Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors

Article abstract of DOI:10.1002/cbic.201700052

Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H⁺/2e^? reduction. IspH contains a 4 Fe?4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe?4 S clusters by η¹ coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H₂O, O₂) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a K_i value of ≈500 nm against Pseudomonas aeruginosa IspH.

Full text of DOI:10.1002/cbic.201700052

Accepted Article
Title: Spectroscopic and Computational Investigations of Ligand
Binding to IspH: Discovery of Non-Diphosphate Inhibitors
Authors: Bing O'Dowd, Sarah Williams, Hongxin Wang, Joo Hwan
No, Guodong Rao, Weixue Wang, J. Andrew McCammon,
Stephen P. Cramer, and Eric Oldfield
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ChemBioChem
10.1002/cbic.201700052
COMMUNICATION
Spectroscopic and Computational Investigations of Ligand
Binding to IspH: Discovery of Non-Diphosphate Inhibitors
[
a]
[b]
[c,d]
[e]
[a]
[e]
Bing O'Dowd , Sarah Williams , Hongxin Wang , Joo Hwan No , Guodong Rao , Weixue Wang ,
J. Andrew McCammon[b,f,g], Stephen P. Cramer[c,d] and Eric Oldfield[a]*
+
Abstract: Isoprenoid biosynthesis is an important area for anti-
infective drug development and one target is IspH, (E)-1-hydroxy-2-
methyl-but-2-enyl 4-diphosphate (HMBPP) reductase, which forms
isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP
in a 2H /2e reduction. IspH contains a 4Fe-4S cluster and here, we
first investigated how small molecules can bind to the cluster using
HYSCORE and NRVS spectroscopies. The results of these as well
as other structural and spectroscopic investigations led to the
conclusion that in most cases, ligands bind to IspH 4Fe-4S clusters
are involved in 2H /2e reductions: IspG converts 2-C-methyl-D-
erythritol-2,4-cyclo-diphosphate (MEcPP, 1, Scheme 1) to (E)-1-
hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP; 2), and
IspH converts 2 to dimethylallyl diphosphate (3) and isopentenyl
diphosphate (4), the building blocks of isoprenoid biosynthesis.
Both enzymes are not produced by humans (who use the
mevalonate pathway for isoprenoid biosynthesis), but are
essential in plants and in many bacteria and protozoa, so IspG
and IspH are of interest as potential herbicide or drug targets. In
earlier work[2] we reported the first structure (PDB ID 3DNF) of
an IspH, from Aquifex aeolicus, finding that one Fe atom in the
cluster was lost during crystallization, and similar results (PDB
+
-
1
th
via  coordination, forming tetrahedral geometries at the unique 4
2
Fe, ligand side-chains preventing further ligand (e.g. H O, O )
2
binding. Based on these ideas, we sought using in silico methods to
find drug-like inhibitors that might occupy the HMBPP substrate
binding pocket and bind to Fe, leading to the discovery of a
[
3]
ID 3F7T) were reported for IspH from E. coli . However, using
computational modeling to add back the unique 4th Fe, together
with computational docking of the HMBPP substrate 1, we were
able to produce[1-2] ligand-bound structures that were very similar
to later 4Fe-4S IspH/1 X-ray structures[ , and these structures
have led to detailed mechanism of action models[
barbituric acid analog having a K
aeruginosa IspH.
i
~ 500 nM against Pseudomonas
4-5]
Introduction. The enzymes IspG ((E)-1-hydroxy-2-methyl-but-2-
enyl 4-diphosphate synthase, also known as GcpE) and IspH
6-8]
for IspH
(
(E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate reductase,
catalysis. In other early work we discovered, based on previous
reports that alkynes could bind to and be reduced by 4Fe-4S
clusters[ , that alkyne diphosphates such as 5 (propargyl
diphosphate, PPP) were low µM inhibitors of IspH (as well as
also known as LytB) are the last two enzymes of the 2-C-methyl-
D-erythritol 4-phosphate (MEP) pathway of isoprenoid
biosynthesis in many bacteria, as well as in some protozoa, and
9-10]
[
1]
[1]
in plants . They are both 4Fe-4S cluster-containing proteins that
IspG, which also contains a 4Fe-4S cubane-like structure) .
