Complexes of bacterial nicotinate mononucleotide adenylyltransferase with inhibitors: Implication for structure-based drug design and improvement

Article abstract of DOI:10.1021/jm100377f

Bacterial nicotinate mononucleotide adenylyltransferase encoded by the essential gene nadD plays a central role in the synthesis of the redox cofactor NAD⁺. The NadD enzyme is conserved in the majority of bacterial species and has been recognized as a novel target for developing new and potentially broad-spectrum antibacterial therapeutics. Here we report the crystal structures of Bacillus anthracis NadD in complex with three NadD inhibitors, including two analogues synthesized in the present study. These structures revealed a common binding site shared by different classes of NadD inhibitors and explored the chemical environment surrounding this site. The structural data obtained here also showed that the subtle changes in ligand structure can lead to significant changes in the binding mode, information that will be useful for future structure-based optimization and design of high affinity inhibitors.

Full text of DOI:10.1021/jm100377f

J. Med. Chem. 2010, 53, 5229–5239 5229
DOI: 10.1021/jm100377f
Complexes of Bacterial Nicotinate Mononucleotide Adenylyltransferase with
Inhibitors: Implication for Structure-Based Drug Design and Improvement†
Nian Huang,‡,^ Rohit Kolhatkar,§,^ Yvonne Eyobo,‡ Leonardo Sorci, Irina Rodionova, Andrei L. Osterman,
Alexander D. MacKerell, Jr.,*,§ and Hong Zhang*,‡
‡Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390,
§Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, Maryland 21201, and
The Burnham Institute for Medical Research, La Jolla, California 92037. ^ These authors contribute equally to this work.
Received March 25, 2010
Bacterial nicotinate mononucleotide adenylyltransferase encoded by the essential gene nadD plays a
central role in the synthesis of the redox cofactor NADþ. The NadD enzyme is conserved in the majority
of bacterial species and has been recognized as a novel target for developing new and potentially broad-
spectrum antibacterial therapeutics. Here we report the crystal structures of Bacillus anthracis NadD in
complex with three NadD inhibitors, including two analogues synthesized in the present study. These
structures revealed a common binding site shared by different classes of NadD inhibitors and explored
the chemical environment surrounding this site. The structural data obtained here also showed that the
subtle changes in ligand structure can lead to significant changes in the binding mode, information that
will be useful for future structure-based optimization and design of high affinity inhibitors.
Introduction
target for developing novel antibiotics due to its crucial role
in synthesizing NADþ; its essentiality has been demonstrated
experimentally in a number of species.5,6 Another attractive
aspect of targeting NadD is that it is highly conserved in the
overwhelming majority of bacterial species including most
pathogens. Therefore, drugs developed based on the inhibition
of NadD have the potential of possessing wide-spectrum
antibacterial activity. Since the first report of the identification
of nadD gene in 2000,16 many biochemical and structural
studies have been conducted on this enzyme. The crystal
structures of NadD from a number of pathogenic bacterial
species, such as Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, and Bacillus anthracis have been
reported.17-22 More recently, by using a structure-based drug
design approach, we have identified for the first time several
inhibitors of NadD and demonstrated that inhibition of NadD
indeed leads to the suppression of bacterial growth.14
Due to the widespread occurrence of drug resistance in
many infectious bacterial pathogens, there is an urgent and
continuing need for developing new antibiotics.1-3 In the
current postgenomics era, the complete genome sequences
of hundreds of bacterial species have become available,
allowing for many potentially new antibiotic targets to be
identified through comparative genomic studies and experi-
mental gene essentiality analysis.4-8 Such capabilities are of
special utility given the significant increase in the number of
bacterial strains resistant to common antibiotics.9-11 An
approach to combat bacterial drug resistance is to develop
new antibiotics against previously unexploited targets that
have emerged from genomics studies.4,12,13 One such target is
the enzyme NaMNa adenylyltransferase encoded by gene
nadD in the biosynthesis pathways of the ubiquitous cofactor
nicotinamide adenine dinucleotide (NADþ).5,14
NADþ is the essential redox cofactor for hundreds of
enzymes and has an impact on nearly all aspects of metabolism
in the cell. The enzyme NaMN adenylyltransferase, or NadD,
occupies a central position in bacterial NADþ biosynthesis
and is required for both de novo and salvage routes to generate
NADþ.15 NadD has been recognized as a promising new
Bacterial NadD, as well as its human counter parts (human
Nmnat isoforms -1, -2, and -3), are members of the HxGH-motif
containing nucleotidyl transferase superfamily and share the
same overall fold.23 However, the sequence identities between
the bacterial and human enzymes are low (∼22%) and the
biochemical properties of the two enzyme subfamilies are also
distinct especially with regard to substrate specificity.18,22,24,25
While the bacterial enzyme almost exclusively prefers nicotinic
acid mononucleotide (NaMN) as substrate, all three human
Nmnat isoforms work equally well on both NaMN and its
amidated form, nicotinamide mononucleotide (NMN). Struc-
tural analyses have revealed conformational differences in the
enzyme’s active sites that may account for their different
biochemical properties.26 These differences have allowed de-
velopment of specific inhibitors against NadD that have no
adverse effects on the activity of human Nmnat isoforms.14
Indeed, among the first NadD inhibitors identified, which
†The atomic coordinates and structure factors (accession codes
3MLA, 3MLB, and 3MMX) have been deposited in the RCSB Protein
Data Bank (www.rcsb.org).
