Structural studies of pterin-based inhibitors of dihydropteroate synthase

Article abstract of DOI:10.1021/jm900861d

Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bi

Full text of DOI:10.1021/jm900861d

166 J. Med. Chem. 2010, 53, 166–177
DOI: 10.1021/jm900861d
Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase†
Kirk E. Hevener,‡,# Mi-Kyung Yun, ,# Jianjun Qi,‡ Iain D. Kerr, Kerim Babaoglu, Julian G. Hurdle,‡ Kanya Balakrishna,‡
Stephen W. White,*, ,^ and Richard E. Lee*,‡,§
‡Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 327 Johnson Building,
Memphis, Tennessee 38163, §Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas
Place, Mail Stop 1000, Memphis, Tennessee 38105, Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis,
Tennessee 38105, and ^Department of Molecular Sciences, University of Tennessee Health Science Center, 658 Madison Avenue, G01 Molecular
Science Building, Memphis, Tennessee 38163. # These authors contributed equally to this work.
Received June 12, 2009
Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the
sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a
decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the
p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass
sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed
virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with
inhibitory activity have been identified, and crystal structures have been determined that show how the
compounds engage the pterin site. The structural studies identify the key binding elements and have been
used to generate a structure-activity based pharmacophore map that will facilitate the development of the
next generation of DHPS inhibitors which specifically target the pterin site.
Introduction
agents can also provide valuable information for the design
and development of the next generation agents.
There is an urgent need for novel antibacterial agents for
treating infections caused by resistant organisms.1 The emer-
gence of bacterial resistance is a pressing concern and has led
to a significant decrease in the clinical utility of many anti-
bacterial agents. One approach to this problem is to identify
new classes of antibacterial agents with novel mechanisms of
action, but this has proven to be extremely difficult in practice
leading to high failure rates.2 An alternative approach is to
characterize the mechanism of resistance in traditional anti-
bacterial drug targets and to design new agents that can
bypass these mechanisms. This approach has proven to be
more productive in recent years, for example, with the success-
ful development of glycylcycline and ketolide antibiotics.3,4
There are several advantagestothisapproach. First, the target
would be prevalidated by the prior clinical use of the earlier
generation agents. Second, key biochemical information
about thetarget andthe mechanisms of resistance are typically
already available to guide the design of the next generation
agents. Finally, clinical experience with the earlier generation
The sulfonamide class of antibacterial drugs has been used
clinically since the 1930s, and it was the first class of synthetic
antibacterial agents to be used successfully.5 Sulfonamides
target the enzyme dihydropteroate synthase (DHPSa) which
catalyzes the addition of p-aminobenzoic acid (pABA) to
dihydropterin pyrophosphate (DHPP) (Figure 1a) to form
pteroic acid as a key step in bacterial folate biosynthesis. The
folate biosynthetic pathway has a key role in nucleic acid
synthesis, and inhibition by the sulfonamides prevents bacter-
ial growth and cell division. The absence of the pathway in
higher organisms makes it a particularly attractive target for
antibacterial drug design. Historically, the sulfonamides have
been successfully used for a variety of Gram-positive and
Gram-negative bacterial infections, and combinations with
inhibitors of dihydrofolate reductase (DHFR) which cata-
lyzes a subsequent step in folate synthesis have proven to be
particularly effective. For example, cotrimoxazole is a com-
monly used sulfamethoxazole-trimethoprim combination.
However, drug resistance has emerged as an important factor
that now severely limits the use of the sulfonamides.6 For
example, previously considered to be a first-line agent, cotri-
moxazole has now been relegated to a second or third line
option for a broad variety of infections. Resistance can be
caused by altered drug uptake or efflux, but the predominant
mechanism is mutation of the FolP gene that encodes DHPS.
†The atomic coordinates of the B. anthracis DHPS cocrystal complex
structures have been made publicly available through the Protein Data
Bank (www.rcsb.org/pdb) with the following PDB IDs: compound 2,
3H21; compound 3, 3H22; compound 4, 3H23; compound 5, 3H24;
compound 6, 3H26; compound 7, 3H2A; compound 10, 3H2C; com-
pound 11, 3H2E; compound 16, 3H2F; compound 17, 3H2M; com-
pound 18, 3H2N; compound 19, 3H2O.
