Polyphosphate-containing bisubstrate analogues as inhibitors of a bacterial cell wall thymidylyltransferase

Article abstract of DOI:10.1039/c4ob02583k

A series of polyphosphate containing sugar nucleotide analogues were synthesized and evaluated as bisubstrate inhibitors of α-d-glucose 1-phosphate thymidylyltransferase Cps2L, the first enzyme in Streptococcus pneumoniael-rhamnose biosynthesis, and a nov

Full text of DOI:10.1039/c4ob02583k

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DOI: 10.1039/C4OB02583K
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ARTICLE
Polyphosphate-Containing Bisubstrate
Analogues as Inhibitors of a Bacterial Cell Wall
Cite this: DOI: 10.1039/x0xx00000x
Thymidylyltransferase
Deborah A. Smithen,a Stephanie M. Forget,a Nicole E. McCormick,b Raymond T.
Syvitski,a and David L. Jakeman*a,b
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
A series of polyphosphate containing sugar nucleotide analogues were synthesized and
evaluated as bisubstrate inhibitors of α-D-glucose 1-phosphate thymidylyltransferase Cps2L,
the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis, and a novel
antibacterial target. WaterLOGSY NMR spectroscopy demonstrated binding of bisubstrate
analogues to Cps2L and a spectrophotometric coupled assay was used to determine apparent Ki
values.
www.rsc.org/
of the substrates, proceeds through a highly negatively charged
trigonal bipyramidal transition state. 8, 10-12
Introduction
Streptococcus pneumoniae is a highly infectious, Gram-positive
bacterium that is considered to be the most important pathogen
in many pneumococcal infections including community-
1
acquired pneumonia, bacteremia and bacterial meningitis.
ꢀ
ꢀ
Management of such infections used to be relatively
straightforward through treatment with penicillin and other
microbial agents; however, in 1967 the first case of penicillin
non-susceptibility in S. pneumoniae was described and by the
1990’s, pneumococcal isolates were found to exhibit high
levels of resistance to penicillin and other β-lactam antibiotics.
ꢀ
2-4
The rapid development and spread of this resistance in
many countries has been of great concern in recent years and so
the development of novel antibiotics that bypass known
mechanisms of resistance is of the utmost importance.
The disruption of cell wall biosynthesis is a known
mechanism of action for many clinically used antibiotics and a
commonly pursued target given that cell wall assembly is
essential for bacterial survival and virulence. Cps2L is a
bacterial
cloned from S. pneumoniae that catalyses the first committed
step in
-rhamnose biosynthesis in many pathogenic bacteria, 5,
thymidylyltransferase
(nucleotidylyltransferase)
L
6
a necessary sugar constituent of the bacterial cell wall. This
Scheme 1. Physiological reaction catalyzed by Cps2L and ordered Bi-Bi
mechanism (inset).
process involves the condensation of α-D-glucose 1-phosphate
(Glc-1-P) and deoxythymidine triphosphate (dTTP) to yield
Cognizant that enzymes function to preferentially stabilize the
transition state that lies between substrates and reaction
products, it would be expected that a species that closely
5, 7
dTDP-glucose and pyrophosphate (PPi), Scheme 1.
Such
nucleotidylyltransferases represent attractive antimicrobial
targets in that they display broad substrate specificity and
homology across various bacterial species. 8, 9
The physiological reaction catalysed by Cps2L (Scheme 1)
is known to follow an ordered Bi-Bi mechanism characteristic
of nucleotidylyltransferases and, following step-wise addition
resembles the transition state, but could not be turned-over,
13
would act as a potent non-covalent inhibitor.
Species
comprising of structural components of both substrates would
also be considered bisubstrate analogues. 14-20 Studies carried
out concerning the transition states of phosphoryl transfer
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J. Name., 2013, 00, 1-3 | 1

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ARTICLE
DOI: 10.1039/C4OB02583K
corresponding sugar nucleotides. Following the reaction,
purification of the crude products was achieved in a step-wise
fashion: a chloroform wash was employed to remove organic-
soluble compounds. Chelex treatment was used to remove
metal cations. Alkaline phosphatase (ALP) affected enzyme-
assisted breakdown of any remaining nucleotide reagent, and
finally, reversed-phase column chromatography over C18 silica
was employed to remove aforementioned breakdown products
and isolate the compounds of interest. Whilst conversion to the
desired sugar-nucleotides appeared to be moderate in each case
enzymes have demonstrated the dominant role of charge
balance with regard to transition state stabilization.