*To whom correspondence should be addressed. For H.Z.: phone,
214-645-6372; fax, 214-645-5948; E-mail, zhang@chop.swmed.edu. For
A.D.M.: phone, 410-706-7442; fax, 410-706-5017; E-mail, amackere@
rx.umaryland.edu.
a Abbreviations: NAD, nicotinamide adenine dinucleotide; NaAD,
nicotinic acid adenine dinucleotide or deamido-NAD; NaMN, nicotinic
acid mononucleotide; NMN, nicotinamide mononucleotide; DMSO,
dimethyl sulfoxide; PEG, polyethylene glycol; PPi, inorganic pyropho-
sphate; rmsd, root-mean-square deviation.
r
2010 American Chemical Society
Published on Web 06/25/2010
pubs.acs.org/jmc

5230 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Huang et al.
Table 1. Chemical Structures of Two Classes of Bacterial NadD
Inhibitors As Represented by Compounds 1_02 and 3_02a
a The numbering of the compounds follows the scheme: compound
class_1st generation analogue_2nd generation analogue.
included two distinct chemical scaffolds (classes 1 and 3,
Table 1) with IC50 values in the low μM range, none of them
have detectable inhibitory activity against human Nmnat.14
These results further validated NadD as a tractable target for
antibacterial therapeutic development.
Figure 1. Inhibitor 1_02 binds between two monomers of baNadD.
(A) The Fo - Fc omit map for 1_02. Two 1_02 molecules, colored
green and yellow, respectively, each with half occupancy are modeled
in the density. (B) 1_02 binds at a baNadD monomer-monomer
interface formed in the crystal of the complex. The two baNadD
monomers are colored cyan and green, respectively.
Previously, we reported the crystal structure of Bacillus
anthracis NadD (baNadD) in complex with product NaAD
and with a class 3 inhibitor (3_02).14 In the current study, we
present crystal structures of NadD in complex with a class 1
inhibitor (1_02) identified in our previous study and two novel
inhibitors (1_02_1 and 1_02_3) designed and synthesized based
on the complex structure of 1_02. Comparison of these complex
structures revealed a common binding region in NadD shared
between the two different classes of inhibitors with distinct
chemical scaffolds. Binding of these inhibitors appears to
stabilize the enzyme in a catalytically impaired conformation
and blocks substrate binding. Interestingly, the overall binding
modes of the inhibitors differ although they all interact via
aromatic groups through a common site. The detailed interac-
tions between the inhibitors and enzyme residues as revealed
from these structures indicate the potential for the identification
of chemically diverse inhibitors that target this region. Such
potential is anticipated to facilitate the structure-based design
of highly potent and specific NadD inhibitors.
the positions of the central anthracene ring overlapping with
each other (Figure 1A,B). These two orientations are in fact
equivalent and physically indistinguishable. It can be viewed
as such that in the complex crystal, half of the protein
molecule population would bind the inhibitor in one orienta-
tion, while the other half bind the inhibitor in the second
orientation. The resulted electron density is the accumulated
average from all the complex molecules in the crystal. There-
fore we modeled 1_02 molecule in two orientations, each
with half occupancy (Figure 1).
baNadD structures have been reported recently in its apo
form, in complex with substrate NaMN, with product
NaAD, as well as with inhibitor 3_02.14,21,22 The overall
baNadD structure contains a Rossman-fold core with a
central six-stranded parallel β-sheet and two or three R
helices on each side of the sheet (Figure 2A). Following the
sixth and the last β strand, two R helices (R6 and R7) form a
small C-terminal subdomain that is characteristic of the
nucleotidyltransferase superfamily. The signature HxGH
motif (15HYGH18) is located in the loop connecting the first
β-strand (β1) and succeeding R helix (R1). This motif is
involved in the interaction with the phosphate groups of
the substrates (ATP and NaMN) and participates in the
catalysis.
In the baNadD-1_02 complex structure determined in the
present work, 1_02 sits at a central cleft between strands β1
and β4 of the β sheet, which is the catalytic and substrate
binding sites of the enzyme (Figure 2A). The compound is
bent at the acylhydrazone linkage and follows the contour of
the crevice of the substrate binding site (Figure 2B,C).
The anthryl rings together with the acylhydrazone of the
compound stack against the side chains of Trp 116, Tyr112,
and Met109 (Figure 2B). A single direct hydrogen bond is
formed between the amide group of the carboxyamide
moiety of 1_02 to the main chain carbonyl of Gly8. The
Results
Structure of baNadD in Complex with Inhibitor 1_02. The
complex of baNadD and 1_02 crystallized in the same space
group P21212 as the previously reported baNadD-3_02
complex14 and the protein conformations in the two inhibi-
tor complexes are also very similar, with root-mean-square
˚
deviation (rmsd) for all CR atoms of 0.175 A; they resemble
the conformation of the enzyme in its apo state rather than
the substrate or product bound state, with rmsd values of
˚
0.494 and 0.833 A, respectively, compared to the apo and
product bound baNadD.14,21,22 Inspection of the electron
density for the bound compound revealed a symmetrically
shaped density much larger than the compound (Figure 1A).
This density is located at a symmetrical interface between
two baNadD monomers where the inhibitor can bind in one
of two different but symmetrically related orientations, with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5231
Figure 3. Comparison of the binding modes of 1_02 (magenta),
3_02 (yellow), and the product deamido-NAD (blue). (A) Super-
position of baNadD bound 1_02 with 3_02 showing the overlapping
binding mode. The protein conformations of the two structures are
essentially identical and a single ribbon diagram is shown. (B)
Superposition of the baNadD-1_02 complex (orange) with the
baNadD-product complex (cyan). 1_02 is in magenta; the product
deamido-NAD is in blue.
chloride of the terminal chlorophenyl group appears to
interact favorably with the side chain of His18 of the HxGH
motif. There are two indirect hydrogen bonds between the
compound and protein atoms. One is formed between the
hydrazone amide and the side chain of Thr85 via a water
molecule (wat2) and the other between the acyl oxygen group
and Asn39 side chain through wat1. The chlorophenyl ring is
also in contact with the side chains of Ile 7 and Ile 21, which
may provide additional stabilizing van der Waals interac-
tions with the compound (Figure 2B).
