Reducing Holomycin Thiosulfonate to its Disulfide with Thiols

Article abstract of DOI:10.1021/acs.chemrestox.8b00243

The dithiolopyrrolone (DTP) natural products contain a unique ene-disulfide that is essential for their antimicrobial and anticancer activities. The ene-disulfide in some DTPs is oxidized to a cyclic thiosulfonate, but it is unknown how the DTP thiosulfonates react with biomolecules. We studied the reactivity of the thiosulfonate derivative of the DTP holomycin, oxo-holomycin, and discovered a unique redox reaction: Oxo-holomycin is reduced to its parent disulfide, while oxidizing small molecule and protein thiols to disulfides. Our work reveals that the DTP core is a privileged scaffold that undergoes unusual redox chemistry. The redox chemistry of the DTP natural products may contribute to their mechanism of action.

Full text of DOI:10.1021/acs.chemrestox.8b00243

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Article
Reducing Holomycin Thiosulfonate to its Disulfide with Thiols
Andrew N Chan, Walter J. Wever, Elisabetta Massolo, Scott E. Allen, and Bo Li
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00243 • Publication Date (Web): 07 Dec 2018
Downloaded from http://pubs.acs.org on December 7, 2018
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Reducing Holomycin Thiosulfonate to its Disulfide with Thiols
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,&
2,&,#
1,‡
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1,*
Andrew N. Chan, Walter J. Wever,
Elisabetta Massolo, Scott E. Allen, and Bo Li
&
These authors contributed equally to this work.
Department of Chemistry, University of North Carolina at Chapel Hill, CB#3290, Chapel Hill, NC 27599
USA)
Department of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University
of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (USA)
Present address: Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical
School, Center for Life Sciences 11096, 3 Blackfan Circle, Boston, Massachusetts 02215 (USA)
Present address: a Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133
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*
Questions should be addressed to boli@email.unc.edu
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Abstract
The dithiolopyrrolone (DTP) natural products contain a unique ene-disulfide that is essential for their
antimicrobial and anticancer activities. The ene-disulfide in some DTPs is oxidized to a cyclic
thiosulfonate, but it is unknown how the DTP thiosulfonates react with biomolecules. We studied the
reactivity of the thiosulfonate derivative of the DTP holomycin, oxo-holomycin, and discovered a unique
redox reaction: oxo-holomycin is reduced to its parent disulfide, while oxidizing small molecule and
protein thiols to disulfides. Our work reveals that the DTP core is a privileged scaffold that undergoes
unusual redox chemistry. The redox chemistry of the DTP natural products may contribute to their
mechanism of action.
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Introduction
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The rise of antibiotic resistance has heightened the pursuit of compounds that display novel
mechanisms of action. The dithiolopyrrolone (DTP) family of natural products exhibits broad-spectrum
antimicrobial activities and unique chemical structures, thus it is of great interest to understand the
molecular mechanism accounting for their biological activities. DTPs are active against both Gram-
positive and Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa,
1
Staphylococcus aureus, Mycobacterium tuberculosis, and Neisseria gonorrhoeae. They are structurally
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characterized by a compact bicyclic core containing an ene-disulfide. Examples include holomycin (1),
1
thiolutin, xenorhabdin I, and thiomarinol A (Figure 1). Thiomarinol A is a hybrid antibiotic featuring the
2
-4
DTP core covalently tethered to the polyketide antibiotic marinolic acid (Figure 1).
Several cellular effects have been reported for
DTPs against bacteria and yeast, including inhibition of
5
-8
transcription, replication, and respiration.
Only
recently have these effects been connected to the DTP
9,10
ene-disulfide.
Our work identified
a
role for
holomycin in the intracellular chelation of essential
metals and inhibition of zinc metalloenzymes in
10
bacteria. Holomycin is a prodrug and its antimicrobial
activity requires intracellular reduction of the ene-
9
disulfide to ene-thiols for metal chelation. A similar
mechanism was also demonstrated for the anticancer
activity of thiolutin, which involves inhibiting the zinc-
dependent protease subunit Rpn11 of the mammalian
1
1
proteasome.
The metal-chelation mechanism of
DTPs against bacteria and human cells suggest
potential applications in both antimicrobial and
anticancer therapy, however, for use as antibiotics the
selectivity of DTPs will need to be improved for
bacteria over human cells.
In addition to the ene-disulfide forms of DTPs, the
more oxidized thiosulfonate derivatives have also been
isolated from bacteria and exhibit a variety of biological
activities. A small amount of thiomarinol B featuring a
Figure 1. Structures of dithiolopyrrolones and their oxidized
thiosulfonate moiety (Figure 1) was isolated from the
thiosulfonate derivatives.