Plus, the 1-amino (6) and
1
-thio (7) analogs of
HMBPP have been found
to inhibit IspH with K ~20-
0 nM[11-12], and we[13] and
others (PDB ID codes:
i
5
3
ZGL,
3ZGN)
their
have
X-ray
are
reported
structures
which
basically the same as
found with the HMBPP
substrate,
binding to the unique, 4
Fe. We also reported[
with
N,S
Scheme 1. Structures of IspG (GcpE) and IspH (LytB) substrates and reaction products, and some ligands/inhibitors of
interest. OPP in 5-7 is diphosphate.
th
14]
[
[
[
[
[
[
[
a] B. O’Dowd, G. Rao, Prof. Dr. E. Oldfield
Department of Chemistry, University of Illinois, 600 South Mathews
Avenue, Urbana, IL 61801, USA
b] S. Williams, Prof. Dr. J.A. McCammon
Department of Chemistry & Biochemistry, University of California at San
Diego, La Jolla, CA 92093, USA
c] Dr. H. Wang, Prof. Dr. S.P. Cramer
Department of Chemistry, University of California, 1 Shields Avenue,
Davis, CA 95616, USA
d] Dr. H. Wang, Prof. Dr. S.P. Cramer
Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley,
CA 94720, USA
e] J.H. No, W. Wang
Center for Biophysics and Computational Biology, 607 South Mathews
Avenue, Urbana, IL 61801, USA
several other IspH-ligand complex structures with, in each case,
a single O-containing ligand (alcoholate or enolate) bound to the
unique, 4th Fe atom with Fe-O bond lengths of ~2 Å. What has
been (and still is) missing is the X-ray structure of a “ligand free”
4 4 4 4
IspH-either reduced ([Fe S ] ) or oxidized ([Fe S ] ), the
problem being that it has not been possible to crystallize the 4-
Fe containing protein. However, the results of 57Fe Mössbauer
indicated the presence of a 5 or 6-coordinate
th iron in “ligand-free” oxidized IspH, with three cluster S and
most likely three additional N/O ligands bound to the 4 Fe.
More recently, Faus et al.[ reported a NRVS (nuclear resonant
vibrational spectroscopy) investigation of oxidized IspH and
suggested that the three non-cluster ligands were
molecules (or presumably OH , or a mixture of both). This
+
2+
[15]
spectroscopy
4
th
16]
f] Prof. Dr. J.A. McCammon
Howard Hughes Medical Institute, University of California at San Diego,
La Jolla, CA 92093, USA
2
H O
g] Prof. Dr. J.A. McCammon
-
National Biomedical Computation Resource, University of California at
San Diego, La Jolla, CA 92093, USA
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ChemBioChem
10.1002/cbic.201700052
COMMUNICATION
ν Fe-Sbr
ν Fe-Xt
δ S-Fe-X
addition, we used HYSCORE (hyperfine sublevel correlation
-
A)
spectroscopy) to investigate how another small ligand, CN ,
-
cluster
torsion
might bind to reduced IspH, of interest since CN can be involved
in -back-bonding with d-orbitals. The results of these
experiments together with an examination of numerous IspH and
IspG structures suggested the importance of  -bonding of
th
ligands to the 4 Fe, together with the presence of a bulky side-
0
100
200 300
400
500
600
chain, for IspH inhibition, so we then used in silico screening of
possible inhibitors, finding interesting new drug-like leads that,
we propose, bind in this manner.