Comparison of the Binding Modes of 1_02 and 3_02.
Comparison of the binding mode of 1_02 and that of 3_02
reported previously14 shows that the nearly coplanar anthra-
cene rings and the hydrazone portion of 1_02 overlaps with
the largely planar 3_02 (Figure 3A). They form similar
stacking and van der Waals interactions with multiple
protein residues including Trp116, Tyr112, Met109, and
Lys115. This shared binding site corresponds to the region
that binds nicotinic acid riboside portion of NaMN substrate
in the absence of the inhibitors (Figure 3B). In particular,
Trp116 would stack against the pyridine ring of NaMN
and is critical for the proper positioning of the substrate.
Figure 2. Interactions of 1_02 with baNadD. (A) Ribbon repre-
sentation of baNadD-1_02 complex. Inhibitor 1_02 is shown as
sticks. (B) Detailed interactions between 1_02 and baNadD resi-
dues. The CR trace of the protein is shown; relevant side chains are
shown as sticks. Hydrogen bonds are shown as dotted lines. Water
molecules are shown as small red spheres. (C) Surface representa-
tion of the inhibitor binding site on baNadD, colored by the
electrostatic potentials. Three water molecules adjacent to 1_02
are shown as green spheres.

5232 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Huang et al.
Figure 4. (A) Structure and activities of representative class 1 compounds. The compounds cocrystallized with baNadD are boxed. (B) Dose
dependent inhibition by compound 1_02_1 against baNadD (left panel) and ecNadD (right panel).
Therefore binding of the inhibitors would prevent NaMN
binding all together. Notably, the two classes of compounds
do not overlap completely and each has additional interac-
tions with the enzyme that are not present in the other com-
pound (Figure 3A). While 3_02 largely overlaps with the
NaMN substrate binding site, 1_02 also intrudes into the
ATP binding pocket and its chlorophenyl group would
overlap with the ribose of ATP (Figure 3B). The inhibitory
efficiencies of the two compounds against baNadD have been
determined previously, with 1_02 having Ki of 9 and 10 μM,
respectively, with regard to NaMN and ATP substrates, while
3_02 has Ki of 18 and 32 μM against NaMN and ATP,
respectively.14 These values are consistent with the structural
observation that 1_02 interferes with binding of both NaMN
and ATP whereas 3_02 mostly interferes with NaMN binding.
Binding of both 1_02 and 3_02 appears to stabilize the
enzyme in a conformation that is significantly different from
its substrate or product bound form and is apparently
catalytic incompetent (Figure 3B). The different conforma-
tion associated with inhibitor binding as compared to the
substrate or product bound conformations has been sug-
gested to lead to mixed inhibition kinetics that contains both
competitive and noncompetitive characters.14
the overlapping position of the anthracene rings, we hypothe-
sized that a symmetrical compound that fit the observed
density of 1_02 would bind to the enzyme with full occupancy
and higher affinity. A compound was designed to retain the
central planar ring system with an acylhydrazone arm and
terminal chlorophenyl ring on each side. The resulting com-
pound, designated 1_02_1 (Figure 4A), was synthesized and
subjected to biochemical and crystallographic analysis. Com-
pound 1_02_1 replaced the anthracene ring with a benzene and
includes a Cl atom at the ortho position of the terminal phenyl
rings; this selection was based on availability of chemical
precursors. 1_02_1 was then tested as a NadD inhibitor.
According to the structure-activity relationship (SAR) data
from a limited set of analogues of class 1compounds (Figure 4A),
it was expected that 1_02_1 should have an improved activity
compared to the different asymmetric “monomeric” com-
pounds. Inhibition assay on 1_02_1 yielded an IC50 of 13 (
2 μM and 16 ( 4 μM against E. coli NadD (ecNadD) and
baNadD, respectively (Figure 4B). Compared to the class
1 analogues shown in Figure 4A, 1_02_1 is significantly better
than those compounds with either a benzene or naphthalene
rings, while its activity is similar to those compounds contain-
ing an anthracene ring, including 1_02. As 1_13 and 1_15
in Figure 4A contain only benzene rings and linkers identical
to 1_02_1, they may be considered as “precursors” of 1_02_1.
Therefore, the design strategy to create a symmetrical
Structure of baNadD in Complex with Inhibitor 1_02_1.
Because 1_02 must adopt either of the two symmetrically
related orientations with half occupancy in the crystal due to

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5233
several rotatable bonds (Figure 5B). 1_02_1 also interacts
with the protein similarly as 1_02. Most van der Waals
interactions, especially the stacking interactions with
Trp116 and Tyr112, are preserved (Figure 5C). However,
due to the difference in the central ring systems and the
restriction of the covalent linkage to the central benzene ring,
the acylhydrazone arms of 1_02_1 displays a slight rigid
body rotation (∼15ꢀ) compared to 1_02. As a result, there are
differences in the hydrogen bond patterns and in the orienta-
tion of the end chlorophenyl group. The hydrogen bond
between the carboxyamide nitrogen of 1_02 and Gly8 main
chain (shown in Figure 2B) is lost in the 1_02_1 complex
structure, whereas a new hydrogen bond is formed between
the carboxyamide oxygen and Gly106 main chain amide
(Figure 5C). The smaller single six-membered central ring of
1_02_1 may lead to a decrease in the van der Waals interac-
tions with the protein as compared to the anthracene ring
in 1_02.
Interestingly, in the 1_02_1 complex structure, two well-
ordered formate molecules are observed mediating specific
interactions between the acylhydrazone amide group and the
main chain amide and side chain hydroxyl of residue Thr85
(Figure 5C). In the NaMN or NaAD complex structure, the
carboxylate group of the nicotinic acid binds in this region
and interacts with the main chain amide of Thr85. Thus, the
formate molecule observed in the 1_02_1 complex structure
mimics the interaction between the nicotinic acid carboxy-
late group of the substrate NaMN and the enzyme.