Phosphotransfer enzymes will prioritize the retention of charge
21-24
at the expense of the preferred native geometry within the
22
active site.
With these paradigms in mind, we set out to
synthesize inhibitors of Cps2L that possess charge
complimentary to that of the binding site, with the hypothesis
being that compounds possessing the greatest amount of
negative charge would act as the best inhibitors. Furthermore,
structurally
related
compounds
including
diuridine
1
(according to analysis of the crude products using H and 31P
tetraphosphate (Up4U) have been approved for clinical use. 25
We herein report the synthesis of a series of sugar
nucleotide analogues that vary in choice of nucleobase and
length of polyphosphate linker with a view to mimicking the
highly anionic environment within the active site. All
compounds were evaluated for their ability to bind Cps2L using
NMR spectroscopy, difficulties relating to the co-elution of
structurally-related impurities during purification using
reversed-phase column chromatography were encountered.
Nevertheless, the isolated yields (8-20%) were comparable to
other recent approaches for phosphate-phosphonate coupling. 27
28
WaterLOGSY NMR
was used to qualitatively confirm
water-ligand
observed
via
gradient
spectroscopy
that sugar nucleotide analogues 2-7 bound Cps2L.
WaterLOGSY NMR measures nOe transfer from irradiated
water to ligands and to non-binding small molecules; protein-
bound water molecules possess an opposite nOe relative to
unbound water molecules free in solution, thus enabling a
distinction between ligands and non binders. The result is a net
opposite phasing in processed spectra for ligands versus
nonbinding small molecules. NMR experiments were carried
out between each of the chemically synthesized compounds (2-
7) and Cps2L in the presence of the enzyme cofactor Mg2+. In
addition, benzoic acid was added to each experiment as a non-
binding control, as it does not interact with Cps2L it was used
to identify the phase of non-binding molecules during
processing (Fig. 1). Analysis of the resulting spectra (Fig. 1)
revealed that all sugar-nucleotide analogues (2-7) demonstrated
binding to Cps2L as a result of signals phasing opposite to the
non-binding control, benzoic acid. dTDP-Glc (2), the natural
product of the physiological reaction catalyzed by Cps2L,
showed the clearest binding interaction of both sugar and
nucleotide moieties as evidenced by strong, well defined peaks.
(WaterLOGSY) NMR and were subsequently examined as
inhibitors of Cps2L using a coupled spectrophotometric
enzyme assay.
O
S
OTf
Ph
N
N
Base
Base
O
P
O
P
O
1
O
O
N
N
O
O
O
O
O
MgCl2, DIPEA
DMF, r.t., 5 min
n
n
OTf
⋅
NBu4
OH
R
OH
R
Base = dT, U, A
n = 1, 2, 3
OH
R = H, OH
O
HO
HO
O
P
HO
OH
O
O
O
⋅
NBu4
O
HO
HO
Base
Glc-1-P
O
P
O-
HO
O
O
O
OH
0 °C - r.t., 30 min
CHCl3 Wash, Chelex,
Alkaline phosphatase,
n
R
Chromatography (C18 silica)
8 - 20 % yield
dTDP−Glc 2
dTTP−Glc 3
dTP4−Glc 4
UTP−Glc 5
UP4−Glc 6
ATP−Glc 7
Scheme 2. Synthesis of sugar nucleotides 2-7 using sulfonylimidazolium salt (1)
as the coupling reagent.
