Synthesis and evaluation of stable substrate analogs as potential modulators of cyclodiphosphate synthase IspF

Article abstract of DOI:10.1039/c2md20175e

Stable IspF substrate analogs were synthesized. In the presence of substrate analogs, the E. coli IspF-MEP complex shows activities distinct from IspF, and bisphosphonates (BP) behave differently than their diphosphate (DP) counterparts. Bisphosphonate an

Full text of DOI:10.1039/c2md20175e

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Synthesis and evaluation of stable substrate analogs as
potential modulators of cyclodiphosphate synthase
IspF†
Cite this: Med. Chem. Commun., 2013,
4, 130
*
J. Kipchirchir Bitok and Caren Freel Meyers
Received 30th June 2012
Stable IspF substrate analogs were synthesized. In the presence of substrate analogs, the E. coli IspF–MEP
complex shows activities distinct from IspF, and bisphosphonates (BP) behave differently than their
diphosphate (DP) counterparts. Bisphosphonate analogs activate and/or stabilize IspF, and only the
closest structural substrate analog weakly inhibits the IspF–MEP complex.
Accepted 19th August 2012
DOI: 10.1039/c2md20175e
www.rsc.org/medchemcomm
In Nature, the distinct mevalonate (MVA)1,2 and 2Cmethyl- possible feed-forward regulatory mechanism. The turnover
D-erythritol phosphate (MEP) pathways3–5 produce dimethylallyl efficiency of IspF (KCmDPME2P ¼ 339 ꢀ 32 mM; kcat ¼ 61 ꢀ 3 minꢁ1
)
pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) increases 4.8-fold in the presence of MEP (in the presence of
for biosynthesis of the essential isoprenoid class of natural 500 mM MEP, KCmDPME2P ¼ 93.8 ꢀ 11; kcat ¼ 80.7 ꢀ 5 minꢁ1 and
MEP
products (Fig. S1†). While the MVA pathway is operational in AC
¼ 133 ꢀ 33 mM), and the IspF–MEP complex displays
50
plants, archaea, fungi and animals, the MEP pathway is utilized altered sensitivity to inhibitors.17
by higher plants, algae, bacteria and parasites.3–5 The MEP
Here we report the design, synthesis and evaluation of stable
pathway is widespread in pathogenic organisms, including M. substrate analogs as potential active site probes and modulators
tuberculosis and P. falciparum, and is therefore being pursued as of IspF activity. In light of our recent ndings, we have also
a potential target for development of anti-infective agents.6–8 evaluated these compounds as modulators of the more active
Cyclodiphosphate synthase IspF catalyzes the h step in the IspF–MEP complex, which may be the more physiologically
MEP pathway, converting 4-diphosphocytidyl-2C-methyl-D-
erythritol 2-phosphate (CDPME2P) to the cyclic diphosphate 2C-
methyl-D-erythritol 2,4-cyclopyrophosphate (MEcPP) (Fig. 1).9,10
In vitro, IspF also catalyzes formation of 2C-methyl-D-erythritol
3,4-cyclomonophosphate (MEcP) from the IspD product, 4-
diphosphocytidyl-2C-methyl-D-erythritol (CDPME, Fig. S2†).10
Importantly, IspF is essential in pathogenic organisms;11–13
however, efforts to target this interesting enzyme have produced
modest inhibitors,14–16 with the most potent thiazolopyrimidine
class exhibiting inhibitory activities in the low micromolar
range.15,16 These efforts by Diederich and colleagues to identify
inhibitors underscore the challenges in targeting the highly
polar active site of IspF.
Our interest in targeting isoprenoid biosynthesis in human
pathogens has motivated studies to understand pathway regu-
lation and to identify new IspF inhibitors. A recent investigation
in our laboratory toward understanding pathway regulation
shows that the upstream IspC product, 2C-methyl-D-erythritol
4-phosphate (MEP), enhances and sustains activity of IspF in a
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University
School of Medicine, Baltimore, MD, 21205, USA. E-mail: cmeyers@jhmi.edu
† Electronic supplementary information (ESI) available: The methylerythritol
phosphate (MEP) pathway; IspF reaction conditions; the synthesis of CBPME,
CBPME2P and MEBP; tabulated and normalized rates of CMP formation; CBP
inhibition of IspF or IspF–MEP complex. See DOI: 10.1039/c2md20175e
Fig. 1 IspF-catalyzed conversion of CDPME2P to MEcPP. C ¼ cytosine.