*To whom correspondence should be addressed. For S.W.W.: phone,
(901) 595 3040; E-mail, stephen.white@stjude.org; address, Department
of Structural Biology, St. Jude Children’s Research Hospital. For R.E.
L.: phone, (901) 595 6617; E-mail, Richard.Lee@stjude.org; address,
Department of Chemical Biology and Therapeutics, St. Jude Children’s
Research Hospital.
a Abbreviations: DHPS, dihydropteroate synthase; pABA, para-ami-
nobenzoic acid; DHPP, dihydropterin pyrophosphate; DHFR, dihy-
drofolate reductase; PCP, Pneumocystis carinii pneumonia; MANIC,
2-amino-6-(methylamino)-5-nitropyrimidin-4(3H)-one; CAS, Chemical
Abstracts Service; HMDP, 6-hydroxymethyl-7,8-dihydropterin.
r
pubs.acs.org/jmc
Published on Web 11/09/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 167
several common pathogenic bacteria. This conservation re-
flects the severe constraints imposed on the pocket by its
substrate specificity, compactness, and structural integrity
within the β-barrel. This contrasts with the pABA site that is
comprised largely of flexible loop residues. Thus, inhibitors of
the constrained pterin binding pocket would be predicted to
have a broad spectrum of activity against both Gram-positive
and Gram-negative bacteria and also be less able to tolerate
resistance mutations.
In the mid-1980s, a series of compounds with inhibi-
tory activity against E. coli DHPS was disclosed by resear-
chers at Burroughs-Wellcome, Inc.16,17 The compounds
were pterin-like, had activity in the low micromolar range
and were presumed to bind within the pterin pocket,
although no structural information was reported. During
our initial investigations into the structure of B. anthracis
DHPS, we were able to resynthesize and structurally ana-
lyze one of these compounds within the DHPS active site.12
The compound, 2-amino-6-(methylamino)-5-nitropyrimidin-
4(3H)-one (MANIC, but herein referred to as 1), engages
the pterin pocket as predicted, and this structure has now
led to the identification of similar inhibitory molecules that
are presented in this report. The identification of these
molecules has progressed in defined stages. The initial com-
pounds were also derived from the Burroughs-Wellcome
studies and include 2, a particularly potent inhibitor of
B. anthracis DHPS that provided valuable design features
for three stages of subsequent virtual screening (VS) studies.
Our final cohort of 12 inhibitory molecules have been
characterized by enzyme kinetics, X-ray crystallography,
and antibacterial activity. This information was then
combined in an initial structure-activity relationship
(SAR) analysis, which allowed us to develop a set of pharma-
cophore hypotheses withwhichtodevelopfuture pterin-based
inhibitors.
Figure 1. Pterin substrate binding pocket of DHPS. (a) The struc-
ture of the natural substrate, DHPP, with ring numbering.
(b) LigPlot18 view of the PtPP substrate analogue bound in the
BaDHPS active site with the key binding interactions displayed.
However, several emerging pathogens have shown universal
susceptibility to cotrimoxazole, and this warrants further
investigation of DHPS as a drug target. Notably, cotrimox-
azole is a recommended agent for treating community-ac-
quired MRSA and the recommended prophylactic agent for
the prevention of Pneumocystis pneumonia (PCP) in adult
HIV patients.7,8
The first crystal structure of DHPS (from Escherichia coli)
was determined in 1997, fully 36 years after the last sul-
fonamide agent entered the market. Since that time, five
additional crystal structures have been resolved, from Staphy-
lococcus aureus, Mycobacterium tuberculosis, Bacillus anthra-
cis, Thermus thermophilus, and Streptococcus pneumoniae and
also one from the fungus Saccharomyces cerevisiae.9-15 These
structures and associated mechanistic studies represent valu-
able new information with which to revisit DHPS as a
therapeutic target. DHPS has a classic (β/R)8 TIM barrel
structure in which the active site is located at the “C-terminal”
end of the barrel and contributed to by elements of the flexible
loops that connect the β strands and R helices. The crystal
structure of B. anthracis DHPS (BaDHPS) with a pteroate
product analogue in the active site is a key structure deter-
mined by our group because it reveals the locations of both the
pterin and pABA binding sites. Although a sulfonamide has
yet to be unequivocally visualized in complex with DHPS,
these molecules appear to bind to the pABA subsite and
inhibit product formation and/or form “dead-end” products
with pterin. Consistent with this notion, mutations that confer
sulfonamide resistance all map to the pABA binding site
locale. Although it has not been established how these muta-
tionsproduceresistance, agents that inhibitthe DHPSenzyme
bybinding to the distinct pterin subsite arepredicted tobypass
these sulfonamide resistance sites. Another advantage of
targeting the pterin site is revealed by Table 1, which reveals
the high conservation of the key pterin-binding residues in
Results and Discussion
