β-Keto and β-hydroxyphosphonate analogs of biotin-5′-AMP are inhibitors of holocarboxylase synthetase

Article abstract of DOI:10.1016/j.bmcl.2014.11.010

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (β-ketoP) and hydroxyphosphonate (β-hydroxyP) analogs of biotin-5′-AMP inhibit holocarboxylase synthetase (HLCS) with IC₅₀ values of 39.7 μM and 203.7 μM. By comparison, an IC₅₀ value of 7 μM was observed with the previously reported biotinol-5′-AMP. The K_i values, 3.4 μM and 17.3 μM, respectively, are consistent with the IC₅₀ results, and close to the K_i obtained for biotinol-5′-AMP (7 μM). The β-ketoP and β-hydroxyP molecules are competitive inhibitors of HLCS while biotinol-5′-AMP inhibited HLCS by a mixed mechanism.

Full text of DOI:10.1016/j.bmcl.2014.11.010

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5568–5571
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
b-Keto and b-hydroxyphosphonate analogs of biotin-50-AMP are
inhibitors of holocarboxylase synthetase
a,
Wantanee Sittiwong a, ,à, Elizabeth L. Cordonier b, , Janos Zempleni b, , Patrick H. Dussault
⇑
⇑
a Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA
b Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0806, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mito-
chondrial carboxylases, nuclear histones, and over a hundred human proteins.
Nonhydrolyzable ketophosphonate (b-ketoP) and hydroxyphosphonate (b-hydroxyP) analogs of biotin-
Received 5 September 2014
Revised 29 October 2014
Accepted 3 November 2014
Available online 7 November 2014
50-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7
l
M and 203.7
M was observed with the previously reported biotinol-50-AMP. The Ki values,
M, respectively, are consistent with the IC50 results, and close to the Ki obtained for
biotinol-50-AMP (7
M). The b-ketoP and b-hydroxyP molecules are competitive inhibitors of HLCS while
biotinol-50-AMP inhibited HLCS by a mixed mechanism.
lM. By compar-
ison, an IC50 value of 7
3.4 M and 17.3
l
l
l
Keywords:
Biotin-50-AMP
b-Ketophosphonate
b-Hydroxyphosphonate
Holocarboxylase synthetase
Biotinylation
l
Ó 2014 Elsevier Ltd. All rights reserved.
Holocarboxylase synthetase (HLCS) is the sole enzyme in the
human proteome capable of catalyzing the covalent attachment
of biotin to lysine residues.1 HLCS localizes in the cytoplasm, mito-
chondria, and cell nuclei.1,2 HLCS catalyzes biotinylation of five car-
boxylases in mitochondria and cytoplasm, which play key roles in
gluconeogenesis, fatty acid metabolism, and leucine metabolism.3
In addition, a recent mass spectrometry screen identified 108 novel
biotinylated proteins; heat shock proteins and enzymes from
glycolysis are overrepresented among these proteins.4 HLCS
orchestrates the assembly of a multiprotein gene repression
complex in human chromatin, partially mediated through HLCS-
dependent methylation of the histone methyltransferase EHMT1
and the nuclear receptor co-repressor N-CoR.5 Consistent with
the importance of HLCS in intermediary metabolism and cell
function, no living HLCS null person has ever been reported, and
persons with HLCS mutations require lifelong treatment with
pharmacological doses of biotin.6
In the second step, the phosphate anhydride serves as an acylating
agent for a target lysine in carboxylases (2), histones (3), and other
proteins,3,4,7 covalently linking biotin to the substrate via an amide
bond.
HLCS-dependent biotinylation involves basically two steps
(Fig. 1). In the first, activation of biotin by ATP generates the mixed
anhydride biotin-50-adenosine monophosphate (Bio-50-AMP) (1).
⇑
Corresponding authors. Fax: +1 402 472 1587 (J.Z.), +1 402 472 9402 (P.H.D.).
E-mail addresses: jzempleni2@unl.edu (J. Zempleni), pdussault1@unl.edu (P.H.
Dussault).
These authors contributed equally to the research.
Current address: Department of Chemistry, Thammasat University, Pathumthani
à
12120, Thailand.
Figure 1. HLCS catalysis.
http://dx.doi.org/10.1016/j.bmcl.2014.11.010
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

W. Sittiwong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5568–5571
5569
Figure 2. (a) Chemical structure of biotin-50-AMP and ketophosphonate (6a) and
hydroxyphosphonate (6b) analogs; (b) reported sulfamoyl, sulfonamide, triazole,
and phosphate inhibitors.