-
1
B)
Energy (cm )
Results and Discussion
In the following we first investigated the interactions of IspH with
the alkyne diphosphate 5, as well as with a small molecule
-
ligand, CN , to see if there were any interactions of the alkyne
-
with the cluster, and whether the anionic species CN bound
since in principle, both might be involved in metal-ligand π-
bonding/back-bonding. Then, using information from these and
other related studies, we used in silico screening to try and find
new, drug-like inhibitor leads.
0
100
200
300
400
500
600
-1
Energy (cm )
NRVS spectroscopy of the IspH-5 complex. NRVS provides
C)
57
Fe-specific information on the partial vibrational density of
states (PVDOS) for Fe-X vibrational modes in a molecule[18]. For
systems containing 4Fe-4S cubane-like clusters, such as IspH,
there are typically four main NRVS features, as shown
[
18-21]
schematically in Figure 1A
: cluster torsional modes occur at
5
-1
-1
<
100 cm ;  (S-Fe-X) bending modes at ~150 cm ;  (Fe-S)
(
(
cluster) stretching modes at ~280 cm-1 and
terminal) stretching modes in the ~320-370 cm-1 range.
 (Fe-Ster)
Although there are of course additional mixed modes, these
NRVS spectral features are present in most if not all species
containing 4Fe-4S as well as 4Fe-3S clusters.[18-21] For example,
the NRVS spectrum of oxidized Pyrococcus furiosus ferredoxin
containing a D14C mutation (which therefore has 4 Cys ligands;
Figure 1B, black line)[20], is very similar to that seen with the
Figure 1: NRVS spectra. A, Cartoon representation of a NRVS
spectrum of a 4Fe-4S cluster-containing σ-bonding ligands (e.g. OR,
NHR, SR, Cl) at a 4 , unique Fe-site. B, Experimental spectra of
th
model compound [Fe
[Fe Cl
]2- cluster in which the four terminal ligands are Cl
Figure 1B, grey line) . Basically the same features are also
4
S
4
Cl
4
](Ph
4
P)
2
,
which contains
a
oxidized P. furiosus D14C ferredoxin (PfFd; black), PfFd+NO (red) and
13
4
S
4
4
[
4 4 4 4 2
Fe S Cl ](Ph P) (grey). C, EcIspH + 5 (red) and [ C]-5 (black).
[
20]
(
structure is surprisingly labile, leading under crystallization
conditions to loss of the 4th Fe. “Ligand-free” IspH is also very
seen in oxidized IspH with HMBPP (2), the amino-analog (6) or
the thiol analog (7) as ligands [16], in which O, N or S are directly
sensitive to O
liganded species are much less sensitive to cluster degradation
by O [17], suggesting that the presence of relatively bulky ligand
side-chains might block the H O/O binding that leads to lability.
For example, in the PPP (5) structure (crystallized from oxidized
2
while HMBPP, PPP as well as an enolate-
th
bonded to the 4 Fe. In sharp contrast, these features are all
less obvious (or absent) in the NRVS spectrum of IspH in the
absence of 2, 6 or 7[16]. In the presence of NO, the 4Fe-4S
2
2
2
[22]
cluster in P. furiosus D14C ferredoxin
is converted to
]- and the same set of peaks
at ~150, 280 and 370 cm ) as seen in the 4-Cys liganded
Roussin's black salt, [Fe
4
S
3
(NO)
-1
7
-
th
IspH), there is a single H
2
O (or OH ) bound to the 4 Fe[14] while
(
the acetylene group is ~3.6 Å from the Fe, acting perhaps as a
barrier to water and oxygen ligands.
In our EPR work on reduced IspH-5, we concluded (based in
4 4
part on early observations of Fe S –acetylene interactions) that
there could be -bonding with the 4th Fe in the reduced cluster,
but with oxidized IspH, S = 0, so the system is not accessible via
EPR and it is not clear if there is any alkyne interaction with the
cluster. We thus used NRVS to investigate the IspH-5 system. In
protein are present. However, there are in addition strong peaks
-1
at ~540, 610 cm , Figure 1B (red line), suggesting a contribution
from Fe-N-O and/or N-Fe-N vibrational modes, due in part to
metal-ligand pi-bonding. We thus investigated the NRVS spectra
of the IspH-5 complex to see whether there might be any
evidence for interaction between the alkyne group and the
oxidized 4Fe-4S cluster (Fe-C bonding) that we previously
proposed to be important in the reduced protein.