The DHPS Pterin-Binding Pocket. The pterin-binding
pocket has been visualized in all the available crystal
structures of DHPS and shown to be highly conserved
(Table 1).9-15 The pocket is located within the TIM barrel,
directly below two flexible loops (loop1 and loop2) that are
known to contain important elements of the active site, and is
bounded by several key conserved residues that recognize the
pterin-pyrophosphate substrate (Figure 2). In BaDHPS,
Asp101, Asn120, Asp184, Lys220, and a structural water
molecule provide a hydrogen bond donor/acceptor constella-
tion thatrecognizes the pterin ring. Arg254 atthe “base” of the
pocket provides a stacking platform for the pterin ring and,
together with His265 and Asn27, also provides an anion-
binding pocket for the β-phosphate of the substrate. A LigPlot
view of this binding site, which is the target of our current
studies, is shown (Figure 1b).18 DHPS catalyzes a strictly
ordered reaction in which pterin-pyrophosphate is the lead
substrate, and Lys220 has an important role to play in this
mechanism. In the apo structure lacking any ligand, Lys220 is
somewhat flexible, but its interaction with the pterin ring
stretches out the side chain. Our structure with the product
analogue pteroic acid reveals that the now rigid side chain
provides a binding platform for the second pABA substrate. In
the absence of definitive structural data, it is generally as-
sumed that loop1 and loop2 clamp down over the two
substrates to complete the active site and promote catalysis.

168 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 1. DHPS Pterin Binding Site Residuesa
Hevener et al.
a Residues differing from B. anthracis target are colored in red.
Figure 2. B. anthracis DHPS enzyme shown with 2 bound. (a) The full protein is shown with homology modeled loops colored. (b) The pterin
binding site with key binding residues and nearby loop residues. Hydrogen bonds are indicated by red dashes. Distances to nearby the loop
residues in their modeled positions are shown.
Known Pterin-Based Inhibitors. The first compounds that
were tested in these studies were selected from a series of
DHPS-targeted inhibitors that were synthesized, analyzed,
and published in the 1980s but for which structural informa-
tion was not generated (Figure 3a).16,17 These formally
include 1 that we resynthesized and structurally analyzed
in an earlier study.12 We demonstrated that this compound
does engage the pterin pocket and interacts with five of the
six pterin recognition elements. Asp101 is the exception
because the electrostatic interaction is blocked by the N-
methyl group. In the absence of the pyrophosphate moiety,
the anion-binding pocket is occupied by a sulfate ion. This
DHPS-inhibitor complex represents the starting point for
our current studies. We selected three additional com-
pounds, 2-4, for further analysis with B. anthracis DHPS
based on a combination of potency, as judged by the
published IC50 values against E. coli DHPS, chemical diver-
sity, commercial availability, and ease of synthesis.
(Table 2). This improvement in activity can be rationalized
by the crystal structure, which reveals that the unsubstituted
amine at the 6-position engages Asp101 in an electrostatic/
hydrogen-bonding interaction that is blocked by the methyl
substitution in 1 (not shown). Compound 2 was shown to be
an effective inhibitor of BaDHPS (IC50 value 19.8 μM), and
the structure of the complex revealed the basis of this potency
(Figure 4a-c). Although the interaction with Asp101 is
blocked by the methyl substitution at the 6-position, the
remaining pterin-binding residues are engaged and the car-
boxyl group provides an additional interaction with the
anion-binding pocket which displaces the sulfate ion. In
addition, there is a van der Waals interaction between the
methyl group on the linker and the ring of Phe189. Finally, 4
resembles the DHPS product in which the pterin moiety is
replaced by 3 and the pABA moiety is attached via an
extended linker. The molecule has an IC50 of 19.3 μM in
BaDHPS (Table 2). In the structure of the complex
(Figure 4d-f), the pterin-like half engages the pterin pocket
in a similar fashion to 3 with two differences: the interaction
with Asp101 is blocked by the linker and the interaction with
the side chain amine of Lys220 is via the nitrogen atom of the
Compound 3, a close analogue of 1, has a nitroso group
substituted for a nitro group and an unsubstituted amine at
the 6-position rather than the N-methyl substitution of 1. In
B. anthracis, 3 shows improved inhibitory activity over 1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 169
Figure 3. DHPS hit compounds as evaluated by enzyme assay (>30% inhibition). Compounds are organized according to how they were
identified. (a) Compounds from previously reported studies. (b) Compounds originating from preliminary screen. (c) Compounds originating
from large scale virtual screens. (d) Compounds originating from the final knowledge-based search. aCompounds for which cocrystal structures
have been determined.