The objective of this study was to develop a new class of synthetic
HLCS inhibitors which could potentially be targeted to distinct cellu-
lar structures. Such an inhibitor would be a useful analytical tool in
studies of HLCS-dependent biotinylation events in the cytoplasm,
mitochondria, and nucleus. We based these studies on analogs of
biotin-50-AMP, namely biotin b-ketophosphonate-50-AMP (b-ketoP)
and biotin b-hydroxyphosphonate-50-AMP (b-hydroxyP), that sub-
stitute a hydrolytically stable phosphonate for the acyl phosphate
found in biotin-50-AMP (Fig. 2a). The use of phosphonates as unreac-
tive isosteres of phosphates is well established.8 For example, nonhy-
drolyzable aminoacyl analogs of aspartyl adenylate exhibit potent
inhibitory activity against Escherichia coli aspartyl-tRNA synthetase.9
There is precedence for the efficacy of structurally analogous com-
pounds sulfamides, sulfonamides, and 1,2,3-triazoles in the inhibi-
tion of a microbial biotin protein ligase, the HLCS ortholog BirA
(Fig. 2b).10–13 We also investigated biotinol-50-AMP, a known phos-
phate analog of biotin-50-AMP which replaces the carbonyl oxygen
with a methylene (CH2).10,14
Scheme 1. Synthesis of ketophosphonate (b-ketoP) and hydroxyphosphonate (b-
hydroxyP) analogs of biotin-50-AMP.
ketone phosphonate was observed (NMR) to readily undergo H/D
exchange upon dissolution in d4-MeOH.
Reduction of the ketone with NaBH4 resulted in formation of
the corresponding alcohol (4b) as a mixture of diastereomers at
the newly formed stereocenter. Stirring the ketone diester (4a) or
the alcohol diester (4b) in pyridine/water resulted in selective
demethylation to afford monoesters 5a or 5b, respectively.
Removal of the acetonide protecting group from the sugar followed
by neutralization with ammonium bicarbonate provided the target
biotin b-ketophosphonate (b-ketoP) 6a in 58% yield for two steps.
The same procedure, when applied to alcohol 5b, furnished bio-
tin-b-hydroxyphosphonate (b-hydroxyP) 6b in 99% yield.
Inhibitor synthesis: The central element of the synthesis is the
formation of a protected version of a biotin ketophosphonate
(4a) via condensation of a biotin-derived ketophosphonic acid (3)
with a protected adenosine (Scheme 1). The synthesis begins with
biotin methyl ester (1), prepared via the acid-catalyzed esterifica-
tion of biotin.15 Reaction with the carbanion derived from methyl
phosphonate was anticipated to offer a convenient route to a pre-
cursor of the desired phosphonates. However, reaction of ester 1
with the lithiated methylphosphonate, generated using lithium
bis(trimethylsilyl)amide (LiHMDS) or n-butyl lithium (n-BuLi),
resulted in poor yields. Fortunately, reaction of the ester with a
large excess of the lithiated phosphonate, followed by quenching
with deionized water to minimize demethylation of the phospho-
nate diester product, produced a 69% yield of dimethyl b-keto-
phosphonate 2.16 Selective monodemethylation with lithium
bromide provides a good yield of mono ester 3. The pyridinium salt
of 3 underwent coupling with 20,30-isopropylidineadenosine (i-PrA)
in the presence of O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluro-
nium hexafluorophosphate (HBTU) to provide a mixed phospho-
nate diester (4a) in 79% yield for two steps.17,18
Inhibition of HLCS: Biotin b-ketophosphonate-50-AMP (b-ketoP,
6a) inhibited HLCS in a dose-dependent manner. The polypeptide
p67 is a substrate for biotinylation by HLCS.19 When recombinant
p67 was incubated with recombinant HLCS and 20
presence of 50–500 M b-ketoBP, HLCS inhibition was maximal at
the highest inhibitor concentration tested. For example, the inhibi-
tion at 500 M b-ketoBP was 81.3% 11.1% (P<0.01; n = 4) com-
lM biotin in the
l
l
pared with vehicle control (Fig. 3A). Data are presented as
mean SD of 4 replicates. Incubation of p67 in the absence of HLCS
produced no detectable signal (lane 6 in Fig. 3B). Under the condi-
tions used here, IC50 and Ki for b-ketoBP equaled 39.7 1.9
2.0 0.1 M, respectively, see Supplementary materials for details
regarding assays and calculations. When tested under identical
lM and
No coupling was observed if N,N0-dicyclohexylcarbodiimide
(DCC) was substituted for HBTU. The acidic methylene of the
l

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W. Sittiwong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5568–5571
Figure 3. Inhibition of HLCS by biotin b-ketophosphonate-50-AMP. (A) HLCS activity was quantified using infrared absorbance in the presence of 20
lM biotin and various
concentrations of biotin b-ketophosphonate-50-AMP (One way-ANOVA; ⁄P<0.05; N = 4; Dunnett’s multiple t-test ⁄p<0.05, ⁄⁄p<0.01, ⁄⁄⁄p<0.001). (B) Representative gel,
depicting biotinylation of p67 in the presence of various concentrations of biotin b-ketophosphonate-50-AMP. Lane 6 shows p67 incubated in the absence of HLCS. (C)
Competitive inhibition of HLCS by biotin b-ketophosphonate-50-AMP.
