Investigation of (S)-(-)-acidomycin: A selective antimycobacterial natural product that inhibits biotin synthase

Article abstract of DOI:10.1021/acsinfecdis.8b00345

The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of (S)-(-)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (minimum inhibitory concentrations (MICs) = 0.096-6.2 μM) but is inactive against nontuberculosis mycobacteria and Gram-positive and Gram-negative pathogens (MICs > 1000 μM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a K_i of approximately 1 μM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosyl-l-methionine (SAM) to generate the toxic metabolite 5′-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult, and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.

Full text of DOI:10.1021/acsinfecdis.8b00345

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Article
Investigation of (S)-(–)-acidomycin: A selective
antimycobacterial natural product that inhibits biotin synthase
Matthew R Bockman, Curtis A. Engelhart, Julia D Cramer, Michael Howe, Neeraj
Mishra, Matthew Zimmerman, Peter Larson, Nadine Alvarez-Cabrera, Sae Woong
Park, Helena I. M. Boshoff, James M Bean, Victor Young, David M. Ferguson,
Veronique Dartois, Joseph T. Jarrett, Dirk Schnappinger, and Courtney C. Aldrich
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00345 • Publication Date (Web): 17 Jan 2019
Downloaded from http://pubs.acs.org on January 19, 2019
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Investigation of (S)-(–)-acidomycin: A selective antimycobacterial natural
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product that inhibits biotin synthase
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Matthew R. Bockman, Curtis A. Engelhart, Julia D. Cramer, Michael D. Howe, Neeraj K. Mishra,
d
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Matthew Zimmerman, Peter Larson, Nadine Alvarez-Cabrera, Sae Woong Park, Helena I. M.
e
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Boshoff, James M. Bean, Victor G. Young Jr., David M. Ferguson, Veronique Dartois, Joseph T.
c
b*
a
Jarrett, Dirk Schnappinger, Courtney C. Aldrich *
aDepartment of Medicinal Chemistry, University of Minnesota, 308 Harvard Street SE, Minneapolis,
MN 55455, USA
Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue,
New York, NY 10021, USA
Department of Chemistry, University of Hawaii at Manoa, 2545 McCarthy Mall, Honolulu, HI 96822,
USA
b
c
d
Public Health Research Institute, Rutgers, The State University of New Jersey, 225 Warren Street,
Newark, NJ 07103, USA
e
Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, NIAID, NIH,
5601 Fishers Lane, Bethesda, MD 20892, USA.
Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York,
f
NY 10065, USA.
g
X-Ray Crystallographic Laboratory, LeClaire-Dow Chemical Instrumentation Facility, Department of
Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, MN 55455, USA.
Corresponding Author Footnote: To whom correspondence should be addressed. C. A. Phone 612
6
25-7956. Email: aldri015@umn.edu; D. S. phone 646-962-6219. Email:
dis2003@med.cornell.edu
The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism
of action and resistance, and pharmacokinetic properties of (S)-(–)-acidomycin are described.
Acidomycin possesses promising antitubercular activity against a series of contemporary drug
susceptible and drug-resistant M. tuberculosis strains (MICs = 0.096–6.2 µM), but is inactive against
non-tuberculosis mycobacteria, gram-positive and gram-negative pathogens (MICs > 1000 M).
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Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical
studies confirmed acidomycin inhibits biotin synthesis with a K of approximately 1 µM through the
i
competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-
adenosylmethionine (SAM) to generate the toxic metabolite 5-deoxyadenosine. Cell studies
demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for
the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to
acidomycin was difficult and only low-level resistance to acidomycin was observed by overexpression
of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a
promising target.
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Keywords: Mycobacterium tuberculosis, tuberculosis, biotin biosynthesis, biotin synthase,
antimetabolite, acidomycin, accumulation
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Tuberculosis (TB) remains the leading cause of bacterial infectious disease mortality from a single
agent, with an estimated 1.6 million deaths worldwide in 2016.1 TB is caused by Mycobacterium
tuberculosis which belongs to the greater Mycobacterium tuberculosis complex, a group of closely-
related human- and animal-adapted strains: M. africanum, M. bovis, M mungi, M. pinnipedii, M. microti,
M. caprae, and M. canettii.2-3 The standard treatment of drug-susceptible TB is challenging, and
necessitates a 6–9 month dosing regimen of the four first-line antitubercular agents: isoniazid, rifampicin,
ethambutol, and pyrazinamide. The emergence of multidrug-resistant (MDR-TB; resistant to isoniazid
and rifampicin) and extensively drug-resistant (XDR-TB; MDR-TB resistant to the fluoroquinolones
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and at least one of the injectable aminoglycosides) further complicates the management of TB. Since
the approval of rifampicin in 1971, bedaquiline has been the only new TB antibiotic approved by the
FDA, illustrating a lack of novel TB antibiotics developed in the last few decades. Motivated to combat
drug-resistant TB, researchers have sought to identify and exploit the inhibition of vulnerable pathways
for antitubercular drug development.
The output of antibacterial drugs and drug classes has been remarkably low in the past 25 years,
stymied by the numerous challenges associated with antibacterial discovery. Effective drug discovery
programs applied two different approaches to the development of novel antibiotics: target-oriented
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screening, and phenotypic or empirical screening. Empirical screening was the predominant method of
antibiotic drug discovery during the “Golden Age” of antibiotics, where natural products and microbial
metabolites were being unearthed at impressive rates with an estimated tens of millions of soil
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microorganisms screened. These screening methods involved the evaluation of growth inhibition of
bacterial organisms for natural products, irrespective of whether the mechanism of action was known.
The method was successful for many years and engendered the discovery of many most common
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antibiotics known today, including the focus of this study, the thiazolidinone antitubercular drug
acidomycin.
Acidomycin (11, ACM, also known as: actithiazic acid, cinnamonin and mycobacidin) was
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discovered in 1952 independently by four different research teams,7-10 and was originally isolated from
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culture filtrates of Streptomyces virginiae, Streptomyces acidomyceticus, Streptomyces lavendulae,
and Streptomyces cinnamonensis.12-13 Acidomycin possessed a considerable degree of selective
antimicrobial activity against M. tuberculosis in vitro, but was inactive against an M. tuberculosis
infection in vivo.7,14 The inhibitory activity of acidomycin depended on the incubation period, amount
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of inoculum, and concentration of biotin in the medium. Concentrations of acidomycin ranging from
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.25 to 10 g/mL (1.2–46.0 M) were required to inhibit the growth of mycobacteria in vitro, and
conversely acidomycin did not inhibit the growth of certain gram-positive and gram-negative bacteria or
fungi at concentrations as high as 1 mg/mL (4.6 mM).7,9 The presence of biotin in growth media as low
as 0.064 g/mL (0.26 M) was shown to fully antagonize the antibiotic activity of acidomycin and was
attributed to the structural similarity between acidomycin and biotin.7,11,13,15-17 Dethiobiotin (DTB, 4)
also affected the inhibitory of acidomycin, albeit to a much lesser degree.16 Taken together, these data
implicated acidomycin as an inhibitor of the biotin biosynthetic pathway.