Benzoic
acid
Glc
Buffer
Water
Nucleotide
°
Results and Discussion
Synthesis of a series of sugar nucleotide analogues (2-7),
varying in choice of nucleobase and length of polyphosphate
linker, were identified as targets. Specifically, thymidine (dT)
polyphosphates 2-4 contain the physiological nucleobase. To
evaluate the role of the nucleobase versus the role of the
polyphosphate linker in determining enzyme affinity, uridine
(U) derivatives 5 and 6, and adenosine (A) derivative 7 were
also synthesized since Cps2L has previously been shown to
turnover both uridine and adenosine nucleoside triphosphates.5
We chose the coupling method described by Mohamady et al.,
26
involving use of a sulfonylimidazolium salt (1) to facilitate
reaction between glucose 1-phosphate and the desired
nucleotide, Scheme 2, due to the impressive isolated yields.
Treatment of a nucleotide with coupling salt 1 is proposed to
produce a highly reactive imidazolium salt intermediate that
reacts immediately with sugar 1-phosphates to generate the
°
° °
2 | J. Name., 2012, 00, 1-3
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ARTICLE
DOI: 10.1039/C4OB02583K
Fig. 1. WaterLOGSY NMR Spectra confirming binding of compounds 2-7 to Cps2L.
The water-derived peak at 4.79 (●) was removed for clarity. Positively-phased
peaks indicate binding, negatively-phased peaks indicate non-binding.
linear phosphate groups from three to four within the
compounds also had a positive effect on the inhibitory activity
of these compounds, with tetraphosphate analogues providing
the most potent Ki values for both the deoxythymidine and
uridine-based compounds (compare entries 2 and 3; and 4 and
5). However, dTDP-Glc (2) demonstrated greater inhibition
than analogue 3. The increase in inhibition observed for the
tetraphosphates over the triphosphate analogues supports the
original hypothesis that either by increasing the number of
phosphate groups, we would more closely mimic the transition
state of the normal physiological reaction or provide a
bisubstrate-like species that provides binding interactions from
both substrates thereby inhibiting Cps2L. The nucleotide-based
inhibitors 2-7 were found to be 5-fold more potent than hexose-
phosphonate based-inhibitors, 30, 31 providing kinetic evidence,
in addition to the WaterLOGSY binding data, that Cps2L
prioritizes binding of the nucleoside base over the hexose.
In conclusion, a series of six sugar nucleotide analogues
were obtained via chemical synthesis and evaluated as
inhibitors of thymidylyltransferase Cps2L. WaterLOGSY NMR
was used to confirm that all compounds bind Cps2L, with the
nucleotide portion of each molecule indicating a strong binding
interaction with the enzyme in the resulting spectra. The most
potent inhibitors possessed a deoxythymidine base and/or a
tetraphosphate linker, and are the more effective Cps2L
inhibitors reported to date. Enhancement of the nucleobase
binding interaction via derivatization, or substitution of the
polyphosphate linker with non-scissile isosteres may produce
more potent inhibitors. Of significance is that the
straightforward 1D WaterLOGSY NMR experiment provided
qualitative insight into the relative importance of the nucleoside
versus hexose binding that was corroborated through a detailed
kinetic study.
Increasing the length of the polyphosphate linker from two
to three units in compound 3 and then four units in compound 4
resulted in the sugar-derived peaks becoming more dispersive,
potentially indicative of a weaker binding interaction, whilst the
nucleotide-derived peaks continued to demonstrate a strong
binding interaction. This can be rationalized by consideration of
the ordered Bi-Bi-mechanism of action reported for Cps2L,
whereby the nucleotide portion of the substrate is proposed to
bind initially. The sugar moiety would then be tethered to the
enzyme substrate complex via the polyphosphate linker,
whereby
a longer linker would likely result in less
magnetization being transferred to the sugar portion of the
molecule, thus producing a weaker nOe effect. A similar trend
was observed with the uridine-based compounds (5 and 6), with
a longer polyphosphate linker resulting in more dispersive
glucose-derived peaks in compound 6. ATP-Glc (7) was
observed to bind Cps2L with similar intensity compared to
tetraphosphates 4 and 6: however, given that ATP is a
5
substantially less reactive substrate than dTTP and UTP,
orientation of 7 is likely non-optimal.