Carboxylate Containing Analogues of 1_02 and 1_02_1.
Motivated by the presence of the formates in the 1_02_1
complex structure, additional analogues were designed.
These analogues (1_02_2 and 1_02_3 in Scheme 3) were
designed to include a carboxylate moiety to approximate
the location of the formates in the baNadD-1_02_1 complex
˚
structure. In that structure, one formate oxygen is 2.76 A
from the side chain hydroxyl group of Thr85 and the other
˚
oxygen is 2.91 A from the backbone amide nitrogen of
Thr85, forming well-defined ion-dipole interactions. Ac-
cordingly, it was hypothesized that the carboxylate moieties
would mimic these interactions, thereby further improving
binding. In addition, the inclusion of the carboxylate moi-
eties would enhance the solubility of the compounds, making
them more suitable for biochemical experiments and poten-
tially enhance their bioavailability. This led to the design and
synthesis of 1_02_2 and 1_02_3, shown in Scheme 3. 1_02_2
was a direct mimic of 1_02_1, while 1_02_3 was designed as
an analogue of 1_02, to test if the presence of the carboxylate
could improve the affinity of the monomeric species.
Experiments were then undertaken on the two new 1_02
analogues to measure the inhibitory activity against ba-
NadD. Surprisingly, 1_02_2 did not inhibit baNadD at
concentrations up to 100 μM, while 1_02_3 only weakly
inhibits baNadD activity (IC50 > 200 μM). Thus, the inclu-
sion of the carboxylates did not lead to improved binding
with the symmetric, dimeric analogue 1_02_02, although
some binding affinity of the monomer analogue, 1_02_3 is
present.
Figure 5. Structure of baNadD-1_02_1 complex. (A) The 2Fo - Fc
map of 1_02_1, the two formate molecules (For) and the surround-
ing regions. (B) Superposition of the enzyme bound 1_02_1 (blue)
with 1_02 in its two orientations (represented in two different shades
of gray). (C) Detailed interactions between 1_02_1 and baNadD
residues.
compound may be considered successful, as a more than
10-fold improvement in activity was achieved. 1_02_1 is also
slightly more active than compound 1_02, which has an IC50
of 25 μM.
To understand the binding mode of 1_02_1, we deter-
mined the crystal structure of baNadD in complex with the
compound. The baNadD-1_02_1 complex has the same
crystal form as the 1_02 complex and retains the crystal
lattice packing involving the same monomer-monomer
interface to which the inhibitor binds. 1_02_1 has well-
defined electron density and is modeled with full occupancy
(Figure 5A). As predicted, 1_02_1 binds at the same site as
1_02 and overlaps with the two orientations of that molecule
(Figure 5B). The conformations of the acylhydrazone arms
of the two compounds are very similar despite the presence of
To understand the unexpected results, both 1_02_2 and
1_02_3 were subjected to crystallographic analysis. All at-
tempts to cocrystallize 1_02_2 with baNadD were unsuccessful;
however, cocrystals of 1_02_3bound to baNadD were obtained
˚
and the complex structure was determined to 2.55 A resolution.
The 1_02_3 complex crystal is in a different space group (C2)
from all other baNadD-inhibitor complexes and contained

5234 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Huang et al.
Figure 6. Structure of baNadD-1_02_3 complex. (A) Compound 1_02_3 (blue sticks) binds between two baNadD monomers (colored cyan
and light cyan), which have a difference interface from that in the 1_02_1 complex. The two monomers of baNadD in the 1_02_1 complex are
colored light pink with one monomer superimposed onto the cyan monomer of the 1_02_3 complex. (B) The Fo - Fc omit map for 1_02_3. (C)
Superposition of the three enzyme-bound class 1 inhibitors showing the common aromatic binding site as well as differences in the binding
mode of each compound. The protein molecules in the 1_02_1 and 1_02_3 complexes are shown in light pink and cyan, respectively. (D).
Detailed interactions between 1_02_3 and baNadD residues.
eight baNadD monomers in the asymmetric unit. Notably, the
baNadD monomer-monomer interface to which 1_02_3 binds
is different from that observed in all other inhibitor complex
structures (Figure 6A), indicating that packing of the enzyme
molecules in the crystal can be influenced by inhibitor binding.
The electron density for 1_02_3 was well-defined, allowing
unambiguous modeling of the compound in the complex
(Figure 6B). Interestingly, the binding mode of 1_02_3 differs
significantly from that of 1_02 and 1_02_1, although some
overlap is present (Figure 6C). In particular, the naphthalene
ring of 1_02_3 binds to the same site as the aromatic rings of
the other class 1 compounds and form similar van der Waals
contacts with Trp116, Tyr112, Met 109, as well as with Lys115.
However, the acylhydrazone arm and the end chlorobenzene
ring of 1_02_3 adopt completely different conformations from
that of compounds 1_02 and 1_02_1 and interact with different
functional groups on the protein. In this binding mode, the
carboxylate group of the compound, although occupying a
similar position as the formate molecule in the 1_02_1 complex,
interacts with the enzyme somewhat differently. One oxygen of
the carboxylate still interacts with the side chain hydroxyl of
groups bind to the sites as predicted based on the 1_02_1
complex. In this mode, the carboxy amide moiety and adjacent
chlorophenyl ring of the compound are largely exposed to the
solvent, while their counterpart in the 1_02 and 1_02_1 com-
plexes binds to the adenosine binding site of the enzyme and is
much less solvent accessible.
The 1_02_3 complex structure provides a possible expla-
nation as to why 1_02_2 does not bind as anticipated. While
the naphthalene ring and carboxylate moieties bind to the
anticipated sites, the geometrical restraints to achieve these
interactions lead to a reorientation of the compound and a
significant change in the overall binding mode of 1_02_3
(Figure 6C,D). Potential binding of 1_02_2 in the same
orientation as 1_02_3 would disallow the second arm of
the hydrazine linker to access the binding pocket occupied by
1_02 and 1_02_1, thereby abolishing binding.