The biotin biosynthetic pathway (Figure 1) has been proposed as a promising target for
antitubercular drug development. Biotin (vitamin H, vitamin B7, or co-enzyme R) is a required cofactor
for the growth of M. tuberculosis, as it is central to cellular metabolism involving the transfer of CO2
during carboxylation, decarboxylation and transcarboxylation reactions. These biotin-dependent
enzymes play important roles in many biological pathways such as: membrane lipid biosynthesis, amino
acid metabolism, and tricarboxylic acid cycle replenishment.18-22 Biotin biosynthesis involves the
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conversion of acyl-carrier protein (ACP) conjugate of pimelate (pimeloyl-ACP, 1) to biotin (5, Figure 1)
via four highly conserved steps, catalyzed by BioF (7-keto-8-aminopelargonic acid synthase), BioA
(7,8-diaminopelargonic acid synthase), BioD (dethiobiotin synthetase), and BioB (biotin synthase).
Biotin is then conjugated onto requisite biotin-dependent proteins by BirA (biotin protein ligase).
Uniquely, M. tuberculosis relies heavily on this pathway as its genome encodes three putative biotin-
dependent acyl-CoA carboxylases (ACCs, 6), which are essential for the synthesis of the extraordinarily
complex mycobacterial cell wall, a quintessential hallmark of virulence.23 The biotin biosynthetic
pathway has also been shown to be essential for bacterial persistence in murine TB infection models,
thus making it a promising target for novel TB therapy.24-25
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AMTB
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(DAPA), 3
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Figure 1. The conserved biotin biosynthetic pathway. Briefly, pimeloyl-ACP (1) is converted to 7-
keto-8-aminopelargonic acid (KAPA, 2) by BioF (KAPA synthetase). Transamination by BioA (DAPA
synthetase) converts 2 to 7,8-diaminopelargonic acid (DAPA, 3), followed by insertion of a carbonyl by
BioD (dethiobiotin synthetase) gives rise to dethiobiotin (DTB, 4). Finally, BioB (biotin synthase) is
responsible for the conversion of 4 to biotin (5). The biological fate of biotin (5) is being ligated onto
biotin-dependent proteins (such as acyl-CoA carboxylases, ACCs) by BirA (biotin protein ligase, BPL)
affording the catalytically active biotinylated holo-ACC (6).
Although innovative screening techniques are important to the development of novel antibiotics, the
gap of new drugs discovered compounded with the urgent need for new antibiotics imposed by the
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looming threat of M(X)DR-TB prompted us to re-investigate acidomycin as a potential candidate for
antitubercular drug development. We hypothesize the disinterest in pursuing acidomycin was the lack
of in vivo biological activity, unexplored mechanism of action, and the abundance of attractive alternate
natural products with potent in vitro and in vivo activity. The goal of this study was to contextualize
acidomycin as a potential antitubercular agent in the modern drug discovery landscape as well as to
elucidate its mechanism of action. Herein we utilize contemporary synthetic, biochemical, and
microbiological techniques to probe the whole-cell activity of acidomycin, to systematically investigate
its mechanism of action in terms of both target specificity and selectivity for mycobacteria, and to
evaluate its pharmacokinetic properties in vivo.
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RESULTS
Synthesis and Stereochemical Determination of Acidomycin. Acidomycin (ACM, 11) is a
structurally simple antibiotic containing a thiazolidinone ring appended to an aliphatic carboxylic acid at
the 2-position. The reported syntheses from the groups at Abbott and Pfizer in 1952–1953 involved a
low yielding (~40%) condensation of 2-mercaptoacetamide with methyl (9) or ethyl pimelate
semialdehyde, the latter of which was prepared in four steps from pimelic acid.14,26-27 We devised an
alternate three-step route to 9 from cycloheptanone 7, which was converted to the corresponding eight-
membered lactone by Baeyer-Villiger oxidation followed by Fischer transesterification to provide
methyl ester 8 (Figure 2A). A mild (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation
afforded methyl pimelate semialdehyde (9) in a 3.5-fold higher overall yield than the route from pimelic
acid.26 The key condensation between 2-mercaptoacetamide and methyl pimelate semialdehyde (9) to
furnish 4-thiazolidinone (±)-10 was achieved in an improved 79% yield employing freshly prepared 2-
mercaptoacetamide.28 Saponification of the methyl ester and recrystallization from water gave racemic
(±)-acidomycin [(±)-11], which was readily resolved in gram quantities via preparative chiral HPLC
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(Chiralpak® IE 5 m LC column) to afford the natural product (–)-acidomycin and the unnatural
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antipode (+)-acidomycin in greater than 97.5% optical purities (Figure 2B). Surprisingly, the absolute
configuration of (–)-acidomycin had never been established. Thus, the stereochemistry of the
levorotatory enantiomer was determined by single crystal X-ray diffraction (XRD) analysis. The low
value of the Flack parameter and its uncertainty unambiguously identified the configuration at C-2 of
(–)-acidomyin as S (Figure 2C, Table S11).
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Figure 2. A) Synthetic route to (±)-11 (acidomycin); B) Analytical chiral HPLC trace of (±)-
acidomycin (purple), with each resolved enantiomer (S)-(–)-acidomycin (red) and (R)-(+)-acidomycin
(
blue) using Phenomenex® Cellulose-1 5 m 250 × 2.0 mm LC column; C) X-ray crystal structure of (–)-
acidomycin. See Supporting Information for further details.
Antimicrobial Activity. Each of the acidomycin enantiomers and racemic acidomycin were initially
evaluated against wild type M. tuberculosis H37Rv in biotin-free 7H9 medium to determine the
minimum inhibitory concentration (MIC) that resulted in complete growth inhibition. (S)-(–)-
Acidomycin had a MIC of 0.6 µM while the unnatural enantiomer (R)-(+)-acidomycin was thirteen-fold
less active with an MIC of 7.7 µM and racemic (±)-acidomycin displayed an MIC of 1.6 µM, about 2.5-
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fold higher than the natural product. (±)-Acidomycin was bacteriostatic and only modestly impacted M.
tuberculosis viability (Figure S1). Pre-cultures grown in the presence or absence of biotin did not affect
the sensitivity of M. tuberculosis to acidomycin indicating biotin carryover from the initial inoculum
was minimal. Further microbiological evaluation against a panel of eight drug sensitive M. tuberculosis
clinical isolates, hypervirulent M. tuberculosis HN878, M. tuberculosis Erdman as well as a panel of 15
phenotypically characterized MDR and XDR M. tuberculosis strains demonstrated (±)-acidomycin
maintained excellent activity with MICs ranging from 0.096 µM to 6.2 µM for these 25 contemporary M.
tuberculosis strains (Table 1, see Table S1 for more details). (±)-Acidomycin was active against other
members of the M. tuberculosis complex including M. africanum and M. bovis BCG with MICs of 0.2
and 1.3 µM, respectively. Given the alarming rise of non-tuberculosis mycobacteria (NTM) such as M.
abscessus and members of the M. avium complex, which together account for greater than 90% of the
total NTM pulmonary diseases in immunocompromised individuals, we also evaluated (±)-acidomycin
against these NTMs.29 However, all NTM strains examined were intrinsically resistant to acidomycin.