conditions, b-hydroxyP inhibited HLCS activity by 67.7 10.0%
(P = 0.0001; n = 4) at concentrations of 500 M inhibitor. The IC50
and Ki values calculated under these conditions were
203.7 3.7 M and 10.4 0.2 M, respectively. b-KetoP is a com-
petitive inhibitor of HLCS, based on competition studies with bio-
tin. In these studies, the concentration of the inhibitor was held
objective of this study was to develop a synthetic HLCS inhibitor
capable of penetrating cell membranes and which could potentially
be targeted to distinct cellular structures. Such an inhibitor would be
a useful analytical tool in studies of carboxylase biotinylation in
cytoplasm and mitochondria, studies of chromatin protein biotinyl-
ation and HLCS-dependent formation of multiprotein gene repres-
sion complexes in nuclei, and studies of newly discovered species
of biotinylated proteins throughout the cell.
l
l
l
constant at 250
lM while that of biotin was varied from 0 to
320 M; a second curve was generated in the absence of inhibitor
l
(Fig. 3C). The apparent Vmax was similar for incubations with and
without inhibitor [31.5 1.6 vs 22.3 1.51 pmol biotinylated
p67/(nmol HLCS x s); n = 4] whereas the apparent Km for biotin
was increased by the addition of inhibitor (77.9 10.3 vs
Consistent with the importance of HLCS in intermediary metab-
olism and epigenetics, no living HLCS null individual has ever been
reported, suggesting embryonic lethality. HLCS knockdown in
Drosophila melanogaster (ꢀ30% residual activity) produces pheno-
types such as decreased life span and reduced heat resistance.21
Mutations and single nucleotide polymorphisms have been identi-
fied and characterized in the human HLCS gene; these mutations
cause a substantial decrease in HLCS activity, aberrant gene
regulation and metabolic abnormalities.6,22 Unless diagnosed and
treated at an early stage, homozygous severe HLCS deficiency is
characteristically fatal.23 Three independent cancer and patent
databases correlate HLCS loss or mutation with an increase in
detected tumors.24
1.6 1.8 lM biotin; N = 4). b-HydroxyP (6b) also acts as a compet-
itive inhibitor as evidenced by similar apparent Vmax values with
and without inhibitor [20.6 1.8 vs 22.6 1.4 pmol biotinylated
p67/nmol HLCS x s); n = 4] whereas reactions incubated with
inhibitor increased the apparent Km for biotin (82.5 20.4 vs
1.9 1.8 lM biotin; n = 4). As a negative control we conducted
incubations with a biotin ketophosphonic acid (compound 3 in
Scheme 1). This substrate incorporates an electrophilic carbonyl
carbon beta to a charged phosphonate but lacks the adenosyl frag-
ment hypothesized as essential for mediating HLCS inhibition.
Consistent with this theory, the ketophosphonic acid compound
did not inhibit HLCS (data not shown).
The results were compared against assays conducted with biot-
inol-AMP, a known phosphate analog of biotin-50-AMP which has
previously been employed for inhibition of BirA (biotin protein
ligase).10,11 Biotinol-AMP reduces HLCS activity by 98.01 0.1% at
Several classes of biotin-50-AMP analogs have been applied to
study the function of biotin protein ligases (BPLs), exemplified by
HLCS as well as BirA, an enzyme catalyzing biotinylation of acyl
carrier protein in prokaryotes.10,13,14 BirA from E. coli has 21%
sequence similarity to HLCS.25 Biotinol-50-AMP, a phosphate ester
lacking the acyl carbonyl of biotin-50-AMP, binds tightly to the
Escherichia coli biotin repressor (KD = 1.5 0.2 nM)11,20 and inhibits
biotin transfer to the acceptor protein.10,14 Biotinol-50-AMP also
concentrations of 500
8.8 3.6 M and 754 303 nM, respectively. When reactions incu-
bated with biotinol-AMP were challenged with increasing amounts
of up to 320 M biotin, the apparent Vmax decreased compared to
lM and has an IC50 value and Ki of
l
binds tightly to Staphylococcus aureus BPL (Ki = 0.03 0.01 lM).