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COMMUNICATION
identical to those found (g = 2.09, 1.94,
1.93) for CN- bound to Shewanella
oneidensis HydG (minus the “dangler
Fe”)[25] involved in formation of the
B)
C)
A)
[
Fe(CO)
2
CN] synthon in hydrogenase
function. Moreover, the HYSCORE
spectrum of [ CN]-SoHydG has a 13C
isotropic hyperfine coupling Aiso = -2.7
MHz, similar to the Aiso = -2.9 MHz in
13
PfFd with bound 13CN[26] and the Aiso
~
N
1
5
2
.5 MHz we find here. Plus, the
13
15
-
Figure 2: HYSCORE spectra of IspH•CN. A, Experimental HYSCORE spectrum of EcIspH•13C15N.
HYSCORE result for [ C N] bound to
SoHydG [25] is extremely similar to that
+
13
Sample is reduced ([Fe
0
4 4
S ] ) with dithionite. B, Simulation of C hyperfine coupling with A = [-3.9, -3.8,
15
.1] MHz, Euler angle = [0, 40±5, 0]°. C, Simulation of N hyperfine coupling with A = [1.1, 1.1, 2.3] MHz,
we observe with IspH. So, IspH,
SoHydG (the 4Fe-4S cluster) as well as
the (wild type) ferredoxin all appear to bind CN to the 4 Fe,
forming a tetrahedral species. It should be noted, however, that
Euler angle = [0, 30±10, 0]°. Euler angle follows zyz convention.
We show in Figure 1C the NRVS spectrum of EcIspH in the
-
th
2+
oxidized state ([Fe
4 4
S ] ) bound to 5 (red line). The spectrum is
very similar to that seen with HMBPP (2) as well as the amino 6
-
CN is actually a very poor IspH inhibitor (IC50>1 mM) and, as
[
16]
and thiol 7 analogs of HMBPP, bound to IspH,
and the
noted by Suess et al.[25], CN- binds only weakly to other
2
-
[16]
[
Fe
4
S
4
Cl
4
]
model compound , consistent with each Fe being
bound to 3 cluster sulfurs and a single 4 atom. We also found
no spectral shifts when a uniformly 13C-labeled analog of PPP
biological[26] as well as synthetic 4Fe-4S clusters, and cysteine
[27]
th
-
[25]
displaces CN from HydG
.
Ligand binding to IspH and IspG: Clues for inhibitor
(
5) was bound to the protein (blue line, Figure 1C). These results
discovery? When taken together, the results shown above
together with other reported work , show that there are several
ways that ligands can bind to IspH, and suggest a potential route
to finding new inhibitors. First, most ligands bind with 1
[
14]
are consistent with the presence of a single water molecule
[1]
th
binding to the 4 Fe—the tetrahedral geometry seen in the other
IspH structures with 2, 6 and 7 [16]—with no significant bonding
between the alkyne group and the cluster. A compilation of the
NRVS spectra of all of the variously ligated protein and model
compound 4Fe-4S cluster-containing systems discussed above,
highlighting their similarities, is shown in Supporting Information
Figure S1. The spectra with 5 are also dissimilar to that
observed with IspH containing 3 H O molecules bound to the 4th
2
Fe, again consistent with the lack of multiple (alkyne, water)
interactions with the 4th Fe.
We next sought to see how other small molecules/ions might
bind to the 4Fe-4S cluster. Attempts to bind CO were
unsuccessful, as determined by UV-VIS and EPR spectroscopy.
