiti, R. R.; Dorich, S.; Banerjee, S.; Deschenes-
Simard, B. J. Org. Chem. 2012, 77, 9458–72.
(9) Knapp, S.; Yu, Y. Org. Lett. 2007, 9, 1359–62.
(10) Malinowski, J. T.; McCarver, S. J.; Johnson, J. S. Org. Lett.
2012, 14, 2878–81.
§ These authors contributed equally.
(1) Bhuyan, B. K. Appl. Microbiol. 1962, 10, 302–4.
(2) White, F. R. Cancer Chemother. Rep. 1962, 24, 75–8.
(3) Taber, R.; Rekosh, D.; Baltimore, D. J. Virol. 1971, 8, 395–401.
(4) Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.;
Nishihara-Tukashima, A.; Shibahara, S.; Kondo, S.; Yamada, H.;
Omura, S. J. Antibiot. (Tokyo) 2010, 63, 381–4.
(5) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-
Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103,
1143–54.
(11) Ito, T.; Roongsawang, N.; Shirasaka, N.; Lu, W.; Flatt, P. M.;
Kasanah, N.; Miranda, C.; Mahmud, T. ChemBioChem 2009, 10,
2253–65.
(6) Dinos, G.; Wilson, D. N.; Teraoka, Y.; Szaflarski, W.; Fucini, P.;
Kalpaxis, D.; Nierhaus, K. H. Mol. Cell 2004, 13, 113–24.
(12) Lu, W.; Roongsawang, N.; Mahmud, T. Chem. Biol. 2011, 18,
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r
10.1021/ol4004614
Published on Web 03/22/2013
2013 American Chemical Society

Figure 1. Identification of genes responsible for the production of pactamycin precursor.
ptmH (a gene that encodes a radical SAM-dependent
protein) (Figure 1A) was inactivated, produces two novel
analogues of pactamycin, TM-025 (de-6MSA-7-demethyl-
7-deoxypactamycin) and TM-026 (7-demethyl-7-deoxy-
pactamycin). These new analogues are chemically more
stable and are produced in 10- to 20-fold higher yield than
pactamycin. More significantly, they showed potent anti-
malarial activity at low nanomolar concentrations, but in
contrast to pactamycin have no significant antibacterial
activity and reduced cytotoxicity against human colorectal
HCT-116 cells.12
To further improve the pharmacological properties of
these analogues, we explored the application of the muta-
synthetic approach to generate fluorinated TM-025 and
TM-026 by blocking the formation of pactamycin biosyn-
thetic precursor in S. pactum and performing precursor-
directed structure modifications. Fluorinated compounds
are rare in nature; however, the fluorine atom has been
widely used in medicinal chemistry to improve drug prop-
erties. An estimated 15À20% of new pharmaceutical leads
contains the fluorine atom in their structures.13
Previously, Rinehart and co-workers demonstrated that
feeding of S. pactum var. pactum with 3-amino-5-fluoro-
benzoic acid resulted in the production of a fluorinated
pactamycin analogue.14 The product was reported to have
a comparable biological activity as pactamycin, but the
production yield was inferior, as the fluorinated precursor
has to compete with the intrinsic precursor 3-aminoben-
zoic acid (Figure 1B) in the cells. Therefore, we set out to
engineer a mutant strain of S. pactum that lacks the ability
to produce 3-aminobenzoic acid.
pathway (either dehydroquinate or dehydroshikimate) in-
volving a transamination reaction.15 However, no genetic
and biochemical data are available to support that notion.
Our recent investigations of the pactamycin biosynthetic
gene cluster revealed the presence of two genes, ptmA and
ptmT, that encode PLP-dependent aminotransferases in the
cluster.11 However, it is unclear if any of these genes is
involved in 3-aminobenzoic acid formation.
To investigate the role of the aminotransferase genes in
pactamycin biosynthesis, we carried out in-frame deletion
of ptmA and ptmT individually from the S. pactum genome
and analyzed the metabolic profiles of the resulting mu-
tants. The mutants were constructed by cloning two 1-kb
PCR fragments upstream and downstream of the ptmA
and ptmT genes, respectively, and separately cloned into
the plasmid pTMN002.12 The products pTMW034 (for
ptmA) and pTMW037 (for ptmT) were individually intro-
duced into S. pactum by conjugation. Apramycin resistant
strains representing single crossover mutants were ob-
tained and subsequently grown on nonselective manni-
tol-soy flouragarcontaining MgCl2 toallow the formation
of double crossover recombinants. Apramycin sensitive
colonies were counter-selected by replica plating onto
MS-Mg agar with and without apramycin, and the result-
ing double-crossover candidate strains were confirmed by
PCR amplification (Figure S1, Supporting Information).