Sugar-nucleotide analogues 2-7 were evaluated as inhibitors
of Cps2L at various concentrations (0-200 µM), against varying
concentrations of dTTP, using a 7-methyl-6-thioguanosine
(MESG)-based coupled spectrophotometric inhibition assay. 29,
30
The kinetic data obtained were fit to standard inhibition
equations by non-linear regression, using GraFit analysis
software, providing a Ki value for each of the compounds (2-7)
evaluated, Table 1. A Lineweaver-Burk plot was also obtained
in each case in order to demonstrate the mode of inhibition best
described for each compound (ESI).
Acknowledgements
Table 1. Inhibition of nucleotidylyltransferase Cps2L by sugar
nucleotide analogues 2-7.
The Canadian Institutes for Health Research and the Natural
Sciences and Engineering Research Council of Canada are
thanked for funding. We thank V. Schramm (Albert Einstein
College of Medicine) for the gift of the PNP plasmid. 32
-
Entry
Analogue
n (PO3 )
Ki (µM)
Kinetic
model
C
C
C
NC
NC
NC
1
2
3
4
5
6
2 (dT)
3 (dT)
4 (dT)
5 (U)
6 (U)
7 (A)
2
3
4
3
4
3
111a
144
85a
710
470
778
Notes and references
a
Department of Chemistry, Dalhousie University, 6274 Coburg Rd., PO
Box 15,000, Halifax, Nova Scotia, B3H 4R2, Canada.
aAverage Ki from two inhibition assays; C: Competitive; Non-Competitive.
b
College of Pharmacy, Dalhousie University, 5968 College St., PO
Box15,000, Halifax, Nova Scotia, B3H 4R2, Canada.
Results from this series of Cps2L inhibition assays revealed all
compounds evaluated (2-7) to be micromolar inhibitors of the
physiological reaction (Table 1) at constant Glc-1-P
concentration of 1 mM with an apparent Km for dTTP of 50 µM
(ESI). Within the series, deoxythymidine-based analogues (2-4,
entries 1-3) demonstrated the highest levels of inhibition (Ki 85
µM, entry 3) and were all found to be competitive-type
inhibitors with respect to the dTTP substrate. The uridine and
adenine derivatives were significantly less potent inhibitors
with non-competitive inhibition. Increasing the number of
Electronic Supplementary Information (ESI) available: HPLC traces and
retention times, Synthetic methods and NMR spectra of synthetic
compounds and kinetic plots. See DOI: 10.1039/b000000x
1 S. L. Kaplan and E. O. Mason Jr, Clin. Microbiol. Rev., 1998, 11, 628-644.
2 J. Linares, C. Ardanuy, R. Pallares and A. Fenoll, Clin. Microbiol. Infect.,
2010, 16, 402-410.
3 T. Strateva and D. Yordanov, J. Med. Microbiol., 2009, 58, 1133-1148.
J. Name., 2012, 00, 1-3 | 3
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Page 4 of 4
ARTICLE
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4OB02583K
4 R. Pallares, P. F. Viladrich, J. Linares, C. Cabellos and F. Gudiol, Microb
Drug Resist, 1998, 4, 339-347.
24 Y. Jin, D. Bhattasali, E. Pellegrini, S. M. Forget, N. J. Baxter, M. J. Cliff,
M. W. Bowler, D. L. Jakeman, G. M. Blackburn and J. P. Waltho,
Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 12384-12389.
5 S. C. Timmons, R. H. Mosher, S. A. Knowles and D. L. Jakeman,
Org. Lett., 2007, 9, 857-860.
25 A. M. Fahmy and D. R. Hardten, Clin. Ophthalmol., 2011, 5, 465-472.
26 S. Mohamady, A. Desoky and S. D. Taylor, Org. Lett., 2012, 14, 402-405.
6 M. S. Alphey, L. Pirrie, L. S. Torrie, W. A. Boulkeroua, M. Gardiner, A.
Sarkar, M. Maringer, W. Oehlmann, R. Brenk, M. S. Scherman, M. McNeil,
M. Rejzek, R. A. Field, M. Singh, D. Gray, N. J. Westwood and J. H.
Naismith, ACS Chem. Biol., 2013, 8, 387-396.