However, in previous work[23] we showed that CN bound to
reduced IspH, yielding an EPR spectrum characterized by g-
coordination with O, N and S binding via -interactions with the
th
-
4
Fe. Second, potential -bonding species (CN , CO, propargyl
alcohol) do not bind strongly. Third, while  as well as 3
coordination is possible , these cases are rare. Fourth, when
2
[
1]
alkyne diphosphates bind to oxidized IspH, the main cluster
th
-
interaction is with a 4
H
2
O (or OH ), the alkyne fragment
molecules. Fifth, the
blocking addition of further
2
H O
diphosphate moiety must contribute in a major way to IspH
inhibition, because propargyl alcohol itself is a very weak
inhibitor (K>10 mM). Sixth, these general patterns of ligand
i
binding are very similar in IspH and in IspG. This is shown in
-
Figure 3A in which we compare PPP (5) binding to IspH and
IspG. In both cases, there is a 4th
H
2
O bound to the unique 4th
values of 2.08, 1.94 and 1.93 (with a small shoulder at g =
2
were unknown. We thus next collected HYSCORE spectra using
[13
there
HYSCORE features that
Fe (dFe-O = 1.9 Å), while the alkyne is more distant (d ~3.6 Å).
The IspH•5 structure is very similar to the structures found with
the amino (6) and thiol (7) HMBPP analogs, the IspH•5 water
.05[ ), but the number of ligands as well as the binding mode
23]
15
-
C N] bound to E. coli IspH. As can be seen in Figure 2A,
are clearly
A)
B)
C)
D)
are
consistent
with
-
binding of a single CN to
the unique, 4th Fe in the
3
.6
cluster.
EasySpin program
simulated
coupling tensors of A( C)
[-3.9, -3.8, 0.1] MHz,
Using
the
we
3.6
3.6
3
.5
[
24]
hyperfine
1
3
=
1
5
Figure 2B, and A( N) =
1.1, 1.1, 2.3] MHz,
Figure 3: Comparisons between IspH/IspG structures with various ligands binding to the unique, 4th Fe site. A, PPP (5)
bound to EcIspH (red) and EcIspG (green) (PDB ID codes 3URK, 4S3E). B, Superposition of EcIspH PPP (5)/H O (red)
and thiolate (cyan, 7) X-ray structues (PDB ID codes 3URK, 4H4E). C, Superposition of PPP (5)/H O IspG (green)
structures with PPP (5)/H O-GcPE reaction intermediate structures (magenta) (PDB ID codes 4S3B, 4S3E). All structures
contain a σ-bonding ligand (H
Fe lead to low stability; a H O and a “bulky” ligand (that prevents binding of additional water molecules) leads to more
[
2
Figure 2C. Interestingly,
the g-values observed[23]
in the EPR spectrum of
IspH•CN (g=2.08, 1.94,
2
2
th
th
2
2
O, RNH , RSH, enolate) at the unique, 4 Fe structure. Three water molecules binding to 4
2
2
stable species. D, Superposition of E307 from AaIspG (blue) and PPP (5)/H O-GcPE structures (green) (PDB ID codes
1.93)
are
virtually
3NOY, 4S3E).
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COMMUNICATION
1
.0
E)
F)
G)
A)
C)
8
ox.
0
.5
PaIspH
red.
K = 0.5 μM
i
IC50 = 22 μM
0
.0
-2
0
2
red. + 9 (5 eq.)
red. + 9 (9 eq.)
log10(inhibitor/μM)
D)
B)1
.0
0
9
0
.5 5
EcIspH
K = 3 μM
i
IC = 23 μM
5
0
2.4 2.2 2.0 1.8
g-factor
1.6
0
.0
0
-
2
0
2
4
log10(inhibitor/uM)
log10(inhibitor/μM)
Figure 4: IspH inhibition by barbiturate analogs. A,B. Dose-response curves for EcIspH inhibition. C, Computational docking structure of 9 binding to oxidized
AaIspH. View from electron transfer side. D, View from substrate binding side. E, Side view. F, Proposed binding of the barbiturate enolate group in 9 to the
th
unique, 4 Fe in the 4Fe-4S cluster in oxidized AaIspH. G, 9 GHz EPR spectra of PaIspH. Oxidized protein (top, orange); dithionite reduce (red); dithionite
reduced plus 5 eq. 9 (green); dithionote reduced plus 9 eq. 9 (blue).