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relevant enzyme form.17 Our results provide further evidence component to bind in the more exible hydrophobic “pocket
that IspF and the IspF–MEP complex display distinct charac- II”.14 We reasoned that the unique methylerythritol scaffold
teristics in the presence of potential modulators. Contrary to our could also drive recognition of selective IspF inhibitors. Thus,
expectations, stable bisphosphonate substrate analogs are not methylerythritol bisphosphonate 3 (MEBP) lacks a cytidyl
potent inhibitors. Bisphosphonate analogs bearing the methyl- component but retains the methyerythritol component that is
erythritol motif enhance and/or sustain activity of IspF, while the also recognized by IspF.10,19 The stable P–C–P linkage was
closest structural homolog bearing a 2-phosphoryl group incorporated as a diphosphate mimic, with the idea that a
exhibits weak inhibitory activity and only against the IspF–MEP bisphosphonate prodrug strategy could eventually be pursued
complex. Further, this study reveals striking differences in the for intracellular delivery of MEBP should it display potent
behaviors of bisphosphonate-containing substrate analogs inhibition of cyclodiphosphate synthase activity.20
compared to their diphosphate counterparts and suggests that
the cyclodiphosphate synthase modulating activities of
bisphosphonates may be driven by the distinct steric and/or
electronic properties of the bisphosphonate scaffold (Fig. 2).
Synthesis
Substrate analogs 1 and 2 were synthesized starting from the
commercially available tetramethyl bisphosphonate 5 ester
(Scheme 1). Removal of a single methyl group by treatment with
PhSH and diisopropylethylamine afforded trimethyl ester 6.21,22
Chlorination of crude 6 using Ghosez’s reagent23,24 and subse-
quent reaction of monochloridate 7 with benzylidine protected
methylerythritol 8 (25) afforded compound 9.
Results
Design of substrate analogs 1 and 2
Structural and biochemical studies support the proposed
mechanisms of cyclization to give MEcPP or MEcP from
CDPME2P and CDPME, respectively;10,18,19 however, little is
known about the transition state structures or conformational
dynamics along these reaction coordinates. Bisphosphonate 2
(CBPME2P) is the closest structural homolog of the natural IspF
substrate, incorporating a stable P–C–P linkage to mimic the
diphosphate group in CDPME2P. Similarly, bisphosphonate 1
(CBPME) is a close structural homolog to IspD product CDPME,
known to undergo IspF-catalyzed conversion to MEcP.10 We
reasoned that 1 and 2 would mimic the binding properties of
CDPME or CDPME2P, respectively, and/or intermediates along
the corresponding reaction coordinates, but would be unable to
complete cyclization to release CMP, and thereby act as inhib-
itors. Further, studies using 1 and 2 as active site probes could
reveal distinctive features of IspF at the transition states toward
formation of MEcP or MEcPP and provide a basis for the
development of transition state analogs as selective inhibitors.
Benzylidine-protected methylerythritol-based precursors
have been successfully converted to MEP and MEcPP.25
However, removal of the benzylidine protecting group from late
stage intermediates en route to substrate analog 1 proved diffi-
cult in this system, consistent with the ndings reported by
Narayanasamy, et al.26 Consequently, intermediate 9 was sub-
jected to hydrogenolysis, and the resulting methylerythritol
bisphosphonate tetraester 10 was acetylated to give interme-
diate 11. Treatment of 11 with PhSH/Et3N followed by cation
exchange afforded bisphosphonic acid 12, which was coupled to
the protected nucleoside 13 (27–30) using Mitsunobu conditions.
Palladium-catalyzed removal of the allyloxycarbonyl (alloc)
protecting group from the 4-amino group of the cytosine base
was accomplished as described27,31 to afford compound 14.
Compound 14 was converted to the IspE substrate analogue
CBPME (1) in three tandem steps, and CBPME (1) was converted
to CBPME2P (2) by the action of kinase IspE (Fig. S3†).32
Substrate analog 3 (MEBP) was prepared using a similar
approach starting from tetrabenzyl bisphosphonate 15
(Scheme 2).33,34 Removal of a single benzyl group in the presence
of quinuclidine,35 followed by conversion to the corresponding
bisphosphonic acid and nally chlorination23,24 afforded inter-
mediate 16. Bisphosphonic monochloridate 16 was coupled to
benzylidene protected methylerythritol 8 (25) to give compound
17. Palladium-catalyzed hydrogenolysis of 17 afforded MEBP (3).
Design of substrate analog 3
Rationally designed IspF inhibitors incorporate the CDP
moiety14 or a cytidyl-like component15 to bind in the rigid, well-
conserved “pocket III” of the IspF active site, and a non-polar
Biochemical evaluation of IspF modulating activity of stable
substrate analogs
Bisphosphonate-containing substrate analogs 1 (CBPME), 2
(CBPME2P), 3 (MEBP) and control compound 4 (CBP) were
evaluated as modulators of IspF activity and the more active
IspF–MEP complex.