nitroso group rather than the oxygen atom seen with 3. The
latter difference is due to a slight repositioning of the pterin-
like moiety in 4 that brings it closer to Lys220 to minimize a
steric clash between the linker and Asp101. The pABA
moiety adopts two conformations in the two molecules of
the asymmetric unit. In molecule A, the moiety points down
to interact with Pro69 in a partially ordered loop2 (shown in
Figure 4d-f), and in molecule B, it points up to interact with
Phe189 (not shown). Both orientations appear to prevent
sulfate binding to the anion-binding pocket.
Preliminary Virtual Screening Results. The first stage of
virtual screening utilized a simple 2D pharmacophore search
with imposed distance constraints between specified donor
and acceptor groups followed by flexible docking of the hit
compounds of the Maybridge and NCI libraries (see Sup-
porting Information Figures S2 and S3). From the hits
identified we obtained three cocrystal structures (Com-
pounds 5, 6, 7; Figure 3b). Compounds 6 and 7 were potent
inhibitors of DHPS. Compound 5, which has a pterin-like
A-ring, did not show inhibition in our DHPS assay. It is unclear
why 5 does not inhibit the enzyme in our assay even though it
was shown to bind in our cocrystal trials. Potent inhibitor 6 is
similar to pterin but has a methyl substitution at position 8 that
prevents interaction with Asp101, and a carboxyl group at
position 6 that forms a novel salt bridge interaction with the
terminal amine of Lys220 (Figure 5a-c). Compound 7 is
structurally very similar to 3, with a nitro in place of a nitroso
group at the 5 position, and the cocrystal structure shows
interactions that are virtually identical (Figure 5d-f).
Large Scale Virtual Screening Results. To expand on the
previous studies, a high-throughput virtual screen of the
ZINC databases was performed. This used the crystal struc-
ture of 2 bound within the pterin pocket receptor for the
docking model. We chose this structure because 2 is a potent
inhibitor that accesses many of the key pterin-pyropho-
sphate binding residues in the pocket and the crystal struc-
ture is well determined. Loops 1 and 2 in our BaDHPS
structure are either disordered or involved in crystal packing
interactions, and although the loops are not believed to play
a major role in binding the pterin substrate, we built an
homology model of their conformations using the E. coli
and M. tuberculosis structures (Figure 2)9,11 and performed a

170 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Hevener et al.
Table 2. DHPS Hit Compounds with Docking Scores and Activitiesa
shown to represent better lead compounds, with more scope
for elaboration and optimization. Although the lower mo-
lecular weight and complexity of the fragment compounds
generally results in lower binding affinity (often high micro-
molar to low millimolar), they are often on par with or exceed
drug like compounds in terms of ligand efficiency (binding
affinity normalized by molecular weight or heavy atom
count).23-25 A further benefit of these selection criteria is
that it increases the likelihood for selecting compounds
with reasonable water solubility, as poor solubility was noted
for some of the analogues identified in the first compound
series.