Discussion
In an effort to develop inhibitors targeting the essential
bacterial NadD enzymes, we have identified two classes of
bacterial NadD inhibitors with distinct scaffolds in a structure-
based in silico screen.14 We have now also obtained the crystals
structures of B. anthracis NadD in complex with inhibitors
from two different chemical classes: 3_02 from class 3(reported
in ref 14) and three different class 1 compounds (1_02, 1_02_1,
and 1_02_3) presented in the current work. The complex
structures of baNadD with different inhibitors revealed a
common binding site near residues Trp117, Try112, and
Met109, as shown in Figure 7, which appears to have an
affinity for aromatic groups from different small molecules.
˚
Thr85 (3.13 A). In addition, there are ion-dipole interactions
of the carboxylate with the main chain amides of Thr85 (3.30 A)
˚
˚
and of Tyr117 (2.81 A) (Figure 6D). These interactions are
reminiscent of those observed in the NaMN substrate complex,
where the carboxylate of the substrate also forms two hydrogen
bonds with the main chain amides of both Thr85 and
Tyr117.14,22 An additional hydrogen bond between the amide
group of 1_02_3 and the main chain carbonyl of Lys115 is also
˚
observed (2.8 A). Overall, these interactions lead to a different
binding mode for 1_02_3 even though the aromatic and acidic

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5235
pated to have minimal adverse effects on the numerous
other NADþ or ATP utilizing enzymes. The noncompeti-
tive character of inhibition by these inhibitors also indicates
that once higher affinity compounds are found, they may
not be strongly influenced by cellular ATP or NADþ con-
centrations, which are on the order of ∼10-103 μM.27-29
Such inhibitors could have better in vivo efficacy than purely
competitive inhibitors.
Although a non-native dimer interface is observed in
several baNadD-inhibitor (e.g., 3_02, 1_02, and 1_02_1)
complex crystal structures, it has become clear that this
dimerization mode is due to crystal lattice packing interac-
tions under specific crystallization conditions because such
dimerization is not observed in solution in an analytical
ultracentrifugation study.14 Crystal structures of the 1_02_3
complex and apo-baNadD obtained in different space groups
also do not have the same dimerization mode.21,22 This
observation partially explains the moderate improvement of
the activity of the much larger dimeric 1_02_1 as compared to
its monomeric precursor. Therefore future inhibitor design
and optimization effort should be focused on engineering
specific direct interactions between the inhibitors and enzyme
monomer. Toward this goal, the complex structures of NadD
with different inhibitors provided useful information on a
common primary target site and the chemical environment of
the vicinity of this site, which can be exploited to improve on
the existing inhibitor scaffolds or design high affinity inhibi-
tors with novel scaffolds for maximum interaction with the
enzyme.
Figure 7. Superposition of the baNadD bound 3_02 (yellow), 1_02
(magenta), 1_02_1 (blue), and 1_02_3 (green). The surface presen-
tation of the enzyme (colored according to electrostatic potentials)
in the 1_02 complex structure is shown. The image also includes
three nearby water molecules observed in the 1_02 complex struc-
ture (cyan spheres) and the formate molecule observed in the 1_02_1
complex structure. The orientation of deamino-NADþ (thin, atom-
colored licorice representation) from the product-complex struc-
ture (PDB code 3E27) is also shown.
This site overlaps but is distinct from the substrate NaMN
binding pocket and may serve as a primary site to be targeted in
future inhibitor design efforts. Such design efforts would target
compounds whose aromatic moieties interact with the identi-
fied “aromatic” site, with the remainder of those putative
molecules sampling various binding modes in the vicinity of
this site.
The complex structures of three class 1 compounds provide
useful information about the chemical characters of the
inhibitor-binding site of NadD. Compounds 1_02 and
1_02_1 bind to the aromatic site with their central anthracene
or benzene rings and hydrazone groups, while the linker and
the terminal chlorobenzene ring intrude into a deep groove on
the enzyme and interact directly with the conserved active site
HxGH motif residues. In addition to this groove, the binding
potentialof a small pocket adjacent tothe primary bindingsite
is highlighted in the 1_02_1 and 1_02_3 complex structures,
where it is revealed that this pocket favors binding of a
carboxylate group. In the 1_02_3 complex structure, binding
of the carboxylate group at this site comes at the expense of
completely reorienting the acylhydrazone arms of the com-
pound, which results in an overall decreased affinity. This
reorientation is also proposed to disallow binding of the
dimeric 1_02_2 to the protein.
Experimental Section
Protein Crystallography. The expression and purification of
Bacillus anthracis NadD (baNadD) has been reported else-
where.14 For cocrystallization of baNadD with compounds
1_02, 1_02_1, and 1_02_3, appropriate amount of the stock
compound solutions (20 mM in DMSO) was mixed with the
protein to the final concentration of 1 mM, while the final
protein concentration is 19 mg/mL. The PEG/Ion Crystalliza-
tion Screening Kit (Hampton Research) was used for the initial
screens of the complex crystals. Hanging drop vapor diffusion
methods were used for the crystallization where equal volume
(1.5 μL) of the complex and reservoir solution was mixed and
equilibrated against the reservoir at 20 ꢀC. The baNadD-1_02
cocrystals were obtained in conditions that contain 0.2-0.25 M
magnesium formate and 20-24% PEG 3350. The baNadD-
1_02_1 complex crystals were obtained from 0.2 M potassium
formate and 20% PEG 3350. Both crystals were cryoprotected
in solutions that contained an increased concentration of PEG
3350 (40%) and original components of the reservoir and frozen
in liquid propane. The baNadD-1_02_3 complex formed crys-
tals in 0.2 M potassium citrate and 20% PEG 3350, and the
crystal was frozen in the cryoprotectant containing original
components of the reservoir supplemented with 10% DMSO
and flash frozen in liquid nitrogen.