27 M. A. Martinez Farias, V. A. Kincaid, V. R. Annamalai and L. L.
Kiessling, J. Am. Chem. Soc., 2014, 136, 8492-8495.
7 S. A. Beaton, M. P. Huestis, A. Sadeghi-Khomami, N. R. Thomas and D.
L. Jakeman, Chem. Commun., 2009, , 238-240.
28 C. Dalvit, G. Fogliatto, A. Stewart, M. Veronesi and B. Stockman, J.
Biomol. NMR, 2001, 21, 349-359.
8 S. Zuccotti, D. Zanardi, C. Rosano, L. Sturla, M. Tonetti and M. Bolognesi,
J. Mol. Biol., 2001, 313, 831-843.
29 M. R. Webb, Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 4884-4887.
30 M. W. Loranger, S. M. Forget, N. E. McCormick, R. T. Syvitski and D. L.
Jakeman, J. Org. Chem., 2013, 78, 9822-9833.
9 S. Singh, G. N. Phillips Jr. and J. S. Thorson, Nat. Prod. Rep., 2012, 29,
1201-1237.
31 S. M. Forget, A. Jee, D. A. Smithen, R. Jagdhane, S. Anjum, S. A. Beaton,
D. R. Palmer, R. T. Syvitski and D. L. Jakeman, Org. Biomol. Chem., 2014, ,
10.1039/c4ob02057j.
10 S. M. Forget, D. Bhattasali, V. C. Hart, T. S. Cameron, R. T. Syvitski and
D. L. Jakeman, Chem. Sci., 2012, 3, 1866-1878.
11 A. Melo and L. Glaser, J. Biol. Chem., 1965, 240, 398-405.
32 A. Lewandowicz and V. L. Schramm, Biochemistry, 2004, 43, 1458-1468.
12 W. Blankenfeldt, M. Asuncion, J. S. Lam and J. H. Naismith, EMBO J.,
2000, 19, 6652-6663.
13 V. L. Schramm, Annu. Rev. Biochem., 2011, 80, 703-735.
14 D. Lavogina, E. Enkvist and A. Uri, ChemMedChem, 2010, 5, 23-34.
15 M. Izumi, H. Yuasa and H. Hashimoto, Curr. Top. Med. Chem., 2009, 9,
87-105.
16 J. Neres, N. P. Labello, R. V. Somu, H. I. Boshoff, D. J. Wilson, J.
Vannada, L. Chen, C. E. Barry III, E. M. Bennett and C. C. Aldrich, J. Med.
Chem., 2008, 51, 5349-5370.
17 M. Izumi, S. Kaneko, H. Yuasa and H. Hashimoto, Org. Biomol. Chem.,
2006, 4, 681-690.
18 D. M. Williams, D. L. Jakeman, J. S. Vyle, M. P. Williamson and G. M.
Blackburn, Bioorg. Med. Chem. Lett., 1998, 8, 2603-2608.
19 I. Kosiova, O. Simak, N. Panova, M. Budesinsky, M. Petrova, D. Rejman,
R. Liboska, O. Pav and I. Rosenberg, Eur. J. Med. Chem., 2014, 74, 145-168.
20 D. Rejman, N. Panova, P. Klener, B. Maswabi, R. Pohl and I. Rosenberg,
J. Med. Chem., 2012, 55, 1612-1621.
21 N. J. Baxter, G. M. Blackburn, J. P. Marston, A. M. Hounslow, M. J.
Cliff, W. Bermel, N. H. Williams, F. Hollfelder, D. E. Wemmer and J. P.
Waltho, J. Am. Chem. Soc., 2008, 130, 3952-3958.
22 M. J. Cliff, M. W. Bowler, A. Varga, J. P. Marston, J. Szabo, A. M.
Hounslow, N. J. Baxter, G. M. Blackburn, M. Vas and J. P. Waltho, J. Am.
Chem. Soc., 2010, 132, 6507-6516.
23 U. Abele and G. E. Schulz, Protein Sci., 1995, 4, 1262-1271.
4 | J. Name., 2012, 00, 1-3
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