The fast-growing non-pathogenic M. smegmatis was slightly less susceptible than M. tuberculosis
H37Rv to (±)-acidomycin requiring 10 µM to achieve ≥90% growth inhibition. As initially reported,7,9
we confirmed acidomycin was highly selective for mycobacteria as (±)-acidomycin was inactive toward
several representative gram-negative (Acinetobacter baumannii, Escherichia coli, Pseudomonas
aeruginosa) and gram-positive pathogens (Staphylococcus aureus, Enterococcus faecalis) cultured in
M9 minimal medium. Importantly, (±)-acidomycin showed no cytotoxicity at 1 mM against HepG2
human liver or African green monkey kidney (Vero) cells providing a large therapeutic index
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(
CC /MIC ) of >600 based on the MIC against M. tuberculosis H37Rv and more than 10,000 against
50 99
the most sensitive DS and XDR M. tuberculosis strains.
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Table 1. Biological activity and selectivity of (±)-acidomycin.
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MIC or EC
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c
(M)
M. tuberculosis H37Rv
M. tuberculosis (8)
M. tuberculosis HN878
M. tuberculosis Erdman
M. tuberculosis (7)
M. tuberculosis (6)
M. tuberculosis (2)
M. africanum
WT
1.56
DS
0.096-6.2
0.60
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DS
DS
1.56
MDR
0.60-6.2
0.20-4.8
0.096-1.2
0.20
MDR+
XDR
MTBC
M. bovis BCG
MTBC
1.30
M. abscessus (4)
NTM
>100
M. avium complex (3)
NTM
>100
10d
>1000d
>1000d
>1000d
>1000d
>1000d
>1000e
>1000e
2
M. smegmatis mc 155
non-pathogenic
gram positive
gram positive
gram negative
gram negative
gram negative
mammalian
mammalian
E. faecalis
S. aureus
A. baumannii
P. aeruginosa
E. coli ATCC 25922
HepG2
Vero
a
Bacterial species and strain or mammalian cell line. The number of strains is indicated in parentheses. Further details of the
b
strain including a complete description of the phenotypic resistance is provided in the Supporting Information. Classification:
WT = wild-type; DS = drug-sensitive Mtb clinical isolates; MDR = multidrug-resistant M. tuberculosis; MDR+ = multidrug
resistant M. tuberculosis that are additionally resistant to ethambutol, pyrazinamide, kanamycin, streptomycin, ofloxacin,
moxifloxacin, and/or levofloxacin (see Supporting Information for details); XDR = extensively drug resistant M. tuberculosis;
MTBC = M. tuberculosis complex; NTM = non-tuberculosis mycobacteria. MIC = minimum inhibitory concentrations that
resulted in complete growth inhibition. MIC90 = minimum inhibitory concentrations that resulted in ≥90% growth inhibition.
EC = effective concentration that resulted in greater than 50% inhibition of cell viability. All MIC experiments were
performed in triplicate for each concentration and repeated independently at least two times. Mammalian cell viability studies
were performed in duplicate for each concentration and repeated independently three times.
c
d
e
Mechanism of Action Studies. Shortly following the discovery of acidomycin, several groups
hypothesized it was a biotin antimetabolite based on the structural similarity to biotin and subsequently
demonstrated biotin fully antagonized its antitubercular activity.7,11,13,30 Later investigations by
Eisenberg and Hsiung using resting cells of an E. coli mutant, which overexpressed the entire biotin
pathway, showed acidomycin inhibited the conversion of DTB to biotin; although these results were
never extended to M. tuberculosis.31 To pinpoint the biochemical step targeted by acidomycin in M.
tuberculosis, we thus performed complementation studies with biotin pathway intermediates to
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determine if growth inhibition by acidomycin could be rescued by upstream exogenous biotin precursors.
The activity of (±)-acidomycin against M. tuberculosis H37Rv was evaluated in glycerol-alanine-salts
(GAS) minimal medium supplemented with either 1 M of 7-keto-8-aminopelargonic acid (KAPA),
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DTB or biotin (note: 7,8-diaminopelargonic acid [DAPA] did not complement a bioA M. tuberculosis
biotin auxotroph presumably due to poor permeability of this highly polar diamino-carboxylic acid
metabolite and hence was not used in our studies).32 Biotin completely rescued growth inhibition by
(±)-acidomycin while DTB shifted the MIC approximately two-fold and KAPA had no effect (Figure
3
A, Table S2). Illustrating the remarkable selectivity, we confirmed supplementation with each of the
biotin pathway intermediates had no impact on the antitubercular activity of rifampicin (Figure 3B).
31
These data are consistent with previous studies, and suggest (±)-acidomycin inhibits the conversion of
DTB to biotin in M. tuberculosis, catalyzed by biotin synthase (BioB). To further corroborate BioB as
the primary target of (±)-acidomycin we analyzed an M. tuberculosis strain carrying two copies of the
bioB gene, which is expected to increase BioB expression. In accordance with the conclusion that BioB
is the primary target of ACM in M. tuberculosis, the merodiploid shows an increase in the MIC of (±)-
acidomycin but not rifampicin (Figure 3CD)
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Figure 3. Impact of extrabacterial biotin and overexpression of BioB on susceptibility of M.
tuberculosis H37Rv to (±)-acidomycin. Panels A and B show MIC curves for (±)-acidomycin (A) and
rifampicin (B) in GAST medium supplemented with 1 µM biotin pathway intermediates (black, biotin;
red, DTB; green, KAPA) or DMSO only (blue). Panels C and D show MIC curves for M.
tuberculosis H37Rv (blue squares) and the BioB merodiploid strain (red squares) against (±)-
acidomycin (C) and rifampicin (D) grown in GAST medium. Normalized growth was calculated at
OD580 at the indicated concentration divided by the OD580 with drug (DMSO). Data are averages (± SD)
of triplicate cultures and are representative of two independent experiments.