The X-ray diffraction data of the baNadD-1_02 complex
crystal was collected at beamline 19BM, Advanced Photon
Source, Argonne National Laboratory, whereas the data for
baNadD-1_02_1 and baNadD-1_02_3, crystals were collected
in-house on a Rigaku FRE rotating anode X-ray generator
equipped with Osmic focusing device and RAXIS IVþþ image
plate detector. The data were further processed using HKL3000
software.30
Both the baNadD-1_02 and baNadD-1_02_1 complexes
were crystallized in the P21212 space group, isomorphous to the
crystals of baNadD-3_02 complex reported recently.14 There-
fore, the model of the baNadD-3_02 (PDB code 3HFJ),
Although the activities of the current NadD inhibitors are
onlyin thelow micromolarIC50 range atbest, there are several
attractive features in their binding modes. Binding of
both classes of inhibitors appears to stabilize the enzyme in a
catalytically incompetent conformation, significantly different
from its substrate or product bound conformation, result-
ing in a mixed inhibition kinetics behavior that contains
both competitive and noncompetitive characters. As such,
the binding pocket can accommodate small molecules with
structures very different from the natural ligands of the
enzymes. Therefore, such small molecule binders are antici-

5236 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 2. Crystal Data and Refinement Statistics
Huang et al.
Scheme 1a
baNadD-
1_02
baNadD
1_02_1
baNadD
1_02_03
3
Data sets
Data Statistics
space group
unit cell dimensions
P21212
a = 88.6 A,
P21212
a = 88.34 A,
C2
a = 295.15 A,
˚
˚
˚
˚
b = 97.53 A,
˚
b = 96.64 A,
˚
b = 46.45 A,
˚
c = 44.30 A
˚
c = 44.13 A
˚
c = 114.95 A,
β = 91.42ꢀ
50-2.55
191091
˚
resolution (A)
50-1.70
230680
74116
50-1.80
73589
47493
total observations
unique reflections
completeness
a (a) benzene-1,4-dicarbaldehyde, ethanol, reflux.
97476
99.6 (100)
99.0 (97.0)
88.8 (79.3)
(outer shell) (%)
sym (outer shell)a
the absorbance was measured at 620 nm. To account for
possible contribution of free phosphate and/or pyrophosphate
(present in the sample or released due to nonspecific hydrolysis
of ATP) as well as of background absorbance (color) of the
tested compounds, parallel reactions were run for each experi-
mental point without addition of NadD enzymes, and their
OD620 values were subtracted from the measurements of enzyme
activity in respective samples. Reaction in the presence of 2%
DMSO but without inhibitor served as the positive control.
For IC50 determination, the initial rate of the enzymatic
reaction was measured at fixed NaMN and ATP concentrations
(equal to 2-fold Km values) in the absence and presence of
various concentrations of inhibitors.The IC50 value was deter-
mined by plotting the rates versus inhibitor concentration and
fitting to the equation 1 using GraphPad Prism.
R
0.040 (0.574)
39.2 (2.6)
0.040 (0.271)
23.8 (2.7)
0.075/0.571
20.53/2.06
I/δ (outer shell)
Refinement
b
Rwork
c
Rfree
0.190
0.230
0.006
1.049
3051
308
0.183
0.232
0.007
1.121
3067
337
0.205
0.270
0.008
1.073
11783
232
˚
rmsd bond length (A)
rmsd bond angle (deg)
protein atoms
water molecules
ligand atoms
average B-factors (A )
85
48
191
2
˚
protein
ligands
water
32.5
39.7
32.6
26.3
24.6
33.2
57.31
67.81
44.19
Ramachandran Plot
vi ¼ v0=ð1 þ ½Iꢀ=IC50Þ
ð1Þ
favored region (%)
allowed region (%)
98.6
100.0
98.6
100.0
97.4
99.8
P
P
P
P
P
P
v0 and vi represent initial rates in the absence and presence of
inhibitors at concentration [I].
a Rsym
Fo|, where Fo and Fc are the observed and calculated structure factors,
=
j|Ij - ÆIæ|/
j|Ij|. b Rwork
=
hkl|Fo - Fc|/
|
hkl
hkl
hkl
Chemistry. Proton NMR spectra were recorded on Varian
500 MHz FT NMR spectrometer. Mass spectra were recorded
on a LCQ mass spectrometer (Finnigan MAT, San Jose, CA).
Element analyses were performed by Atlantic Mircolab, Inc.
(Norcross, GA). Flash column chromatography was performed
using Silica Gel 60 (230-400 mesh) from Thomas Scientific
(Swedesboro, NJ). Analytical thin layer chromatography (TLC)
was performed on precoated glass backed plates from Analtech
Inc. (Newark, DE) (TLC uniplates, Silica gel GHLF, 250 μ).
Plates were visualized using ultraviolet, iodine vapors, phos-
phomolybdic acid, or ninhydrin. Compound 1 was available
from a commercial supplier. The purity of the final compounds
tested for biological activity was determined by elemental
analysis (Atlantic Microlab) and confirmed by HPLC. The
purity was found to be g95%.
respectively. c Five percent randomly selected reflections were excluded
from refinement and used in the calculation of Rfree
.
excluding ligand and solvent molecules, was used as the initial
model for the refinement of both new complexes using the
program Refmac of the CCP4 package.31-33 The solution of
baNadD-1_02_3 complex was found by the molecular re-
placement method of Phaser34 using apo baNadD as the starting
model. Model inspection and adjustment was performed with
Coot.35 The electron densities for compound 1_02, 1_02_1, and
1_02_03 were clearly visible in the early stage of the refinement.