1
2
thiomarinol A producer. The regiochemistry of the
oxidation in thiomarinol B was determined by X-ray crystallography. Thiomarinol B inhibits the growth of
1
2
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3
many infectious bacteria as effectively as the disulfide counterpart thiomarinol A. The thiosulfonate
derivatives of thiolutin and xenorhabdins have been isolated as thiolutin dioxide and xenorxides (Figure
1
4,15
1), respectively, and possess antibacterial, antimycotic and antineoplastic properties.
It is unknown whether the DTP thiosulfonates exhibit the same mechanism of action as the DTP
disulfides, given the significant difference in reactivity of disulfides and thiosulfonates. Generally,
thiosulfonates are subject to nucleophilic attack on sulfur, resulting in heterolytic cleavage of the sulfur-
sulfur bond and generation of a sulfite anion, which can then be trapped with an alkyl halide to form a
1
6-18
sulfone.
Our initial efforts focused on testing physiologically relevant nucleophiles for reactivity with
DTP ene-disulfide and thiosulfonate. We report a unique redox reaction—reduction of the DTP
thiosulfonate to the parent disulfide by thiols.
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Results
We synthesized the thiosulfonate derivative of
holomycin—oxo-holomycin (2)—as a model compound
to investigate the reactivity of DTP thiosulfonates.
Using a similar procedure to that of oxidizing
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thiomarinol A to thiomarinol B, oxo-holomycin was
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TM
prepared from holomycin using Oxone as an oxidant
(
Figure S1a, S2–S10). Oxo-holomycin exhibits
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identical H and C NMR shifts as those reported for
the DTP thiosulfonate moiety of thiomarinol B (Table
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S1, S2 and Figure S7, S8, S11, S12), indicating that
oxo-holomycin contains the same thiosulfonate moiety
as thiomarinol B. This structure is further supported by
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2
-dimensional ( H, C) HSQC and HMBC experiments
(
Figure S3–S18). Thus, oxo-holomycin is a true mimic
of the natural DTP thiosulfonates. Holomycin absorbs
UV light strongly at 395 nm, resulting in its
Figure 2. Oxo-holomycin is converted to holomycin by N-
acetyl-L-cysteine under acidic conditions. This reaction is time
dependent as shown by the change in UV spectrum over
time: the absorbance at 300 nm for oxo-holomycin decreases
and the absorbance at 395 nm for holomycin increases.
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characteristic yellow color. Oxo-holomycin exhibits a
λ
max of ~300 nm and reduced yellow color (Figure
S1b), suggesting that the electronics of the DTP core
have been significantly altered.
UV-vis experiments were conducted to examine
the reactivity of oxo-holomycin in the presence of
biologically relevant nucleophiles. Oxo-holomycin was
separately treated with N-acetyl-L-cysteine (NAC, 3) as
a model thiol or glycine methyl ester as a model amine
for nucleophilic addition. These reactions were
assayed and monitored for changes in UV
absorbance, which would indicate changes to the DTP
chromophore. To probe the effect of acidic or basic
conditions on reactivity, each reaction was examined
at pH 4.5, 7.0, and 8.2, using a 50 mM sodium
phosphate buffer at room temperature. Incubation of
0
.1 mM oxo-holomycin with 10 equivalents (eq.) of
glycine methyl ester or in buffer alone did not alter the
UV absorbance over 14 hours (Table S3), indicating
that oxo-holomycin does not react with amine
nucleophiles or water. Treatment of oxo-holomycin
with 10 eq. of NAC resulted in an increased
absorbance at 395 nm, the signature λmax for
holomycin (Figure 2). Liquid-chromatography coupled
high-resolution
mass
spectrometry
(LC-HRMS)
comparison with a synthetic standard confirmed that
the product is holomycin (Figure S19). This reaction is
accelerated at acidic pH, though a moderate amount of
reactivity can be observed at neutral pH (Table S3). In
contrast, holomycin shows no significant UV shifts
when incubated with water, amine or thiol nucleophiles
at the examined pH values (Table S4), suggesting that
the reactivity with thiols is unique to the thiosulfonate
Figure 3. Conversion of oxo-holomycin to holomycin under
different equivalents of N-acetyl-L-cysteine (NAC). The total
wavelength chromatograms from LC-HRMS analysis are
shown for oxo-holomycin incubated with (a) 4 eq. of NAC, (b)
2
eq. of NAC, (c) 1 eq. of NAC, and (d) no NAC. Full traces
are shown in Figure S21. (e) Proposed structures of the
reaction intermediates (6) and (7).