To biochemically validate these findings, we next overexpressed and purified recombinant M.
tuberculosis BioB (MtBioB) and attempted to reconstitute activity in an anaerobic chamber (< 5 ppm O2)
using E. coli flavodoxin (FLD) and flavodoxin reductase (FLDR) as described.33 Rather than use the
initially disclosed high performance liquid chromatography (HPLC)-based assay to monitor MtBioB
activity, we developed a more sensitive and specific liquid chromatography-mass spectrometry (LC-MS)
assay employing biotin-d as an internal standard. However, we never observed product formation
4
under any assay condition examined. As a control, we also overexpressed and purified E. coli BioB
(EcBioB) and assayed it under identical conditions (see methods). EcBioB was selected because it has
been subject to detailed steady-state and pre-steady-state kinetic analyses as well as structurally
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characterized with bound substrates SAM and DTB.34-48 Unlike MtBioB, EcBioB displayed time-
dependent formation of biotin with kinetic parameters nearly identical to the reported values.37 Both
BioB homologues were purified as stable homodimers and exhibited a characteristic brown color from
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the [2Fe-2S] cluster, thus we hypothesized the inability of MtBioB to turnover substrate was due to
failure of the E. coli FLD to promote electron transfer. Unfortunately, the M. tuberculosis genome does
not encode for any flavodoxins; consequently, further studies will be required to identify the putative
mycobacterial enzyme(s) responsible for single electron transfer to MtBioB. In lieu of a functional
assay for MtBioB, we elected to use EcBioB to evaluate inhibition by acidomycin since our homology
model of MtBioB (see discussion) indicates both active sites are highly conserved.
-1
EcBioB is marked by slow turnover (k ≈ 0.2 min ) and product inhibition (e.g. 5′-deoxyadenosine
cat
and methionine).37 As a result inhibition studies with acidomycin were conducted at a relatively high
enzyme concentration (5 M dimer, Figure 4) under single-turnover conditions at fixed saturating
concentrations of DTB (10 µM) and SAM (100 µM).49 Under these conditions (S)-(–)-acidomycin
inhibited 50% of EcBioB activity at 13.3 ± 1.8 M while and (R)-(+)-acidomycin showed less than 30%
inhibition at 100 M. These results are consistent with the observed relative whole-cell antitubercular
activities of each enantiomer (Figure 4A). Since BioB is a radical SAM enzyme, we hypothesized
acidomycin could potentially act as a mechanism-based inhibitor forming a reactive intermediate that
subsequently inactivates the enzyme. To exclude this possibility, we confirmed acidomycin did not
exhibit time-dependent inhibition by pre-incubating BioB with all substrates and cofactors for various
times (5–30 minutes) followed by initiating the reaction with DTB. To determine the modality of
inhibition as well as inhibition constant (K ) for (±)-acidomycin, initial velocity was measured as a
i
function of DTB concentration (5–30 µM) at several concentrations of (±)-acidomycin ranging from 5 to
3
0 µM that provided the saturation curves (Figure 4B) and the corresponding double-reciprocal (Figure
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D) which shows (±)-acidomycin is a competitive inhibitor with respect to DTB. While this assay
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allowed us to operate under initial velocity conditions, the data cannot be interpreted according to
Michaelis-Menten kinetics, which assumes substrate concentration is much larger than enzyme
concentration ([S] >> [E]), and the concentration of the enzyme-substrate complex does not change over
the course of the assay. Instead, we conducted a simultaneous non-linear regression, modeled according
to Segel’s treatment of multi-site enzymes, taking into account tight-binding behavior giving a
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calculated K of 2.0 ± 1.3 M for (±)-acidomycin or approximately 1 µM for the active S-enantiomer of
i
acidomycin (Figure 4C).50
Figure 4. Inhibition of EcBioB by acidomycin. A) Concentration-response plots of the fractional initial
velocity of EcBioB as a function of acidomycin concentration. (R)-(+)-acidomycin (solid circles) shows
weak inhibition (IC of >100 M) while the calculated IC for (S)-(–)-acidomycin (solid diamonds) is
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50
1
3.3 ± 1.8 M. B) Saturation curve of initial velocity of EcBioB as a function of DTB concentration at
different fixed concentrations of (±)-acidomycin. Each curve was fit to Segel’s model for enzymes with
multiple catalytic sites. C) Data from panel B replotted and fit to a modified version of the Morrison
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equation to obtain K for (±)-acidomycin (K = 2.0 ± 1.3 M). D) Lineweaver-Burke double reciprocal
i
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plot of initial velocity versus free DTB concentration at different concentrations of (±)-acidomycin. The
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intersection near the y-axis exhibits some curvature because the free ligand and steady-state assumptions
are not valid with the implemented assay conditions.
Careful analysis of the reaction byproducts revealed acidomycin significantly enhanced production
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of 5′-deoxyadenosine (22) by BioB in the absence of DTB compared to the background cleavage rate
(Figure S7). We speculate the binding of (S)-(–)-acidomycin to BioB may stimulate unproductive
cleavage of SAM to generate 5′-deoxyadenosine, which if continuously produced could accumulate to a
sufficient concentration to inhibit BioB and as well as other metabolic processes.
Accumulation Studies. We hypothesized the selective microbiological activity of acidomycin for
mycobacteria, despite potent inhibition of EcBioB, may be caused by differential cellular accumulation.
We therefore utilized the detailed method described by Hergenrother employing liquid chromatography
tandem mass spectrometry (LC-MS/MS) with slight modification to quantitatively determine antibiotic
levels in E. coli and M. tuberculosis.51 Rifampicin, thioridazine and tetracycline were used as control
compounds and results are given in Table 2 and Figure 5. The compound accumulation data are
12
expressed in units of nanomoles per 10 CFU to be consistent with Hergenrother’s data as well as
intracellular concentration, which was calculated based on the average volume of cells as detailed in
Table 2. The intracellular concentration (IC) divided by the extracellular concentration (EC), which was
5
0 µM for all compounds, provided the calculated IC/EC ratios. Consistent with previous studies, we
observed rifampicin accumulated to low levels in E. coli resulting in an IC/EC ratio of 2.1 while
tetracycline displayed high accumulation providing an IC/EC value of 62. The accumulation patterns of
tetracycline in M. tuberculosis aligned with the results with E. coli providing an IC/EC ratio of 88.
Thioridazine does not accumulate in M. tuberculosis and was thus selected as control for a low
accumulator. As expected, the IC/EC value for thioridazine was only 0.7 in M. tuberculosis.52 However,
(±)-acidomycin showed a substantial difference reaching an intracellular concentration of 2200 µM in M.
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tuberculosis and only 80 µM in E. coli resulting in IC/EC ratios of 52 and 1.6, respectively. Thus, (±)-
acidomycin accumulated to approximately 30-fold higher levels in M. tuberculosis than in E. coli. We
hypothesized that biotin could potentially compete with transport of (±)-acidomycin in M. tuberculosis
and thus repeated all studies in the presence of 1 M biotin; however, exogenous biotin levels had little
impact on cellular accumulation of (±)-acidomyin in M. tuberculosis as well as the other antibiotics
examined. These data suggest M. tuberculosis is uniquely susceptible to (±)-acidomycin because of
selective accumulation that provides an intracellular concentration more than 50-times greater than the
extracellular concentration.