CBPME (1) and CBPME2P (2). The stable IspF substrate
analogs CBPME and CBPME2P are, in principle, incapable of
completing the cyclization reaction to release CMP and were
therefore expected to exhibit inhibitory activity against the
enzyme. Contrary to our expectations, the closest structural
Fig. 2 Stable substrate analogs as potential modulators of IspF activity. CBP (4)
serves as a bisphosphonate control and potential mimic of CDP.
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Scheme 1 Synthesis of stable substrate analogs 1 and 2. Conditions: (a) PhSH, iPr2Net, 12–14 h; (b) DOWEX 50WX8 H+ form; (c) (CH3)2C]C(Cl)N(CH3)2, CH2Cl2, 40 ꢃC;
(d) iPr2Net, DMAP, 51–65% over 3 steps; (e) Pd–C, H2 (g), MeOH: (f) Ac2O, iPr2NEt, DMAP, 72–82% over 2 steps; (g) PhSH, Et3N; (h) DOWEX 50WX8 H+ form, 76%; (i)
Ph3P, DIAD, THF, 62–78%; (j) PD(Ph3P)4, pTSO2Na, THF/ddH2O, 88–94%; (k) (i) PhSH, Et3N, DMF, 70 ꢃC, 9d, (ii) MeOH/H2O, pH 2.0, 24 h, (iii) 2 : 1 : 0.5
MeOH : H2O : Et3N, 18 h, 39%; (l) IspE, ATP, PEP, PK, 1 h, 43%.
through a 30 minute pre-incubation (column k). This is also in
contrast to its diphosphate counterpart, CDPME, which neither
enhances activity nor stabilizes IspF (columns b and k in
Fig. 3).17 CBPME appears to subtly enhance activity of the IspF–
MEP complex, producing a modest 1.3-fold increase in the rate
Scheme 2 Synthesis of MEBP 3. Conditions: (a) (i) Quinuclidine, toluene, 100 ꢃC,
1 h; (ii) 5% HCl; (iii) (CH3)2C]C(Cl)N(CH3)2, CH2Cl2, 40 ꢃC; (b) 8, iPr2NEt, DMAP,
51–65% over 3 steps; (c) H2, Pd/C, MeOH, 70–80 p.s.i., 2.5 h, 66%.
homolog to the natural substrate, CBPME2P, lacks inhibitory
activity against IspF (column d, Fig. 3). Rather, CBPME2P
prevents loss of enzyme activity to some degree (column l,
Fig. 3), as evidenced by a retention of ꢂ60% activity following a
30 minute pre-incubation (a 7.6-fold enhancement in the rate of
CMP formation, compared to control aer the same pre-incu-
bation, Table S1†). In contrast, CBPME2P exhibits weak, time-
dependent inhibition of the IspF–MEP complex (37% inhibition
following 30 min pre-incubation, column p in Fig. 3, Table S1†).
These results are consistent with our previous ndings that IspF
and the IspF–MEP complex display distinct sensitivities to
inhibitors,17 and suggest CBPME2P exhibits a surprisingly weak
affinity for the enzyme.
CBPME (1) is a close structural analog of the IspE substrate
CDPME, also known to undergo IspF-catalyzed cyclization to
give MEcP (Fig. S2†).10 In contrast to its phosphorylated coun-
Fig. 3 Effects of CDPME, CBPME or CBPME2P on the rate of CMP formation
terpart (CBPME2P), CBPME enhances IspF activity 2.2-fold
catalyzed by IspF or the IspF–MEP complex, in the presence of 100 mM CDPME2P.
(column c in Fig. 3, Table S1†), but fails to stabilize the enzyme Substrate analogs and MEP are added to a final concentration of 500 mM.