HPLC (Figure 2) and MS analyses of the extracts of
those mutants showed that both mutants lack the ability to
produce pactamycin or its intermediates. While the results
suggest that both ptmA and ptmT are critical for pactamy-
cin biosynthesis, it was not clear if ptmA or ptmT are
involved in the formation of 3-aminobenzoic acid.
On the basis of isotope incorporation studies using
13C-labeled glucose, it has been proposed that 3-aminoben-
zoic acid is derived from an intermediate of the shikimate
(14) Adams, E. S.; Rinehart, K. L. J. Antibiot. (Tokyo) 1994, 47,
1456–65.
(15) Rinehart, K. L., Jr.; Potgieter, M.; Delaware, D. L.; Seto, H.
J. Am. Chem. Soc. 1981, 103, 2099–2101.
(13) Chan, K. K.; O’Hagan, D. Methods Enzymol. 2012, 516, 219–35.
Org. Lett., Vol. 15, No. 7, 2013
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To investigate if any of the genes are involved in the
formation of 3-aminobenzoicacid, we carriedout chemical
complementation experiments with 3-aminobenzoic acid.
The compound was pulse-fed to the cultures of ΔptmA and
ΔptmT mutants, and the products were harvested and
analyzed by HPLC and MS. The results showed that feeding
ΔptmT mutant with 3-aminobenzoic acid did rescue the
production of pactamycin (detected as pactamycate),11
whereas that of ΔptmA mutant did not (Figure 2).
and analyzed by MS. The results showed that only feeding
experiments with 3-aminobenzoic acid gave the expected
products TM-025 and TM-026 (Figure S2B, Supporting
Information), whereas feeding with 2- and 4-aminobenzoic
acids did not give any corresponding products (Figures S2A
and S2C, Supporting Information), suggesting substrate
rigidity of some of the enzymes in the pathway.
Figure 2. HPLC analysis of EtOAc extracts of the wild-type and
the mutant strains of S. pactum.
The results suggest that ptmT, not ptmA, is the one
involved in the formation of 3-aminobenzoic acid. To
confirm this incorporation result, we subsequently fed
the mutant with [7-13C]-3-aminobenzoic acid, and MS
analysis of the EtOAc extract of the culture broths revealed
the incorporation of 13C in the final products (Figure 3C),
confirming the direct involvement of 3-aminobenzoic acid
in pactamycin biosynthesis and PtmT is a dedicated en-
zyme for 3-aminobenzoic acid biosynthesis.
To produce fluorinated TM-025 and TM-026 as well as
other possible analogues by a mutasynthesis approach, we
generated a ΔptmT/H double mutant strain. This mutant
strain was obtained by introducing the ptmT knockout
plasmid pTMW037 into ΔptmH mutant (which produces
TM-025 and TM-026)12 by conjugation. The product
(ΔptmT/H mutant) was cultured in the production medi-
um and confirmed for its lack of TM-025 and TM-026
production (Figure 3D). Further chemical complementa-
tion experiments with 3-aminobenzoic acid resulted in the
recovery of TM-025 and TM-026 production (Figure 3E).
The result confirmed full functionality of the biosynthetic
machinery in this mutant except for the genes that have
been inactivated.
Figure 3. Mass spectral data for EtOAc (AÀC) and n-BuOH
(DÀF) extracts of S. pactum mutants with and without chemical
complementations.
To determine if fluorinated TM-025 and TM-026 ana-
logs can be produced by mutasynthesis, we fed ΔptmT/H
mutant with 2-fluoro-5-aminobenzoic acid and 5-fluoro-4-
methyl-3-aminobenzoic acid. Both compounds were che-
mically prepared from commercially available 2-fluoro-5-
nitrobenzoic acid and 5-fluoro-4-methyl-3-nitrobenzoic
acid, respectively, by catalytic hydrogenation (Figure S3,
Supporting Information). The ΔptmT/H mutant was pulse-
fed with the compounds, and the products were analyzed.