From the ZINC screening libraries, 5093 compounds
matched the pharmacophore requirements, and when the
UNITY hit lists were merged, the total number of unique
compounds was 3104, indicating some redundancy in the
ZINC databases. All 3104 compounds were then docked and
scored by the Surflex docking tool within the Sybyl 7.3
molecular modeling suite.21,22 We previously reported a
docking validation study of the DHPS pterin site which
concluded that Surflex-Dock performs well in this particular
active site.26 The top 2% of the ranked compounds (62
compounds) were eventually selected for testing in the DHPS
enzyme assay. Of this number, 17 compounds were no longer
available from suppliers and the remaining 45 compounds
were procured and tested. The compounds were tested at
500 μM concentration (250 μM if very poorly soluble) and
a percentage inhibition was obtained. Eight compounds
showing greater than 30% inhibition, an acceptable stan-
dard when dealing with fragment-like compounds, and
suitable solubility were taken into crystallography trials
(Compounds 8-15, Figure 3c).27,28
Scaffold Search Results. To maximize the return of our
studies, a simple and rapid 2D scaffold search of all commer-
cially available compounds in the CAS registry was per-
formed using the key pharmacophoric elements discovered
in our previous studies. The key elements of the scaffold
search are shown in Figure 6. On the A-ring, the C2 nitrogen
and the nitrogens at the 1 and 3 positions were required to be
unsubstituted, and a carbonyl or the tautomeric phenol was
required at the 4 position. The B-ring allowed more flex-
ibility in the search; double or single bonds were permitted at
the 5,6 and 7,8 positions and the 6 position substituent had
no restrictions imposed. Finally, the substituent at the 8
position was restricted to an N-methyl group or unsubsti-
tuted nitrogen.
Using this scaffold search, 43 compounds were identified,
of which 19 were marked as interesting and selected for
procurement and testing. However, only 10 of these were
commercially available for immediate testing. Seven com-
pounds had activities above our 30% threshold and were
advanced into crystallography trials (Compounds 16-22,
Figure 3d), and four of these generated cocrystal structures.
All four compounds have the same A-ring structure seen in
the natural pterin substrate plus a nitrogen atom at the 5
position, and they engage the pocket in the expected fashion.
16 and 17 both have methyl substitutions at the N8 position
which prevent interaction with Asp101, but this interaction is
possible in 18 and 19, where the N8 is unsubstituted.
Compounds 17 and 19 each have a side chain at the 6 position
of the B-ring and both interact with the active site locale. In
17, the OH group interacts with a sulfate in the anion binding
pocket. Compound 19 is very similar to the product analogue
pteroic acid that we have already visualized in the active site,
a Yellow highlight indicates compounds for which crystal structures
have been determined. b 6-Hydroxymethyl-7,8-dihydropterin (HMDP),
was used as the control compound. c IC50 determination of 14 was not
possible due to a limited availability of the compound.
100 ps molecular dynamics simulation to refine their posi-
tions.
The virtual screening was performed using the UNITY
and Surflex programs available in the Sybyl 7.3 molecular
modeling suite of Tripos, Inc.19-22 We first prepared the
UNITY databases for screening from the ZINC libraries
that, at the time, contained nearly 5 million compounds and
included protonation variants and tautomers for the medium
pH range of 5.75-8.25. We then prepared the pharmaco-
phore filter from the 2 complex structure. The filter con-
tained three elements. The first was a surface volume
constraint created by including all residues surrounding the
˚
pterin pocket within 8 A of the bound 2 with a van der Waals
˚
tolerance of 1 A (Supporting Information Figure 1a). Part of
this volume included residues from the modeled loops 1 and 2
which represented their primary contribution to the overall
pharmacophore filter. The second element was the ligand-
based hydrogen-bonding constellation of 2 (Supporting In-
formation Figure 1b). These parameters were derived from
test runs to derive a hit-to-failure ratio that generated a
reasonable number of candidate compounds for the next
stage of molecular docking. The final element of the screen
was a molecular weight cutoff of 350 D and a maximum of
five rotatable bonds. We applied this filter to generate lower
molecular weight “fragment-like” molecules that have been

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 171
Figure 4. DHPS pterin site binding interactions of compounds 2 (a-c) and 4 (d-f). (a,d) Fo - Fc electron density maps contoured at 3σ using
Pymol.52 Maps were calculated from models refined after removing the compounds to avoid bias. (b,e) Details of the interaction using
MolScript.53 (c,f) LigPlot18 diagram of binding interactions (arrows near benzoic group in (f) indicate positional uncertainty of this group).
Figure 5. DHPS pterin site binding interactions of compounds 6 (a-c) and 7 (d-f). (a,d) Fo - Fc electron density maps contoured at 3σ using
Pymol.52 Maps were calculated from models refined after removing the compounds to avoid bias. (b,e) Details of the interaction using
MolScript.53 (c,f) LigPlot18 diagram of binding interactions.
and the binding is virtually identical (Figure 7a-c) .12 The
side chain engages the acyl chain of Lys220, and the terminal
carboxyl group interacts with the OH of Ser221. As shown in
Table 2, 16, 17, and 18 have relatively weak and equivalent
potencies as inhibitors, but 19 is exceptional, which probably
reflects its close similarity to the product.