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of CMP formation compared to the IspF–MEP complex alone inhibitory activity against the IspF–MEP complex (IC50
¼
(column g in Fig. 3, Table S1†), and this enhanced activity is not 343.4 ꢀ 24 mM, Fig. S4†). Further, CDP displays apparent time-
signicantly affected following pre-incubation of CBPME with dependent inhibition of the IspF–MEP complex (46% inhibition
the IspF–MEP complex (column o, Fig. 3). This is again in following pre-incubation, column o) whereas the more
contrast to its diphosphate counterpart, CDPME, which has no pronounced inhibitory activity of CBP against the IspF–MEP
apparent effect on the IspF–MEP complex (Fig. 3, column n).
complex does not appear to be time-dependent (69% inhibition
MEBP (3) and CBP (4). MEBP (3) lacks the cytidyl component at 0 minutes versus 74% inhibition following pre-incubation
recognized by “pocket III” in the IspF active site, but retains the with IspF–MEP).
unique methylerythritol group recognized by “pocket II” and
was therefore expected to display inhibitory activity. CBP (4) was
used as a bisphosphonate-containing control and stable coun-
Discussion
terpart for the known inhibitor CDP. Interestingly, MEBP The MEP pathway is indispensable in many pathogenic organ-
enhances and sustains activity of IspF. At a nal concentration isms;11,12 thus the enzymes of this metabolic pathway present
of 500 mM, MEBP modestly increases the rate of CMP formation new targets for the development of anti-infective agents. The
by 1.6-fold (Table S2†). However, in the presence of 1000 mM h enzyme, cyclodiphosphate synthase IspF, has been the
MEBP, IspF activity is further enhanced to 1.8-fold above focus of efforts to develop inhibitors of isoprenoid biosyn-
control (column b, Fig. 4), comparable to enhancement thesis.11–16 Rationally designed IspF inhibitors incorporate
observed by the structurally related activator MEP.17 At this recognition elements for the cytidine binding pocket whereas
higher concentration, MEBP also exhibits profound stabilizing compounds identied from a high throughput screen inhibit
effects, retaining 54% activity (column j, Fig. 4) and represent- via an unknown mechanism.16 The present study attempted
ing an 8.7-fold increase in the rate of CMP formation compared new approaches for the design of IspF inhibitors, and resulted
to the control experiment aer the same pre-incubation. in unexpected yet enlightening outcomes. Despite a lack of
Notably, MEBP does not modulate activity of the IspF–MEP potent inhibition by any of the IspF substrate analogs presented
complex (Fig. 4 and Table S2†).
here, this study emphasizes several important points that
As expected, CBP inhibits IspF (IC50 ¼ 797.3 ꢀ 57 mM, should be considered in the design and evaluation of future
Fig. S4†) with an activity comparable to CDP (IC50 ¼ 768 mM).17 IspF inhibitors. First, and consistent with our previous nd-
However, while the introduction of MEP ablates inhibitory ings,17 we provide new examples which highlight the striking
activity of CDP (column g in Fig. 4),17 CBP exhibits enhanced differences in the behaviors of IspF and the IspF–MEP complex
in the presence of modulators. It is clear that a better under-
standing of the mechanism and physiological relevance of IspF
activation by MEP is required to fully appreciate IspF as a
potential drug target, and attention should be given to the
conditions under which IspF inhibitors are screened.
Second, although we postulated that incorporation of the
methylerythritol element in potential IspF modulators could
direct binding to “pocket II” of the IspF active site and cause
inhibition, it appears the methylerythritol component instead
serves as a recognition element to enhance and/or sustain IspF
activity. In the present study, this is demonstrated best by
MEBP, which appears to behave as a lower affinity MEP analog
to enhance and sustain IspF activity.
Finally, incorporation of a stable bisphosphonate scaffold, a
technique commonly used to study phosphoryl transfer
enzymes, offers little to impart potent inhibitory activity
through stabilizing a transition state conformation around the
penta-coordinate intermediate in IspF catalysis. Direct
comparisons of bisphosphonate to diphosphate counterparts
(CDPME vs. CBPME and CDP vs. CBP) suggest that the
bisphosphonate linkage is not a suitable isostere for diphos-
phate in this system. The pKa values for the rst and second
ionizations are well below physiologic pH for diphosphate and
bisphosphonate scaffolds; thus, we presume CDPME and
CBPME are both dianionic under the assay conditions. The
different activities of CDPME and CBPME might therefore arise
from differences in steric properties of diphosphate and
bisphosphonate linkages;36 this phenomenon might also
Fig. 4 Effects of MEBP (1000 mM) and CBP (700 mM) and CDP (700 mM) on the
rate of CMP formation catalyzed by IspF or the IspF–MEP complex in the presence
of 100 mM CDPME2P. *No activity was observed in the presence of CDP following
a 30 minute pre-incubation.
explain the unexpected weak inhibition by CBPME2P against
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