The results showed that feeding experiments with 2-fluoro-
5-aminobenzoic acid gave rise to fluorinated TM-025 and
TM-026, designated as TM-025F and TM-026F (Figures 3F
and 4). These two compounds gave quasimolecular ions m/z
413 and 547, respectively, 18 atom mass unit (amu) higher
than those of TM-025 and TM-026 (Figure S4, Supporting
Information). Further confirmation of the products was ca-
rried out by comparing their MS/MS fragmentation patterns
with those of TM-025 and TM-026 (Figure S4, Support-
ing Information) and by NMR analyses (Figures S9ÀS12,
Supporting Information). Whereas 2-fluoro-5-aminobenzoic
acid was incorporated in relatively high yields, giving
rise to 4À5 mg/L of each TM-025F and TM-026F,
To explore the possibility of using ΔptmT/H mutant to
produce pactamycin analogues with different amino posi-
tions of aminoacetophenone, we fed the mutant with 2-, 3-,
and 4-aminobenzoic acids individually. The mutant was
pulse-fed with the compounds (50 μmol) at 16, 28, 40, 52,
and 64 h, and the products were isolated using n-BuOH
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Org. Lett., Vol. 15, No. 7, 2013

Table 1. Antimalarial Activity of Pactamycin Analoguesa
IC50 (nM)
compd
D6
Dd2
7G8
pactamycin
TM-025
4.9
7.7
3.9
4.2
7.1
10.5
14.0
16.8
39.1
114
TM-026
11.5
12.3
26.5
10.6
9.1
TM-025F
TM-026F
chloroquine
12.4
24.9
89.5
a Key: D6, chloroquine-sensitive P. falciparum; Dd2 and 7G8, multi-
drug-resistant P. falciparum.
concentrations used (Figure S13, Supporting Information),
suggesting their high selectivity for plasmodial cells, but not
for bacteria.
Figure 4. Chemical structures of pactamycin analogues.
In conclusion, we have used an efficient mutasynthetic
strategy to generate fluorinated TM-025 and TM-026, two
promising pactamycin analogues, in S. pactum. Similar to
the parent compounds, the fluorinated analogues maintain
excellent activity and selectivity toward chloroquine-sensi-
tive and multidrug-resistant strains of P. falciparum. The
results also provide insight into the biosynthesis of 3-ami-
nobenzoic acid, which is relatively obscure, as well as
substrate preferences of some of the enzymes in the pathway.
feeding experiments with 5-fluoro-4-methyl-3-aminoben-
zoic acid did not give any new products (Figure S2F,
Supporting Information).
To test the antimalarial activity of TM-025F and
TM-026F, the compounds were subjected to an in vitro
antimalarial activity assay. The compounds, along with
TM-025, TM-026, and pactamycin were tested against
chloroquine sensitive (D6) and multidrug-resistant (Dd2
and 7G8) strains of Plasmodium falciparum. The results
showed that TM-025F and TM-026F maintain strong
activity against malarial parasites with IC50 in low nM
concentrations (Table 1). The results also revealed that
fluorine atom substitution in the aminoacetophenone
moiety of TM-025 and TM-026 does not significantly
affect their antimalarial activity in vitro.
To examine if the fluorinated analogues retain the tar-
get selectivity of TM-025 and TM-026, antibacterial
activity tests were performed against both Gram-positive
(Mycobacterium smegmatis, Staphylococcus aureus, Bacil-
lus subtilis) and Gram-negative (Pseudomonas aeruginosa
and E. coli) bacteria in an agar diffusion assay. Whereas
pactamycin is active against all bacteria tested, neither
TM-025F nor TM-026F showed any significant activity at the
Acknowledgment. This work was funded by the general
research funds from the OSU College of Pharmacy. This
publication was made possible, in part, by the Mass
Spectrometry Facilities and Service Core of the Environ-
mental Health Sciences Center, Oregon State University,
Grant No. P30 ES00210, National Institute of Environ-
mental Health Sciences, NIH.
Supporting Information Available. Experimental proc-
edures, Table S1, Figures S1ÀS13. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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