DHPS Binding Order Studies. Previous kinetic analyses
of S. pneumoniae DHPS have shown that the enzyme cata-
lyzes a strictly ordered reaction in which DHPP is the lead
substrate followed by pABA.29 Our inhibitors all engage the
pterin-binding pocket. Thus it is probable that pterin-based
inhibitors can bind in the absence of other ligands. However,
in this study it was noticed that a sulfate ion in the anion-
binding pocket is present in all our DHPS-inhibitor com-
plexes, apart from compound 2, where the sulfate is dis-
placed by the anionic carboxylate group. This raises the
question if the binding of this inhibitor class may require that
the anion binding pocket also be occupied. To investigate

172 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Hevener et al.
Figure 6. Key pharmacophore elements and scaffold search criteria for pterin-like compounds that access the pterin pocket of DHPS.
Figure 7. DHPS pterin site binding interactions of compound 19. (a) Fo - Fc electron density map contoured at 3σ using Pymol.52 Map was
calculated from model refined after removing the compound to avoid bias. (b) Details of the interaction using MolScript.53 (c) LigPlot18
diagram of binding interactions.
Figure 8. Isothermal titration calorimetry. (a) Titration of 500 μM pABA into a protein solution of 20 μM B. anthracis DHPS. No heats of
binding could be detected. (b) Titration of 500 μM pABA into a 20 μM protein solution containing 10 mM sodium pyrophosphate. Dissociation
constant, Kd, is 5.62 ꢀ 10-6 M. (c) Titration of 500 μM compound 6 into a 20 μM protein solution. Kd is 5.26 ꢀ 10-6 M.
this possibility, we used an isothermal titration calorimetry
(ITC) approach to confirm binding order and study the
requirements for inhibitor binding. First it was verified that
the B. anthracis DHPS catalyzes an ordered reaction. Co-
incubation experiments show that pABA does not bind to the
enzyme in the absence of pyrophosphate, which was used to
mimic the presence of DHPP (Figure 8a), while pABA does
bind tightly to DHPS that has been preincubated with
pyrophosphate (Figure 8b). Thus, the B. anthracis DHPS
catalytic mechanism is indeed ordered. Then the requirement
for phosphate or sulfate anions to be present for the binding
of pterin-based inhibitors was examined using representative
inhibitor 6 for which a sulfate had been clearly resolved in its
complex structure (Figure 5b). Sulfate and phosphate ions
were carefully removed from the enzyme and inhibitor
samples prior to addition of the inhibitor to DHPS sample
in the ITC cell. The ITC still showed a positive isotherm
(Figure 8c), clearly demonstrating that occupancy of the
anion binding pocket is not required for binding of pterin
targeted inhibitors.

Article
Developed Pharmacophore Model. Using the activity and
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 173
Table 3. Calculated rmsd Values for Docked and Crystal Structure Hit
Compounds
structural data obtained from pterin pocket inhibitors iden-
tified in our studies, we have derived an initial SAR (or
pharmacophore) map based on the pterin two-ring structure
of the natural substrate (Figure 6). The A ring, particularly
the N1, C2, N3, and C4 positions that access the conserved
residues deep in the pterin pocket, is least tolerant to
modification. A number of compounds with A ring substitu-
tions were tested, but only seven showed sufficient activity
for structural studies. Compounds 8, 9, 13, 14, and 15 failed
to produce costructures, and 11 did not engage the pterin
pocket and instead formed a stacking/covalent interaction at
a remote surface location. We conclude that all six com-
pounds have little or no affinity for the pterin pocket, which
is consistent with the observed low activity of these com-
pounds in our assay (Table 2). Although 10 also has low
activity and shares minimal structural features with pterin,
we were successful in visualizing it within the pterin pocket.
Its interactions with the pocket residues are quite unique and
it appears to represent a novel low molecular weight scaffold
that we intend to pursue.