The activity of this analog would appear to suggest the key role
of the phosphate moiety and the relative lack of importance of
hydrogen-bonding interactions to the acyl carbonyl of biotin-50-
AMP. However, other work has demonstrated that a sulfamoyl-
containing bisubstrate analog, replacing the acyl phosphate with
an acyl sulfonamide, strongly binds Mycobacteria tuberculosis BPL
(MtBPL); a co-crystal revealing multiple hydrogen-bonding inter-
actions between the protein and the acyl sulfonamide.12 More
recently, a biotin-50-AMP analog replacing the acyl phosphate with
l
reactions without inhibitor (22.6 1.4 vs 5.9 1.4; n = 4) and Km
increased (1.9 1.8 vs 146 79; n = 4) indicating the biotinol-
AMP most likely acts as a mixed inhibitor.
There remains limited knowledge regarding the structure of HLCS
and the mechanism of catalysis.11,13b,20 Similarly, little is known
about the basis for selectivity between the classic carboxylase targets
of HLCS and novel targets in chromatin and other proteins. The

W. Sittiwong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5568–5571
5571
a 1,2,3-triazole was found to bind tightly to BPL and exhibit >1100-
fold selectivity for the S. aureus BPL over the human homologue.13
This suggests the possibility of designing potent inhibitors of bac-
terial BPL. However, no similar approach has been used to study
the function of HLCS or human BPL.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
A model of the HLCS/biotin-50-AMP complex as well as the crys-
tal structure of biotin-50-AMP with BPL from Pyrococcus horikoshii
OT3 (pdb:1wqw) suggests the importance of hydrogen bonding
involving the carbonyl and phosphonate oxygen (Fig. S1).13b,22b
The b-ketophosphonate and b-hydroxyphosphonate analogs intro-
duced here maintain the natural charge state of biotin-AMP and
place a basic oxygen atom beta to the phosphonate group. How-
ever, in contrast to the BirA inhibitors described above, the keto-
phosphonate (b-ketoP, 6) incorporates an electrophilic carbon at
the location of the original acyl group in biotin-50-AMP. Although
the reduced activity of the new inhibitors compared with bioti-
nol-AMP suggests that preservation of an electrophilic center
(C@O) or hydrogen bonding group (CHOH) beta to phosphonate
is of limited importance in inhibitor design, we note that the
1,2,3-triazole analogs completely lacking a carbonyl group show
no inhibition toward human BPL. It is also possible that conforma-
tional differences between the acyl phosphate of biotin-50-AMP
and the phosphonate of 6a and 6b might also contribute to the
reduced binding observed.
1. (a) Chiba, Y.; Suzuki, Y.; Aoki, Y.; Ishida, Y.; Narisawa, K. Arch. Biochem. Biophys.
1994, 313, 8; (b) Suzuki, Y.; Aoki, Y.; Ishida, Y.; Chiba, Y.; Iwamatsu, A.; Kishino,
T.; Niikawa, N.; Matsubara, Y.; Narisawa, K. Nat. Genet. 1994, 8, 122.
2. (a) Bailey, L. M.; Wallace, J. C.; Polyak, S. W. Arch. Biochem. Biophys. 2010, 496,
45; (b) Bao, B.; Wijeratne, S. S.; Rodriguez-Melendez, R.; Zempleni, J. Biochem.
Biophys. Res. Commun. 2011, 412, 115.
3. Biotin; Zempleni, J., Wijeratne, S. S. K., Kuroishi, T., Eds.Present Knowledge in
Nutrition; Erdman, J. W., Jr., Macdonald, I., Zeisel, S. H., Eds.; International Life
Sciences Institute: Washington, D.C., 2012; 10, p 587.
4. Li, Y.; Malkaram, S. A.; Zhou, J.; Zempleni, J. J. Nutr. Biochem. 2014, 25, 475.
5. (a) Li, Y.; Hassan, Y. I.; Moriyama, H.; Zempleni, J. J. Nutr. Biochem. 2013, 24,
1446; (b) Xue, J.; Wijeratne, S.; Zempleni, J. Epigenetics 2013, 8(5), 504; (c) Liu,
D.; Zempleni, J. Biochem. J. 2014, 461, 477.