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Figure 5. Differential accumulation in M. tuberculosis (blue bars) vs. E. coli (red bars) grown in the
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absence of biotin. Accumulation levels (represented in nmol / 10 CFUs) are shown for acidomycin,
high accumulating antibiotic (tetracycline) and low accumulating antibiotics (rifampicin and
thioridazine).
Table 2. Cellular accumulation of antibiotics in E. coli and M. tuberculosis.
Accumulation
Intracellular
IC/EC
Ratio
1
2
Antibiotic
Organism
(nmol/10 CFUs)
biotin +biotin
80 ± 20 61 ± 2
Concentration,
b
a
-
-biotin (µM)
E. coli
80 ± 20
2600 ± 800
3100 ± 120
4400 ± 400
110 ± 60
1.6 ± 0.4
52 ± 16
(±)-Acidomycin
M. tuberculosis 1300 ± 400 1150 ± 40
E. coli 3100 ± 120 1600 ± 100
M. tuberculosis 2200 ± 200 2220 ± 50
E. coli 110 ± 60 144 ± 4
62 ± 2
Tetracycline
Rifampicin
88 ± 8
2.14 ± 0.06
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Thioridazine
M. tuberculosis
18 ± 1
5.2 ± 0.4
36 ± 2
3
0.72 ± 0.04
a
Cell concentrations were calculated based on a cell volume of 1.0 µm for E. coli53 and 0.5 µm for M. tuberculosis54-55;
IC/EC ratios were calculated by dividing the intracellular concentrations by the incubation concentration of 50 µM.
3
b
Resistance Studies. We were unable to isolate any resistant mutants of (±)-acidomycin by plating at
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least 1 × 10 colony forming units (CFUs) of M. tuberculosis H37Rv on biotin-free 7H9 agar plates
containing (±)-acidomycin at 10-, 25-, and 50-fold its agar MIC (12 M), indicating the frequency of
-8
resistance (FOR) for (±)-acidomycin is less than or equal to 1 × 10 . To favor isolation of resistant
mutants, we next plated M. tuberculosis H37Rv in both the presence and absence of 2 µM DTB and
decreased the amount of (±)-acidomycin to be four times its agar MIC. We were unable to isolate any
mutants in plates lacking DTB but identified a single mutant with an MIC approximately 16-fold higher
than that of WT M. tuberculosis H37Rv in plates containing DTB (Figure S2). Whole-genome
sequencing identified a chromosomal duplication spanning from 1750550 to 1807050 which includes
the bioAFD- and bioB-containing operons (SRA accession number: PRJNA507782). This suggested
that resistance was due to overexpression of proteins in the biotin biosynthetic pathway. Western blot
analysis probing for BioA and BioB revealed the mutant to have higher levels of these two enzymes
compared to WT H37Rv (Figure S3).
Additional Mechanism of Action Studies. As a biotin antimetabolite, we hypothesized acidomycin may
be recognized by biotin protein ligase (MtBPL), encoded by birA, in M. tuberculosis where it could
either act as an inhibitor or potentially act as a substrate. Given the high intracellular concentration
achieved by acidomycin, a secondary mechanism of action could be expected to contribute substantially
to the observed antitubercular activity; moreover, inhibition of biotin synthase depletes biotin levels,
which would serve to potentiate the inhibition of MtBPL by acidomycin. Therefore, we investigated the
effect of (S)-(–)-acidomycin on global biotinylated protein levels in M. tuberculosis using a far-western
blot assay and discovered acidomycin had very little impact on the degree of protein biotinylation in the
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time-frame of the assay, even at concentrations that were 10-fold its MIC (Figure 6A, Figure S9). This
result contrasted with a known MtBPL inhibitor, 5ʹ-[N-(D-biotinoyl)sulfamoyl]amino-5ʹ-deoxyadenosine
(Bio-AMS), which showed a noticeable decrease in protein band intensity as a function of inhibitor
concentration.56 Furthermore, we evaluated the binding affinity of (S)-(–)-acidomycin to MtBPL by
isothermal titration calorimetry (ITC) in a direct titration experiment. We observed no heat generated
when (S)-(–)-acidomycin was titrated into MtBPL, relative to the binding enthalpy generated when a
known substrate (biotin) was titrated into MtBPL (Figure 6B, Figure S10). These data indicate (S)-(–)-
acidomycin does not bind to MtBPL, and thus does not act as an antimetabolite in protein biotinylation .
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Figure 6. A) Far-western blot of the biotinylated proteome of M. tuberculosis grown in biotin-free 7H9
media, in the presence of acidomycin. Concentration of acidomycin (M) is depicted at the bottom of
the blot. Lane L: Protein ladder; Lanes 1–3: M. tuberculosis grown in the presence of acidomycin at 0
(lane 1), 1 (lane 2) and 10 (lane 3) its MIC. This experiment was performed twice, independently. B)
Isothermal titration calorimetry thermogram of (S)-(–)-acidomycin (750 M, red) and biotin (750 M,
black) titrated into MtBPL (50 M), with the binding data for biotin shown in black. This experiment
was repeated twice, independently.
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Pharmacokinetics of Acidomycin. To assess the pharmacokinetic (PK) parameters of acidomycin
and compare them to previous pharmacological studies, we administered CD-1 mice a single dose of
either natural (S)-(–)-acidomycin or unnatural (R)-(+)-acidomycin (Table 3). The mediocre aqueous
solubility of acidomycin (< 4 mM) prevented formulations at higher concentrations without using
DMSO, which prompted optimization of the counterion to further improve the solubility. Using the 2-
amino-2-methylpropan-1,3-diol (AMP2) salt form of acidomycin, a known counterion used in the
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formulation of carboxylic acid drugs, we were able to improve its aqueous solubility substantially to
greater than 50 mM. We therefore used this formulation for the PK experiments. The plasma
concentrations of (S)-(–)-acidomycin and (R)-(+)-acidomycin at various time points after a single dose
were determined by LC-MS/MS (Figure 7). Each compound was orally (p.o.) dosed at 25 mg/kg in
three mice, or intravenously (i.v.) dosed at 5 mg/kg. From these studies, the area under the
concentration-time curve (AUC) for each dose and absolute oral bioavailability (F) were determined.
Additional parameters that were determined from these experiments were the volume of distribution (Vd),
terminal elimination half-life (t ) and clearance (CL). Oral administration (p.o.) of (S)-(–)-acidomycin
1
/2
and (R)-(+)-acidomycin gave an oral bioavailability of 8 and 16%, respectively. Following intravenous
i.v.) administration, both (S)-(–)-acidomycin and (R)-(+)-acidomycin exhibited monoexponential
(
elimination with half-lives of 14.4 and 19.2 min, respectively. Consequently, the unnatural (R)-(+)-
acidomycin enantiomer possessed roughly the same PK profile, but improved oral bioavailability
relative to the natural (S)-(–)-acidomycin.
