C O M M U N I C A T I O N S
A similar strategy was employed in the synthesis of the sulfamate
analogues. D-Pantothenic acid was protected as a PMB acetal and
converted to NHS ester 5. Sulfamoyl tribenzoyl cytidine, obtained
by sulfamoyl chloride treatment of tribenzoyl cytidine, was then
linked to the activated NHS ester in the presence of Cs2CO3.18-20
Compound 6 was subjected to the aforementioned sequence of PMB
deprotection, phosphitylation and oxidation, and global deprotection
to generate the sulfamate analogues 7 and 8.
Phosphodiester 3 proved to be the most potent PPCS inhibitor,
showing nanomolar IC50 toward both Types I and III bacterial
enzymes and 145-1000-fold selectivity for bacteria PPCS over the
human enzyme (Table 1). Similar selectivity is seen with compound
4, which differs from 3 by the cyclization of the terminal phosphate
moiety, albeit with a large decrease in potency. Both compounds 7
and 8, containing the internal sulfamate linkage, display micromolar
IC50 toward bacterial PPCS with 20-740-fold selectivity for the
bacterial enzymes.
The compounds reported herein represent the first reported
inhibitors of PPCS. While very effective against the isolated
enzymes, these compounds exhibit no inhibitory effects against
bacterial growth, most likely due to lack of cellular penetration as
a result of their physiochemical properties. However, in vitro these
compounds show a marked selectivity toward both types of bacterial
PPCS, providing a foundation for the possible development of broad
spectrum antimicrobial agents. Efforts to cocrystallize these inhibi-
tors with all three types of PPCS are currently being investigated.
With these studies we hope to gain insight into the binding
determinants of selectivity and potency which could be capitalized
upon to design the next generation of inhibitors. Also, previous
attempts at obtaining crystal structures of PPCS with substrate
L-cysteine bound at the active site have not been successful.10
Because our compounds mimic the phosphopantothenoyl cytidylate
intermediate but are catalytically incompetent, it is possible that
we could capture a ternary crystal complex with PPCS, inhibitor,
and L-cysteine, which would provide a clear depiction as to the
mechanism of PPCS’s selectivity for L-cysteine.21
Table 1. IC50 of Compounds against Types I, II, and III PPCSsa
hsPPCS (II)
efPPCS (III)
spPPCS (III)
ecPPCS (I)
Acknowledgment. We thank Prof. Bruce Palfey for helpful
discussions. This work was supported by the University of
Michigan, College of Pharmacy (UM-COP). J.D.P. was supported
in part by a National Institutes of Health Chemistry and Biology
Interface Training Grant and in part by the Fred Lyons, Jr.
Fellowship administered by UM-COP. J.Y. was supported in part
by a U.S. Department of Homeland Security Fellowship adminis-
tered by the Oak Ridge Institute for Science & Education.
3
4
7
8
10 µM (1)
65 nM (9)
18 µM (4)
2.7 µM (0.2)
181 µM (7)
10 nM (2)
13 µM (4)
3.9 µM (0.2)
279 µM (27)
68 nM (9)
3.0 µM (0.3)
270 nM (3)
16 µM (5)
2.7 mM (0.2)
200 µM (11)
5.9 mM (0.6)
a hs ) human, ef ) E. faecalis, sp ) S. pneumoniae, ec ) E. coli.
Assays were performed in triplicate. Standard error shown in ( ).
Since a full steady state kinetic characterization of efPPCS has
recently been performed, this enzyme was used to determine the
kinetic mechanism and inhibition constant for the most potent
inhibitor, 3.9 Upon addition of various concentrations of 3 to the
PPCS assay, a pattern of slow-onset inhibition was observed (Figure
3). Subsequent plotting of kobs against inhibitor concentration
resulted in data points with a linear relationship. This is indicative
of a single-step enzyme inhibition mechanism characterized by slow
Supporting Information Available: Complete ref 4, synthetic and
biochemical experimental procedures, compound spectroscopic char-
acterization, and equations for inhibition constant determination. This
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Figure 3. Slow-onset, tight-binding inhibition of E. faecalis PPCS by
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