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form. Oxo-holomycin is also converted to holomycin by glutathione (Figure S20). Thus, the thiosulfonate
of oxo-holomycin can be converted to a disulfide using biologically relevant thiol nucleophiles under mild,
aqueous conditions.
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We conducted LC-HRMS experiments to determine the stoichiometry of thiol consumed in converting
oxo-holomycin to holomycin and to identify potential reaction intermediates. Incubation of oxo-holomycin
with 4 eq. of NAC results in complete conversion to holomycin with N,N’-diacetyl-L-cystine (4) as the
major by-product (Figure 3a, S21). N-acetyl-L-phenylalanine (5) was used as an internal standard for
LC-HRMS analysis. Incubation of oxo-holomycin with 1 or 2 eq. of NAC resulted in incomplete
consumption of oxo-holomycin and accumulation of a major intermediate (6, Figure 3b, 3c, S21). This
intermediate displays a mass and a UV profile that correspond to a bis-disulfide between reduced
holomycin and two NAC molecules (6, Figure 3e, S22). A holomycin thiosulfinate intermediate (7) was
also identified (Figure 3b, 3c, 3e, S21, S22). In the absence of NAC, the amount of oxo-holomycin is
unchanged, confirming that oxo-holomycin is stable under the assay conditions (Figure 3D, S21). Using
standards for holomycin, oxo-holomycin, NAC, and N,N’-diacetyl-L-cystine, we quantified the amount of
N,N’-diacetyl-L-cystine and holomycin products (Figure S23). In the presence of 4 eq. of NAC, 2.52 ±
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.02 eq. (average ± SD) of NAC is oxidized per 1 eq. of oxo-holomycin (Figure S21, Table S5, S6).
We monitored the timecourse for the reaction of oxo-holomycin with 4 eq. of NAC. LC-HRMS
experiments revealed that the bis-disulfide intermediate 6 forms rapidly and is consumed over time,
coinciding with the formation of holomycin and N,N’-diacetyl-L-cystine (Figure S24). The holomycin
thiosulfinate 7 appears to be an early stage intermediate (Figure S24). Bis-disulfides similar to 6 have
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been shown to form from reduction of thiosulfinates with thiols. Therefore, 6 may arise from a similar
reaction with 7 and NAC reacts directly with oxo-holomycin.
To further characterize oxo-holomycin reactivity,
we investigated the effect of oxo-holomycin on redox-
active cysteines in proteins. Arginine phosphatase
YwlE in Bacillus subtilis contains two cysteine residues
in its active site that are critical for catalysis and
2
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sensitive to oxidation. These cysteines are also the
only cysteines in the entire protein and intramolecular
disulfide formation between them deactivates the
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enzyme. We used LC/HRMS to directly probe the
effect of oxo-holomycin on the oxidation state of YwlE.
Treatment of YwlE with oxo-holomycin resulted in a 2
Da decrease in the protein mass, consistent with
disulfide formation (Figure 4a, 4b, S25). Holomycin
was detected as a product of the reaction between
YwlE and oxo-holomycin (Figure S26), indicating that
oxo-holomycin has been reduced to holomycin while
oxidizing YwlE. Directly treating YwlE with holomycin
also resulted in disulfide formation in YwlE (Figure 4a,
Figure 4. YwlE is oxidized to its disulfide form by oxo-
holomycin and holomycin at pH 7.4. The deconvoluted
protein spectra are shown for 200 µM YwlE treated with (a)
DMF control, (b) 0.5 mM oxo-holomycin, or (c) 2 mM
holomycin. YwlE mass: 17233 Da (reduced), 17231 Da
(oxidized).
4
c). However, the reduced, dithiol form of holomycin
could not be detected, likely due to rapid re-oxidation
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to holomycin in air. Oxidation of YwlE by oxo-
holomycin and holomycin occurs rapidly at both pH 4.5 and 7.4 (Figure 4, S25), suggesting that
reactions of DTPs with protein cysteines can occur under both acidic and physiological pH. Notably, both
2 2
holomycin and oxo-holomycin promote selective disulfide formation, while H O produces various
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oxidized forms of sulfur such as sulfenic and sulfinic acids (Figure S25); thus, DTPs facilitate disulfide
formation while avoiding alternative cysteine oxidation states.