The PRODRG server36 was used to generate the models for the
compounds to be included in the complex structure. Final
rounds of refinements were performed using PHENIX,37,38
and the model geometry was monitored by Molprobity.39
The crystal data and refinement statistics of these complexes
are listed in Table 2. The coordinates have been deposited in the
Protein Data Bank40 with accession codes 3MLA, 3MLB, and
3MMX.
Enzyme Inhibition Assay. A general phosphate detection
assay method using Malachite Green reagent was adapted to
measure the activity of NaMN adenylyltransferase.14 Briefly,
the byproduct of NadD catalyzed reaction, inorganic pyropho-
sphate (PPi), was hydrolyzed by inorganic pyrophosphatase and
the resulting orthophosphate was detected by the Malachite
Green dye. The reaction mixture contained 2.3 nM ecNadD (or
1.2 nM baNadD) in 100 mM Hepes, pH 7.5 buffer, 0.2 mM
ATP, 0.07 or 0.2 mM NaMN, 10 mM MgCl2, 0.1 mg/mL bovine
serum albumin, 0.2 U inorganic pyrophosphatase. Appropriate
amount of inhibitors were added to the reaction mixture to
assess their effect on enzyme activity. After preincubation of the
enzyme with the compounds for 5 min at room temperature, the
reaction was started by adding NaMN substrate. The reaction
was quenched with two volumes of Malachite Green reagent in
1.2 M sulfuric acid prepared as described by Cogan et al.41 After
20-30 min incubation to allow for complex/color formation,
Synthesis of N-(2-Chloro-phenyl)-3-(4-{[3-(2-chloro-phenylcar-
bamoyl)-propionyl]-hydrazonomethyl}-benzylidene-hydrazinocar-
bonyl)-propionamide (1_02_1, Scheme 1). Benzene-1,4-dicarbal-
dehyde (0.01 g, 0.07 mmol) and N-(2-chloro-phenyl)-3-hydrazi-
nocarbonyl-propionamide 1 (0.036 g, 0.14 mmol) in ethanol
(5 mL) were heated to reflux for 2 h. After cooling to room
temperature, the precipitate was filtered off and washed with
1
ethanol to give 1_02_1 as a pale-white solid (0.03 g, 69%). H
NMR(500MHz, DMSO-d6) 12.31 (2H, s), 8.21 (2H, s), 7.94 (4H,
s), 7.60-8.20 (8H, br). MS Anal. mol wt 580.14 (604.2 M þ Na).
Elemental Anal. Calcd for C28H26Cl2N6O4 0.4H2O: C, 57.13; H,
3
4.58; N, 14.27. Found: C, 57.36; H, 4.49; N, 14.00.
Synthesis of (N0-tert-Butoxycarbonyl-hydrazino)-acetic Acid
Ethyl Ester (4, Scheme 2). Ethyl bromoacetate 3 (6.97 mL, 62.8
mmol) was added to a stirred solution of tert-butylcarbazate 2
(24.9 g, 188.6 mmol) in water (25 mL) at room temperature. The
mixture was stirred for 30 min. Water layer was then extracted
with ethyl acetate (3ꢁ). Ethyl acetate extracts were pooled
together and washed with brine. Ethyl acetate was evaporated
under vacuum to get crude product which was purified by flash
column chromatography using hexane:ethyl acetate (70:30) as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5237
Scheme 2a
a (a) water; (b) succinic anhydride, DMF, 70 ꢀC; (c) HBTU, DIPEA, DMF; (d) TFA/CH2Cl2; (e) ethanol, 1N NaOH.
Scheme 3a
a (a) Napthalene-1-carbaldehyde, ethanol, reflux; (b) benzene-1,4-dicarbaldehyde, ethanol, reflux.
an eluent (yield 70%). 1H NMR (500 MHz, CDCl3) 1.28 (3H,
CH2-CH3, t), 1.45 (9H, C-CH3, s), 3.64 (2H, NH-CH2-CO, s),
4.20 (2H, CH2CH3, q). MS Anal. mol wt 218.25 (M þ 1).
Synthesis of 4-(N0-tert-Butoxycarbonyl-N-ethoxycarbonylmethyl-
hydrazino)-4-oxo-butyric Acid (5, Scheme 2). Into a solution of
(N0-tert-butoxycarbonyl-hydrazino)-acetic acid ethyl ester 4
(1.85 g, 18.5 mmol) in DMF (30 mL) was added succinic
anhydride (4.84 g, 22.2 mmol) and the mixture was stirred at
75 ꢀC for 18 h. DMF was evaporated and the crude mixture was
purified by flash column chromatography using hexane:ethyl-
(0.3 g, 0.7 mmol) was dissolved in 5 mL of 20% TFA in dichloro-
methane and the solution was stirred for 1 h. TFA was then
evaporated under vacuum, and the crude mixture was purified
by flash column chromatography using ethyl acetate as an
eluent (yield 87%). 1H NMR (500 MHz, CDCl3) 1.27 (3H,
CH2-CH3, t), 2.75 (2H, N-CH2-CH2-CO, t), 3.11(2H, N-CH2-
CH2-CO, t), 4.20 (2H, CH2CH3, q), 4.36 (2H, NH-CH2-CO, s),
7.00 (1H, ArH, t), 7.23 (1H, ArH, t) 7.33(1H, ArH, d) 8.24-8.40
(2H, ArNH, ArH, m). MS Anal. mol wt 327.76 (M þ 1).