being in the same position as the 6, 7 NH
2
and SH groups, and
occupy the large substrate-binding site seen in the early X-ray
structures, with the enolate form of the barbiturate reacting with
the Fe, similar to the binding of related barbiturate enolates to
the diphosphate groups overlap, Figure 3B. The IspH•5 water
also co-locates with the ligand-bound oxygen in the IspG-
MEcPP (1) 1st reaction intermediate[28], Figure 3C, and the
IspG•5 bound water is in the same position as a carboxylate
oxygen in E307, in IspG (Figure 3D). So, in the vast majority of
cases the 4th Fe has a tetrahedral coordination geometry with O,
N or S binding to Fe. Unfortunately, these potent diphosphate-
containing inhibitors are not active in cells, presumably because
the diphosphate groups are very highly charged, reducing cell
penetration. These observations led us to try and find more
lipophilic, drug-like species that might bind to Fe, while also
occupying the relatively large substrate-binding pocket.
Zn2+ in other metalloproteins [32-33]
.
Interestingly, we were unable to obtain EPR spectra of 9
bound to IspH (from the pathogen Pseudomonas aeruginosa),
ligand addition resulting in loss of signal intensity, Figure 4G,
due perhaps to a shift in redox potential on ligand binding, or a
change in relaxation behavior but in either case, 8, 9 appear to
represent interesting new IspH inhibitor leads for further
development since they are far more lipophilic than the
[
34]
diphosphates, they do not violate Lipinski's rules and they are
not PAINS compounds[35]
.
We thus next used an in silico approach to screen a library of
drug-like compounds from the ZINC and NCI libraries against
Conclusions
both Aquifex aeolicus IspH (PDB ID 3DNF) and E. coli IspH
The results we have shown above are of interest for a number of
reasons. First, we find that NRVS spectra of the alkyne
diphosphate inhibitor 5 bound to oxidized IspH are very similar
to those found for binding of HMBPP 2, as well as the amino and
thiol analogs of HMBPP, 6 and 7. There is no evidence for any
alkyne-cluster interaction. Second, we show (using HYSCORE)
th
(
PDB ID 3F7T). In both cases, the 4 Fe was reconstituted
[
2]
computationally as described previously , and we used Glide
docking[29] again as described previously[30]
Using this
,
.
approach we obtained and tested 15 potential hits (Figure S2)
that were commercially available, for IspH inhibition. 14 out of
the 15 compounds were inactive (IC50 > 1 mM), but 8 was active.
We then obtained and tested 11 commercially available analogs
of 8 (Figure S3) and tested them against E. coli IspH and
Pseudomonas aeruginosa IspH, both organisms (unlike A.
aeolicus) being important pathogens. The most active
-
that CN binds to IspH (but is a very weak inhibitor), and that the
EPR/HYSCORE spectra of the CN- bound protein are very
similar to those found with CN- binding to SoHydG and P.
furiosus ferredoxin, consistent with binding of a single cyanide in
all three cases. Third, when compared with all known IspH and
IspG structures, it is clear that in most cases, O, N and S-
bonding ligands bind to the unique 4th Fe in the cluster forming
tetrahedral geometries. Fourth, based on the results noted
above, we sought to find novel IspH inhibitors that might bind to
Fe in the active site and be more drug-like than the diphosphate
inhibitors. Using in silico screening we found that the barbiturate
i
compounds were 8 and 9 with 8 having a K = 500 nM against P.