Discussion
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Based on the identified reaction intermediates, we propose a mechanism for the reduction of
holomycin thiosulfonate to holomycin (Scheme 1). Holomycin thiosulfonate 2 reacts with NAC and is
converted to the thiosulfinate 7. Further reaction of 7 with NAC produces the bis-disulfide 6 with
concurrent release of water. Rearomatization of
6
generates N,N’-diacetyl-L-cystine and
holomycin. Alternative mechanisms may also exist. The conversion of oxo-holomycin to holomycin
involves a net 4-electron reduction and the observation that less than 4 eq. of NAC are oxidized
suggests the overall reduction involves latent transformations. For example, the thiosulfinate 7 may
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decompose to holomycin or undergo disproportionation to generate oxo-holomycin and holomycin.
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We have been unable to discern these side reactions. The overall reaction is accelerated at acidic pH,
suggesting that the rate-limiting step is acid catalyzed. The detailed reaction mechanism will be the
subject of future studies.
YwlE oxidation by holomycin is likely accompanied with holomycin reduction to its ene-dithiol form.
Our previous work showed that metal chelation by
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holomycin requires holomycin reduction,
but the
mechanism for this reduction has remained elusive.
Holomycin cannot be reduced by NAC or glutathione
(
Table S4), and previously we have shown that the
reduction of holomycin requires tris-(2-carboxyethyl)-
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phosphine or dithiothreitol. This study suggests that
holomycin is reduced by protein cysteines.
This newly found reactivity of oxo-holomycin with
small molecule and protein thiols may contribute to
how oxo-holomycin exerts its action in cells. Oxidation-
sensitive cysteine residues play important regulatory
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roles in protein function, therefore oxo-holomycin
may exert its inhibitory mechanism by perturbing
protein oxidation states. In addition, reduction of oxo-
holomycin to holomycin by glutathione or proteins
could converge on the same mechanism of action as
holomycin that involves disrupting metal-dependent
processes. Consistent with this proposal, thiolutin
Figure 4. YwlE is oxidized to its disulfide form by oxo-
holomycin and holomycin at pH 7.4. The deconvoluted
protein spectra are shown for 200 µM YwlE treated with (a)
DMF control, (b) 0.5 mM oxo-holomycin, or (c) 2 mM
holomycin. YwlE mass: 17233 Da (reduced), 17231 Da
dioxide, the thiosulfonate derivative of thiolutin, has
15
been described to inhibit neurolysin,
a
zinc
metalloprotease implicated in neurodegenerative
27
diseases.
(
oxidized).
The reduction of oxo-holomycin to holomycin is
different from the typical reactivity of unactivated
thiosulfonates with thiols. The sulfur-sulfur bond in
oxo-holomycin may be cleaved by a thiol, generating a
Scheme 1. Proposed reaction mechanism for the reduction of
oxo-holomycin to holomycin. The fate of oxygen remains to
be determined.
ring open form containing a disulfide and a sulfinic acid, and the sulfinic acid is further reduced to a
sulfenic acid and then to a thiol, eventually reforming the disulfide in holomycin. Reduction of sulfinic
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acids to thiols is extremely rare in biology and requires an ATP-dependent repair enzyme, whereas
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seleninic acids can be reduced to selenols with thiols.
In contrast, while sulfoxides are readily
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1
oxidized to sulfones, the oxidation of selenoxides to selenones are much more difficult. The reactivity
difference is presumably due to the lower electronegativity and larger size of selenium and weaker bonds
compared to sulfur. The reduction chemistry of the DTP sulfinic acid resembles that of a selenic acid and
is likely due to the unique electronics of the DTP scaffold.
In summary, we report the unusual reactivity of oxo-holomycin with thiols. Oxo-holomycin is reduced
to holomycin while oxidizing thiols to disulfides. Oxo-holomycin oxidizes glutathione and the redox-
sensitive enzyme YwlE to disulfides. The interesting redox chemistry of oxo-holomycin may contribute to
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32,33
its biological activity. Many natural products contain cyclic thiosulfinates and thiosulfonates.
Our work
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highlights that understanding the reactivity of the disulfide-containing natural products will likely reveal
new redox chemistry relevant to their mode of action.
Acknowledgements
The authors thank Albert Bowers for helpful discussions, Eric Allen (UCSD) for sharing the thiomarinol
producer Pseudoaltermonas luteoviolacea 2ta16, Timothy Schwochert for suggestions on HR-LCMS
quantification, and Ashley Kretsch, Erinn O’Neill, and Jon Patteson for assistance with NMR analysis.
This work was supported by the National Institutes of Health (R00 GM099904), the Packard Fellowship
for Science and Engineering, and the University of North Carolina at Chapel Hill.
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Conflicts of interest
There are no conflicts of interest to declare.
Supporting Information
Detailed experimental procedures, supplementary figures and tables.
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