Synthesis of {N-[3-(2-Chloro-phenylcarbamoyl)-propionyl]-
hydrazino}-acetic Acid (8). Compound 7 was dissolved in 10
mL of ethanol followed by addition of 1.2 mL of 1N NaOH. The
mixture was stirred for 1 h. Ethanol was then evaporated under
vacuum to obtain crude compound which was dissolved in water
and the solution was neutralized using 1N HCl. Evaporation of
the water followed by separation of salt by precipitation in
ethanol yielded compound 8 (yield 82%), which was used with-
out any purification for next step. 1H NMR (500 MHz, CD3OD)
2.50-3.21 (4H, N-CH2-CH2-CO, m), 4.18-4.44 (2H, NH-CH2-
CO, m), 7.14 (1H, ArH, t), 7.27 (1H, ArH, t) 7.43(1H, ArH, d)
7.78 (1H, ArH, s).MS Anal. mol wt 299.07 (M þ 1).
1
acetate (1% acetic acid) as an eluent (yield 50%). H NMR
(500 MHz, CDCl3) 1.28 (3H, CH2-CH3, t), 1.48 (9H, C-CH3, s),
2.55-3.00 (6H, NH-CH2-CO, N-CH2-CH2-CO, m), 4.20 (2H,
CH2CH3, q). MS Anal. mol wt 318.25 (M - 1).
Synthesis of {N0-tert-Butoxycarbonyl-N-[3-(2-chloro-phenylcar-
bamoyl)-propionyl]-hydrazino}-acetic Acid Ethyl Ester (6, Scheme 2).
Into a mixture of compound 5, HBTU and DIPEA in DMF was
added 2-chloroaniline and the solution was stirred for 48 h. DMF
was evaporated under vacuum, and the mixture was dissolved in
ethyl acetate and washed with water (2ꢁ), 1 M KHSO4 (2ꢁ), and
water (2ꢁ). Ethyl acetate was then evaporated to get crude
compound which was purified by flash column chromatography
Synthesis of {N0-(4-{Carboxymethyl-[3-(2-chloro-phenylcar-
bamoyl)-propionyl]-hydrazonomethyl}-benzylidene)-N-[3-(2-chloro-
phenylcarbamoyl)-propionyl]-hydrazino}-acetic Acid (1_02_2,
Scheme 3). Benzene-1,4-dicarbaldehyde (0.006 g, 0.05 mmol) and
compound 8 (0.032 g, 0.10 mmol) in ethanol (5 mL) were heated to
reflux for 12 h. After cooling to room temperature, the precipitate
was filtered off and washed with ethanol to give 1_02_2 as a pale-
white solid (0.015 g, yield 44%). 1H NMR (500 MHz, DMSO-d6)
2.72 (4H, N-CH2-CH2-CO, t), 3.15 (4H, N-CH2-CH2-CO, t), 4.52
1
using hexane:ethyl acetate (50:50) as an eluent (yield 38%). H
NMR (500 MHz, CDCl3) 1.27 (3H, CH2-CH3, t), 1.48 (9H,
C-CH3, s), 2.58-3.06 (6H, NH-CH2-CO, N-CH2-CH2-CO, m),
4.20 (2H, CH2CH3, q), 7.01(1H, ArH, t), 7.22-7.27(1H, ArH, m)
7.34(1H, ArH, d), 8.06 (1H, ArNH, s), 8.33(1H, ArH, d); MS
Anal. mol wt 427.88 (M þ 23).
Synthesis of {N-[3-(2-Chloro-phenylcarbamoyl)-propionyl]-
hydrazino}-acetic Acid Ethyl Ester (7, Scheme 2). Compound 6

5238 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Huang et al.
(4H, NH-CH2-CO, s), 7.15 (2H, ArH, t), 7.29 (2H, ArH, t),
7.47(2H, ArH, d) 7.65-7.77(6H, ArH, m), 9.56(2H, Ar-CHdN).
MS Anal. mol wt 696.15 (M - 2). Elemental Anal. Calcd for
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C32H28Cl2N6O8Na2 1.5H2O: C, 50.01; H, 4.06; N, 10.93. Found: C,
3
50.07; H, 4.09; N, 11.10.
Synthesis of {N-[3-(2-Chloro-phenylcarbamoyl)-propionyl]-
N0-naphthalen-1-ylmethylene-hydrazino}-acetic Acid (1_02_3,
Scheme 3). Naphthalene-1-carbaldehyde (0.015 g, 0.10 mmol)
and compound 8(0.031 g, 0.10 mmol) in ethanol (5 mL) were
heated to reflux for 12 h. After cooling to room temperature, the
precipitate was filtered off and washed with ethanol to give
1_02_3 as a pale-white solid (0.02 g, yield 47%). 1H NMR (500
MHz, DMSO-d6) 2.64 (1H, N-CH2-CH2-CO, t), 2.79 (1H,
N-CH2-CH2-CO, t), 3.17 (1H, N-CH2-CH2-CO, t), 3.23 (1H,
N-CH2-CH2-CO, t), 4.89-4.97 (2H, NH-CH2-CO,m), 7.17
(1H, ArH, t), 7.31 (1H, ArH, t), 7.48(1H, ArH, d) 7.56-7.68
(3H, ArH, m), 7.75(1H, ArH, d), 7.96-8.04 (3H, ArH, m), 8.50
(1H, ArNH, s), 8.70 (1H, ArH, t), 9.56(2H, Ar-CHdN). MS
Anal. mol wt 437.11 (M - 1). Elemental Anal. Calcd for
C23H20ClN3O4 0.8H2O: C, 61.07; H, 4.81; N, 9.29. Found: C,
3
61.06; H, 4.81; N, 9.27.
Acknowledgment. We thank Dominika Borek for help
with X-ray data collection at APS synchrotron, Chuo Chen
for helpful discussion. This work is supported by grants from
the NIH (AI059146), the Welch Foundation (I-5105), and the
University of Maryland Computer-Aided Drug Design Center.
Results shown in this report are derived from work performed
at Argonne National Laboratory, Structural Biology Center,
at the Advanced Photon Source. Argonne is operated by
University of Chicago Argonne, LLC, for the U.S. Department
of Energy, Office of Biological and Environmental Research
under contract DE-AC02-06CH11357.
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