aeruginosa IspH, Figure 4A, and the benzyl analog 9 having a K
i
=
3 µM, against E. coli IspH, Figure 4B. AaIspH was also
i
inhibited by 9 with a K=700 nM. These compounds are
barbituric acid analogs that have some structural similarity to
anti-infectives developed by Pharmacia[31], and are predicted to
bind to the 4th Fe of the 4Fe-4S cluster. A view of the AaIspH•9
complex (obtained by docking) from the electron-transfer side of
the protein is shown in Figure 4C; a bottom-view (substrate side)
in Figure 4D, and a side-view in Figure 4E. A close up view of
the ligand interacting with the 4th Fe in the cluster (dFe-N = 2.6 Å;
analogs 8, 9 had K
barbiturate enolate moiety to the 4 Fe, the hydrophobic
domains occupying the substrate-binding site.
i
~ 0.5-3 µM, binding we propose, via the
th
Experimental Section.
Chemical Synthesis: General Methods
dFe-O = 2.7 Å) is shown in Figure 4F. The ligand can clearly
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8
and its analogs were purchased from Vitas-M Laboratory (Hong Kong)
square avalanche photodiode. Total data aquisition time was 19 hours.
and used without further purification. Compounds were analyzed by
LC/MS and were >97% pure. Other compounds were from Aldrich,
Asinex, Enamine, NCI/Developmental Therapeutics Program Open
Chemical Repository (dtp.cancer.gov/), or TimTec. All chemicals for the
re-synthesis of 9 were purchased from Sigma-Aldrich. 1H NMR spectra
were obtained on Varian Unity spectrometers at 400 and 500 MHz. High
resolution MS and elemental analyses were carried out in the University
Data reduction was performed by using the PHOENIX software
package[
38]
where the observed raw NRVS spectra were calibrated
(aligned) to the nuclear resonant peak, normalized to the I0, then
summed and converted to the 57Fe partial vibrational density of states
(PVDOS). The spectral conversion was optimized when the observed
Stokes/anti-Stokes imbalance matched the imbalance calculated using
the entered temperature as a variable. The real sample temperature
obtained by using this procedure was ~60 K.
CW-EPR/ENDOR/HYSCORE spectroscopy. All CW (continuous wave)-
EPR experiments were performed on a Varian E-line 122 X-band
spectrometer with an Air Products helium cryostat. Typical data
acquisition parameters were: microwave frequency = 9.05 GHz; field
center = 3250 G; field sweep = 1000 G; modulation frequency = 100 kHz;
modulation amplitude = 5 Gauss; time constant = 32 ms; temperature = 8
–
20 K. HYSCORE spectra were obtained on a Bruker ElexSys E-580-10
FT-EPR EPR spectrometer equipped with an Oxford Instruments CF935
cryostat. HYSCORE used a four-pulse sequence π/2mw − τ − π/2mw − t
1
−
πmw − t
2
− π/2mw − echo; π/2mw = 16 ns and πmw = 32 ns, 128 points for
both t
1
2
and t , each using 24 ns steps. Time-domain data were baseline
corrected using a 3rd order polynomial, then Hamming windowed,
followed by zero-filling, 2D-Fourier transformation, and symmetrization.
Parameters were typically: microwave frequency = 9.65 – 9.72 GHz,
temperature = 8 – 15 K, microwave power attenuation = 6.5 – 9 dB.
Spectral simulations. HYSCORE spectra were simulated by using the
Scheme 1. Synthesis of 9.
of Illinois Mass Spectrometry and Microanalytical Laboratories.
-amino-3-benzyl-2,3,4,4a-tetrahydro-1H,2'H,6H-spiro[pyrazino[1,2-
a]quinoline-5,5'-pyrimidine]-2',4',6'(1'H,3'H)-trione (9). The synthesis
of 9 was based on the synthesis of a morpholine analog[36], and is
illustrated in Scheme 1. 1-benzylpiperazine (9a). Piperazine (59 mmol,
8
EasySpin program[24]
.