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Table 3. In vivo Pharmacokinetic Parameters of (R)-(+)-acidomycin and (S)-(–)-acidomycin in CD-1
mice (n = 3).
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Pharmacokinetic indicesa (R)-(+)-acidomycin
(S)-(–)-acidomycin
Dose i.v., p.o. (mg/kg)
AUC0–∞ (p.o., ng·hr/mL)
AUC0–∞ (i.v., ng·hr/mL)
Vd (i.v. L/kg)
5, 25
5, 25
1591 ± 718
2055 ± 564
1.12 ± 0.37
2433 ± 482
19.2 ± 2.4
16 ± 7
890 ± 106
2116 ± 115
0.82 ± 0.05
2363 ± 126
14.4 ± 0.1
8 ± 1
0
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0
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2
3
4
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7
8
9
0
CL (i.v., mL/kg·hr)
t (i.v., min)
1
/2
F (%)
a
Values are reported as the mean of three replicates. AUC0–∞, are under the plasma concentration-time curve from time 0 to
infinity; V , volume of distribution; CL, clearance; t1/2, terminal elimination half-life; F, relative oral bioavailability
calculated as follows: F = 100 × [(AUCp.o. × Di.v.)/(AUCi.v. × Dp.o.)] where D is the dose.
d
Figure 7. Mean plasma concentration versus time curves after single p.o. (25 mg/kg, blue) and i.v. (5
mg/kg, red) administration of acidomycin to CD-1 mice. Error bars represent standard deviation of the
mean (n = 3); A) (R)-(+)-acidomycin; B) (S)-(–)-acidomycin.
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DISCUSSION
Since its discovery, acidomycin was recognized as an antimetabolite of the biotin biosynthetic
pathway due to the resemblance of the thiazolidinone ring to that of the tetrahydrothiophene/ureido rings
of biotin.7-8,13,16,30 Early biochemical characterization and elucidation of the mechanism of acidomycin
showed accumulation of biotin pathway intermediates (termed “vitamers”) increased by two- to seventy-
fold when acidomycin was added to the culture media of various microorganisms, as determined by a
Saccharomyces cerevisiae-based bioassay.58-59 Conversely, the total amount of biotin detected post-
assay decreased substantially when given acidomycin, which strongly suggest it directly affects biotin
synthesis.58-59 Corroborating these data, we also discovered biotin to rescue activity when M.
tuberculosis was grown in the presence of acidomycin, but that other pathway intermediates could not
complement growth (Figure 3) suggesting that acidomycin inhibits biotin synthase (BioB), the enzyme
responsible for synthesizing biotin (5) from DTB (4).
0
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0
BioB catalyzes the final step of the biotin biosynthetic pathway (Figures 1 & 8). First characterized
6
0
in Escherichia coli by Ifuku and coworkers in 1992, BioB is part of the radical SAM superfamily and
is a homodimeric protein responsible for the formation of the tetrahydrothiophene ring in biotin via
insertion of a sulfur atom between the C-6 and C-9 carbons in DTB. Like most radical SAM enzymes,
+
BioB utilizes a reduced [4Fe–4S] cluster to cleave SAM (20) into methionine and a high-energy 5-
deoxyadenosyl radical (21, Figure 8A).61-63 This reactive, transient radical is responsible for a single
hydrogen atom abstraction from the C-9 carbon and allows for the insertion of sulfur via a newly formed
+
44
C–S bond originating at a second [2Fe–2S] cluster (Figure 8B).
The tetrahydrothiophene ring is
closed from subsequent generation of a second 5-deoxyadenosyl radical (21), abstracting the hydrogen
atom from C-6, and thereby closing the ring (Figure 8B). As a result, BioB requires two equivalents of
SAM (20) per complete turnover.44,64 The direct donation of the [2Fe–2S] sulfur atom thus makes
+
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BioB appear to act as a substrate in vitro, rather than a catalytically functional enzyme, with some
studies reporting less than one turnover per protein monomer.65-66 Thus, BioB distinctively possesses a
layer of complexity when considering it as a target for drug development that is absent in most
enzymatic systems. In the E. coli BioB system, once a successfully completed turnover has occurred,
during which the [2Fe–2S]2+ cluster is destroyed, BioB has an increased propensity for oxidative
degradation via ATP-dependent proteolysis.40 This begs the question of whether BioB acts as a “true”
prototypical enzyme capable of multiple turnovers, or a complex substrate facilitating the conversion of
DTB to biotin and dismantling itself in the process.66 However, the extremely tight regulation of biotin
production (vis-à-vis BioB degradation) could prove to be promising for antibiotic development
targeting BioB, such as acidomycin.
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1
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A
2
+
+
2+
2+
S
S
S
S
Fe
S
Fld-FMNH-
Fld-FMNH
Fe
Fe
S
Fe
Fe
S
Fe
S
Fe
S
Fe
S
Fe
S
Fe
S
Fe
S
Fe
S
S
S
Fe
Fe
Fe
Fe
0
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0
H3C
OH
H3C
OH
H3C
OH
O2C
O2C
O2C
H C
3
O2C
S
NH2
S
NH2
S
NH2
S
NH2
Ad
Ad
HO
Ad
HO
H
H
H
H
O
O
O
HO
Ad
H
H
SAM, 20
O
HO
OH
21
B
Ad
HO
H
O
H
H
O
O
O
O
O
OH
22
HN
NH
NH
O
NH
O
NH
O
21
22
O
S
HN
H
HN
H2C
HN
OH
OH
OH
OH
9
6
5
4
3
2
S
CH2
H C
2
Ad
HO
S
S
S
Cys
Cys
Biotin, 5
H
H
Arg NH2
FeII
FeIII
DTB, 4
Cys
S
OH
S
Cys
Cys
S
S
S
Arg NH2
Cys
Cys
FeIII
FeIII
Arg NH2
21
FeII
FeII
S
Cys
S
Productive radical
propagation
S
Cys S
S
dissociates
Ad
HO
H
C
O
H
H
O
O
OH
O
22
NH
O
NH
O
H
H
S
OH
S
OH
Ad
HO
H
H
ACM, 11
11
OH
21
Unproductive radical
termination
Figure 8. Proposed mechanism of BioB and inhibition by acidomycin (11). A) A single electron from
flavodoxin reduces the [4Fe–4S] cluster, promoting the homolytic cleavage of SAM (20) into
methionine and a high-energy 5′-deoxyadenosyl radical (21). B) Under normal circumstances, the 5′-
deoxyadenosyl radical (21) abstracts the C-9 hydrogen atom of the native substrate DTB (4), forming 5′-
deoxyadenosine (22) and allows for the insertion of sulfur from the [2Fe–2S] cluster (labeled as
+
+
“
productive radical propagation”). Another 5′-deoxyadenosyl radical (21) is responsible for the C-6
hydrogen atom abstraction, completing the thiophane ring in biotin (5). C) When acidomycin (11) is
bound to the active site instead, the lack of a C-6 hydrogen atom prevents the highly reactive 5′-
deoxyadenosyl radical (21) from H-atom abstraction, leading to the radical to be unproductively
quenched as a result (labeled as “unproductive radical termination”).