6. Suzuki, Y.; Yang, X.; Aoki, Y.; Kure, S.; Matsubara, Y. Hum. Mutat. 2005, 26, 285.
7. Lane, M. D.; Young, D. L.; Lynen, F. J. Biol. Chem. 1964, 239, 2858.
8. (a) Thatcher, R. J.; Campbell, A. S. J. Org. Chem. 1993, 58, 2272; (b) Engel, R.
Chem. Rev. 1977, 77, 349.
9. Bernier, S.; Akochy, P. M.; Lapointe, J.; Chênevert, R. Bioorg. Med. Chem. 2005,
13, 69.
10. Brown, P. H.; Cronan, J. E.; Grøtli, M.; Beckett, D. J. Mol. Biol. 2004, 337, 857.
11. Brown, P. H.; Beckett, D. Biochemistry 2005, 44, 3112.
12. Duckworth, B. P.; Geders, T. W.; Tiwari, D.; Boshoff, H. I.; Sibbald, P. A.; Barry, C.
E.; Schnappinger, D.; Finzel, B. C.; Aldrich, C. C. Chem. Biol. 2011, 18, 1432.
13. (a) Soares da Costa, T.; Tieu, W.; Yap, M.; Pendini, N.; Polyak, S.; Pedersen, D.;
Morona, R.; Turnidge, J.; Wallace, J.; Wilce, M.; Booker, G.; Abell, A. J. Biol. Chem.
2012, 287, 17823; (b) Paparella, A. S.; Soares da Costa, T.; Yap, M. Y.; Tieu, W.;
Wilce, M. C. J.; Booker, G. W.; Abell, A. D.; Polyak, S. W. Curr. Top. Med. Chem.
2014, 14, 4.
In conclusion, we have described a new class of inhibitors of
holocarboxylase synthetase HLCS based upon replacement of the
ester of biotin-50-AMP with a ketone or a secondary alcohol. The
analogs produce significant levels of inhibition with isolated
enzyme. Efficacy of the new inhibitors in vivo has not been tested
and further investigations are warranted.
14. Wood, Z. A.; Weaver, L. H.; Brown, P. H.; Beckett, D.; Matthews, B. W. J. Mol.
Biol. 2006, 357, 509.
15. Slavoff, S. A.; Chen, I.; Choi, Y. A.; Ting, A. Y. J. Am. Chem. Soc. 2008, 130, 1160.
16. Maloney, K. M.; Chung, J. Y. L. J. Org. Chem. 2009, 74, 7574.
17. Balg, C.; Blais, S. P.; Bernier, S.; Huot, J. L.; Couture, M.; Lapointe, J.; Chênevert,
R. Biorg. Med. Chem. 2007, 15, 295.
18. Campagne, J.-M.; Coste, J.; Jouin, P. J. Org. Chem. 1995, 60, 5214.
19. Kobza, K.; Sarath, G.; Zempleni, J. BMB Rep. 2008, 41, 310.
20. Naganathan, S.; Beckett, D. J. Mol. Biol. 2007, 373, 96. http://dx.doi.org/10.1016/
j.jmb.2007.07.020.
21. Camporeale, G.; Giordano, E.; Rendina, R.; Zempleni, J.; Eissenberg, J. C. J. Nutr.
2006, 136, 2735.
22. (a) National Center for Biotechnology Information, Online Mendelian
Inheritance in Man. 2008; http://www.ncbi.nlm.nih.gov/omim (accessed: 10/
29/2014).; (b) Esaki, S.; Malkaram, S. A.; Zempleni, J. Eur. J. Hum. Genet. 2012,
20, 428.
23. Thuy, L. P.; Belmont, J.; Nyhan, W. L. Prenat. Diagn. 1999, 19, 108.
24. UniProt UniProtKB. www.uniprot.org/uniprot/P50747 (accessed: 10/29/2014).;
Massague, J.; Bos, P. Metastasis promoting genes and proteins. www.faqs.org/
patents/app/20100029748 (accessed: 10/29/2014).; Institute for Biomedical
Technologies. Genes-to-system breast cancer database. www.itb.cnr.it/
breastcancer/php/showMostCorrelated.php?id=6664 (accessed: 10/29/2014).
25. Hassan, Y. I.; Moriyama, H.; Olsen, L. J.; Bi, X.; Zempleni, J. Mol. Genet. Metab.
2009, 96, 183.
Acknowledgments
A contribution of the University of Nebraska Agricultural
Research Division, supported in part by funds provided through
the Hatch Act (to P.D. and J.Z.). Additional support was provided
by NIH grants DK063945 and P20GM104320 (to J.Z.). Research
was conducted, in part, in facilities remodeled with support from
NIH (RR016544).
Supplementary data
Supplementary data (synthetic protocols, spectral listings, and
assay conditions) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.11.010.