In silico screening. In order to find new inhibitors, we carried out in
5
.09 g) was dissolved in 26 mL of THF by heating. Benzyl bromide (8.4
silico screens of AaIspH and EcIspH using ZINC and NCI libraries and
Glide docking, basically as described previously[
.
30]
mmol, 1mL) was added dropwise to a refluxing solution of piperazine in
THF. After stirring overnight at reflux, the reaction was cooled, and THF
removed by evaporation. The resulting residue was washed with aq.
Acknowledgments. This work was supported by the
United States Public Health Service (NIH grants GM065307 to
EO and GM-65440 to SPC) and by the Department of Energy
Office of Biological and Environmental Research (SPC). The
NRVS measurements were performed at APS (proposals 39192/
43032) and at SPring-8 (2015B1134). The APS is supported by
the DOE Office of Basic Energy Sciences. The EPR
instrumentation was supported by NIH Grants S10-RR15878
and S10-RR025438. Work at UCSD was supported by NIH, NSF,
HHMI, NBCR, and SDSC. W.W. was supported by a Predoctoral
Fellowship from the American Heart Association, Midwest
Affiliate (award 10PRE4430022). We would like to thank Drs. J.
Zhao, M. Hu and E. E. Alp at APS for assistance with the NRVS
measurements, and Mark J. Nilges for assistance with the EPR
measurements.
K
2
CO
NaCl (10 mL x 1), dried with Na
vacuum (9a, 1.29 g, 87%). 9a (207 mg, 1.174 mmol) was dissolved in 3
mL MeCN and 1 mL Et N. Then, 2-fluoro-5-nitrobenzaldehyde (200 mg,
.18 mmol) was added and the solution stirred at reflux overnight. The
3
(20 mL), extracted with EtOAc (10 mL x 3), washed with satd.
2
SO , then evaporated to dryness under
4
3
1
reaction mixture was then diluted with EtOAc (10 mL) and washed with
water (10 mL). The aqueous layer was extracted with EtOAc (10 mL x 2),
dried with Na SO , then solvent removed under vacuum. The crude
2 4
mixture was purified by column chromatography (2:1 Hex:EtOAc, 9b, 272
mg, 71% yield). 9b (192.77 mg, 0.592 mmol) was dissolved in 10 mL
MeOH. To this solution, barbituric acid (79.76 mg, 0.623 mmol) was
added and the mixture heated to reflux and stirred overnight. The crude
reaction mixture was loaded onto a column and purified with 2:3
EtOAc:Tol (9c, 77.33 mg, 30%). 9c (30 mg, 0.069) was dissolved in 8 mL
AcOH and Zn dust (52 mg, 0.795 mmol) added. The reaction mixture
was stirred at room temperature for 2 hours and then quenched with
K
2
CO
3
(20 mL). Next, the mixture was diluted with EtOAc (20 mL) and
CO (20 mL), satd. NaCl, dried with Na SO4, then
washed with aq. K
2
3
2
solvent was removed under vacuum. The crude mixture was purified by
Keywords: Isoprenoid • IspH • NRVS • HYSCORE • in silico
preparative TLC (100% EtOAc) to yield an orange powder 9 (3.92 mg,
9
%). ESI-HRMS: Calc: 406.1879, found: 406.1868
C
22
H
24
N
5
O
3
.
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ChemBioChem
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Bing O'Dowd, Sarah Williams, Hongxin
Wang, Joo Hwan No, Guodong Rao,
Weixue Wang, J. Andrew McCammon,
Stephen P. Cramer and Eric Oldfield*
Page No. – Page No.
0
100
200 300
Energy (cm-1
400
500
65 00 00
)
Spectroscopic and Computational
Investigations of Ligand Binding to
IspH: Discovery of Non-Diphosphate
Inhibitors
Using EPR, HYSCORE and NRVS spectroscopy together with in silico screening
we investigated how ligands bind to the isoprenoid biosynthesis enzyme IspH
finding novel barbiturate inhibitors with K as low as 500 nM.
i
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