Our efforts to probe the inhibitory effect of acidomycin on BioB thus led us to the discovery that
acidomycin is a competitive inhibitor with respect to DTB (Figure 4D). We resorted to using EcBioB
for our inhibition assay as we were unable to develop a functional assay for MtBioB. Although
3
3
enzymatic activity of MtBioB was obtained previously, we were unable to replicate these conditions
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using the E. coli FLD and FLDR electron transport system. We surmise the electron donation system
for BioB is highly tuned to be species-specific and thus the E. coli FLD and FLDR proteins are not
functionally active with MtBioB. Genomic mining of M. tuberculosis failed to identify any candidate
flavodoxins, which might facilitate the analogous reactions in mycobacteria suggesting a different
electron transport mechanism in M. tuberculosis versus E. coli. This is not uncommon, and other
organisms are known to utilize ferredoxins in lieu of a flavoprotein for single electron transfer
reactions.67 We found five putative ferredoxins in the M. tuberculosis genome that could potentially act
as the electron donor: Rv0763c, Rv1177 (potentially FdxC), Rv1786, Rv2007c (potentially FdxA), or
Rv3503c (potentially FdxD). Of these, Rv1786 was recently expressed and characterized to couple with
flavoprotein reductase A (FprA) and ferredoxin reductase A (FdrA), but its function in vivo is still
unknown.68 Investigations into the expression and biochemical evaluation of the MtBioB electron
transport partner(s) are ongoing.
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1
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1
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To reasonably extrapolate the EcBioB inhibitory data of acidomycin to MtBioB, we therefore
constructed a homology model to investigate any differences in the binding site of both EcBioB and
MtBioB using the X-ray crystal structure for EcBioB (PDB: 1R30).34 The two sequences have greater
than 75% similarity near the binding pocket and some non-conserved residues such as Ser212 (Cys221)
and Met246 (Phe253) have backbone interactions with DTB resulting in a nearly identical active site
(Figure 9A). The structural similarity between DTB and acidomycin is evident in our model, as it shows
that substantial overlap in the active site is possible (Figure 9B). Assuming DTB and acidomycin bind
to BioB as illustrated by the model, it is reasonable to extrapolate the EcBioB inhibitory data of
acidomycin to MtBioB.
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Figure 9. A) Homology model of EcBioB (grey, faded) aligned to MtBioB (blue, solid) depicting DTB
purple) and SAM (green) bound to the active site. All key interactions with residues remain the same
between species. B) EcBioB with comparison of acidomycin and DTB (green and purple, respectively)
(
aligned in the active site.
The modest K (~1 M) of acidomycin against EcBioB (and assuming similarity in the inhibitory
i
concentrations between EcBioB and MtBioB), could indicate off-target binding or secondary/indirect
effects. In our efforts to explain the difference between the whole-cell activity and enzymatic activity of
acidomycin, we discovered an increase in the amount of 5′-deoxyadenosine (22) produced compared to
baseline levels in assays containing acidomycin (Figure S7). This unproductive cleavage of SAM does
+
not affect the [2Fe-2S] cluster of BioB, which is responsible for the insertion of sulfur into DTB. This
implies cleavage of SAM to be catalytic, and thus not obstructed by the Fe-S cluster regeneration
requirement that plagues the natural reaction mechanism. Furthermore, the X-ray co-crystal structure of
EcBioB and DTB (PDB: 1R30), previously reported by Berkovitch and coworkers, depicts DTB
sandwiched between the [4Fe–4S] and [2Fe–2S] clusters with the C-9 only 4.6 Å away from the closest
bridging sulfide in the [2Fe–2S] cluster.34 This presumably requires a slight structural change in the
conformation of BioB to enable the abstraction of the C-9 H-atom from the SAM-derived 5′-
deoxyadenosyl radical (21). With acidomycin bound instead, the reductive homolytic cleavage of SAM
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(20) could still take place in the active site of BioB in an unproductive manner, whereby the highly
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reactive 5′-deoxyadenosyl radical (21) is quenched before reacting with acidomycin (Figure 8C).
Although it is known that the H-atom abstraction and subsequent sulfide insertion is step-wise, it is still
0
1
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unclear as to whether or not the binding event of SAM and DTB occur in any ordered fashion.42
Therefore, acidomycin could bind to the active site in a competitive manner followed by a “pseudo
mechanism-based” inhibition of BioB via depletion of the biologically active SAM. The utilization of
the methylthioadenosine/S-adenosylhomocysteine nucleosidase MtnN in our in vitro assays prevented
this known substrate inhibition through hydrolysis of the 5′-deoxyadenosine derived from SAM.69 If
pools of SAM are depleted in vivo, however, it should come as no surprise that this could be problematic
for the survival of the organism, as many essential enzymes – even within the biotin biosynthetic
pathway – are SAM-dependent such as BioC and BioA.70 Additionally, M. tuberculosis lacks the ability
to scavenge exogenous SAM and intermediates of SAM/methionine biosynthesis, emphasizing a major
reliance on intracellular levels of SAM.71 Interestingly, a recent study showed potentiation of para-
aminosalacylic acid (PAS), a selective antimetabolite of the folate metabolic pathway, to biotin
deprivation by utilizing a biotin auxotrophic strain of M. bovis BCG with a disruption in the bioB gene.72
7
3
Methionine is a known potent antagonist of PAS in mycobacteria, and thus the connection between
PAS, methionine and biotin biosynthesis inhibition could be attributed to SAM as well.74
We hypothesized acidomycin may possess a secondary mechanism of action, wherein it would act as
a substrate for MtBPL and become incorporated into the biotin carrier carboxylase protein (BCCP)
domain of acyl-CoA carboxylases (ACC) instead of biotin (Figure 1). This modification would not only
inactivate biotin-dependent proteins, but also prevent canonical activation, as only non-ligated apo-
proteins are recognized by BPLs. Acidomycin ligated to proteins would be unable to facilitate carboxyl-
group transfer, as it lacks the proper ureido nitrogen responsible for fixing CO . The unique bicyclic
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ring system of biotin is thought to have evolved specifically for carboxylation, as the presence of the
sulfur atom reduces unproductive hydrolysis by blocking the approach of water from one side, greatly
improving the efficiency of biotinylated enzymes.75-76 The importance of the sulfur in biotin is evident
from an evolutionary standpoint as BioB is a highly regulated enzyme with a complex mechanism for
sulfur insertion. Therefore, any deviation from this highly specialized ring system (even similar
molecules such as DTB) would presumably result in a non-functional enzyme. However, both our
whole-cell biotinylated proteome assays (Figure 6A) and direct binding assays (Figure 6B) showed
acidomycin does not bind to MtBPL, and thus acidomycin does not appear to possess this intriguing
secondary mechanism of action.
0
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Antibiotics with poor cellular accumulation was a major problem faced during many early discovery
programs.77-78 As such, the ability to quantify intracellular accumulation of drugs has become a recent
trend in the evaluation of antibiotics, new and old.51,79-80 In an effort to reconcile the disparate whole-
cell inhibitory activity of acidomycin against E. coli and M. tuberculosis, even though we presented
direct inhibition of EcBioB, we chose to look at the levels of intracellular accumulation for these two
organisms. Alas, we discovered a thirty-fold increase in intracellular accumulation of acidomycin in M.
tuberculosis compared to E. coli, which is a trend we expected, but does not fully explain the >100-fold
difference in whole cell activity. These measurements account for drug association within the cells as
well as (in the case of M. tuberculosis) embedded in the cell envelope, and these concentrations
represent concentrations after a five-minute incubation period. Efflux pumps in E. coli and M.
tuberculosis could act on these drugs at different rates, which could also affect the intracellular
concentration at a given time. However, solely relying on accumulation to explain the difference in
activity oversimplifies the mechanism of selectivity between these two organisms.
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A promising result in our studies was the observation of the very low frequency of resistance of
acidomycin, consistent with previous studies.7 When M. tuberculosis was grown in the presence of
acidomycin and 2 M DTB, we observed a 16-fold increase in the MIC. The rationale for including
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DTB was to “force” a mutation of bioB, however when DTB concentrations were reduced to 1 M, only
a two- to three-fold increase in MIC was observed. Grundy and coworkers discovered resistance to
acidomycin was not easy acquired, as a meager two-fold increase in inhibitory concentrations were
achieved after growth in the presence of acidomycin.7 Sequencing of the mutant identified
chromosomal duplication leading to the overexpression of enzymes required for biotin synthesis. The
difficulty in obtaining a viable resistant mutant suggests simple point mutations to BioB are unable to
confer resistance to acidomycin.
Although acidomycin displayed promising in vitro selective inhibitory activity against mycobacteria,
it was shown in this study and in previous studies to be pharmacodynamically inert.11,81-82 The inability
of acidomycin to exert an antitubercular effect in vivo prompted researchers to hypothesize that biotin
present in tissues antagonized the in vivo activity of acidomycin, when dosed to mice and rats.11,30
Schnappinger and coworkers definitively proved this to not be the case, as their conditionally-regulated
bioA knockdown mutant was not only unable to establish an infection in mice, but also chronic
infections were cleared when biotin biosynthesis was inhibited, indicating M. tuberculosis requires de
novo biotin biosynthesis for the persistence in vivo.24,83 Conditional genetic inactivation of BPL further
exemplified that not only de novo biotin biosynthesis but also protein biotinylation is required by M.
tuberculosis to sustain an infection.25 Our own PK experiments showed rapid elimination of acidomycin
from mice (Table 3). Biotin concentrations in mammalian tissues (including mice) are well under the
amount required to antagonize acidomycin, implying the lack of in vivo activity could be explained by
rapid elimination. To that end, one study observed approximately 30 % of the intravenous (i.v.)
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acidomycin dose was recovered from the urine of mice, dogs, cats, and rabbits within 60 minutes, as
well as provide evidence that acidomycin is partially inactivated by the abdominal organs.82 Further
early in vivo experiments also showed elevated levels of biotin in the urine of rabbits treated with
acidomycin.11,15,82 Acidomycin did not significantly change the measured degree of biotin deficiency in
egg white-fed rats but rather increased the amount of what was then-termed “avidin uncombinable biotin”
and “SC factor,” which was later elucidated to be biotin pathway intermediates.81,84 Combined with our
PK data, we can establish the reason for its inactivity to be due to rapid elimination from the host rather
than biotin antagonism. Further improvement of these parameters would necessitate the design of
prodrugs to increase permeability and extend its terminal half-life.
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In addition to the antimicrobial and pharmacological experiments performed, several structurally
related acidomycin analogues were synthesized and evaluated for antimicrobial activity against both M.
tuberculosis and M. avium.7-8,13,16,30,85-86 The optimal chain length is five methylene units, and removing
the carboxylic acid ablates activity.86 Any modifications to the N3 position, as well as oxidizing the
sulfur also diminishes activity.85
The primary amide and lower alkyl esters showed improved
antitubercular activity, but this could be due to increased cellular permeability.86 The activity of
acidomycin derivatives seem to be sensitive to minor changes, indicating a limited scope of SAR.
Going forward, it would be prudent to improve the PK properties of acidomycin through prodrug design
(via alkyl esters and amides), rather than through analogues of the parent compound.
CONCLUSION
We have explicitly demonstrated the mechanism of acidomycin against M. tuberculosis using both
whole-cell and in vitro biochemical assays. Biotin was the only biotin pathway intermediate to
antagonize the inhibitory activity of acidomycin against M. tuberculosis. This was further confirmed as
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(
±)-acidomycin displays competitive inhibition against EcBioB, with an inhibitory constant, K of 2.0
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
M, which is analogous to MtBioB due to the high sequence similarity in the active sites of EcBioB and
MtBioB. The purified natural product (S)-(–)-acidomycin shows a 14-fold or greater increase in activity
over the non-natural (R)-(+)-acidomycin enantiomer in both whole-cell and in vitro biochemical assays.
We observed a 30-fold increase in the amount of accumulated acidomycin in M. tuberculosis over E.
coli, owing to its selectivity for M. tuberculosis. The low FOR of acidomycin against M. tuberculosis
initially suggested a secondary mechanism of action, but none of our experiments showed evidence of
acidomycin acts as a biotin antimetabolite or binds to MtBPL. We also discovered the lack of in vivo
activity of (S)-acidomycin in mice was caused by poor PK properties as it is rapidly eliminated with a
half-life of 14.4 min. Overall, this study provides the most detailed study of acidomycin’s mechanism
of action and selectivity in M. tuberculosis and demonstrates future efforts should focus on improving
the pharmacokinetic properties of acidomycin.
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METHODS
General materials and methods for synthesis. Chemicals and solvents were purchased from Acros
Organics, Alfa Aesar, Sigma-Aldrich, and TCI America and were used as received. An anhydrous
solvent dispensing system using two packed columns of neutral alumina was used for drying MeOH,
toluene, THF and CH Cl , while two packed columns of molecular sieves were used to dry DMF, and
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the solvents were dispensed under argon gas (Ar). EtOAc and hexanes were purchased from Fisher
Scientific. All reactions were performed under an inert atmosphere of dry argon gas (Ar) in oven-dried
(180 °C) glassware. TLC analyses were performed on TLC silica gel 60F254 plates from EMD
Chemical Inc. and were visualized with UV light. Optical rotations values were obtained on a
polarimeter using a 1 dm cell. Purification by flash chromatography was performed using a medium-
pressure flash chromatography system equipped with flash column silica cartridges with the indicated
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