In contrast, the B ring that binds closer to the opening of
the pterin pocket is far more tolerant of modifications and
provides more opportunities for optimizing the potency of
pterin-based inhibitors. Compounds with both six- and five-
membered B rings and open B rings were visualized in our
structural studies. Compound 5 was the only five-membered
ring compound identified in our screens, and this showed
little or no inhibition of the enzyme. We also tested a 5
homologue in which the SH group is replaced with an OH
group, and this was also shown to have minimal activity
(data not shown). We therefore concentrated on the six-
membered and open B ring compounds and identified three
features that improve potency. First, an acceptor at the 5
position is required to form a second hydrogen-bonding
interaction with conserved Lys220. An Sp2 nitrogen per-
forms this task in the natural pterin substrate, but carbonyl,
nitro, or nitroso groups appear to be superior based on our
structures and assay data. The second favorable feature is a
carboxyl group attached to the C6 position. This feature is
present in 2 and 6, which are both potent inhibitors. In 6, the
carboxyl group is directly attached to C6 and forms a salt
bridge with Lys220, and a sulfate ion is present in the anion-
binding pocket. However, in 2, the carboxyl group is at-
tached via a short linker, which allows it to engage Arg254 in
a salt bridge/hydrogen bonding interaction, and it displaces
the sulfate from the anion-binding pocket. In addition, the
methyl group on the linker makes van der Waal interactions
with the conserved Phe189. The potencies of 2 and 6 are
equivalent, and it is unclear which of the two carboxyl
interactions is superior. However, the 2 costructure suggests
that extension of the linker by one or two carbon atoms
would enable the carboxyl to more fully engage the anion-
binding pocket. The final favorable feature is a hydrogen-
bonding interaction between the conserved Asp101 and a
donor at the N8 position. This interaction is possible in 3, 5,
7, 18, and 19 as well as the natural pterin substrate, but not
possible in 2, 6, 16, 17, and 4 (the latter for steric reasons). We
have direct evidence that this feature increases potency; 1 and
7 are identical compounds apart from a methyl substitution
at the N8 position in 1, and 7 is the more potent compound.
In addition, 19 is our most potent compound but introducing
a double bond at N8 which removes the donor hydrogen, as
in 21, significantly reduces potency (Table 2).
˚
rmsd value (A)
compd
1
1.100
0.906
2
3
2.134
4
4.73 (1.148)a
1.003
0.837
5
6
7
0.585
0.651
10
16
17
18
19
0.496
0.658
1.412
5.121 (0.509)a
a Values in parentheses reflect calculated rmsd values for only the
pterin ring substructure of these two compounds.
Crystal versus Docked Structures. The 12 crystal structures
presented here, together with the compound 1 structure
reported earlier12 provide an alternative test of the docking
procedure, namely calculating the heavy atom rmsd values
between the crystal structures and their corresponding
docked poses. As can be seen in Table 3, the docked poses
generally correspond very closely to the crystal structure
positions, and we attribute this success to the prior validation
that was performed to select the optimal docking software, in
this case, SURFLEX-Dock.26 In these docking validation
˚
studies, we used an rmsd value of 1.5 A as the cutoff for
success and, applying the same criteria here, it can be seen
that eight of the 11 docking poses accurately predict the
crystal structures. Can we rationalize the three docking
failures? The reason why 3 failed is not clear, especially
because the predicted pose of the similar 1 closely matches
the crystal structure. One possible explanation is that the
position of the compound in the docked pose is influenced by
an electrostatic interaction between the 6-amino group and
Asp61, which results in the loss of the electrostatic interac-
tion with Lys220. Compound 3 is also the smallest hit
compound studied, and it can sterically adopt poses that
are not accessible to the larger compounds. Regarding 4 and
19, the docked positions of the pterin ring substructure were
essentially correct (see numbers in parentheses in Table 3),
and the deviations occurred in the pABA-like moieties that
are predicted to engage the flexible loops with associated
docking uncertainty.
Limitations of Pterin-Based Inhibitors. With one excep-
tion, all of the hit compounds are similar to the natural pterin
substrate. Considering the high specificity and conserved
nature of the pterin-binding pocket, this selectivity is not
surprising, and it has been noted that the pocket does not
easily accommodate compounds with alternate scaffolds.16
However, from a drug discovery perspective, there are two
drawbacks to pterin-like compounds. First, pterin-like com-
pounds tend to be poorly soluble due to their planar char-
acter, which results in high crystal lattice energy.30 This has
led to some degree of experimental difficulty and, in some
cases, necessitated activity testing at a lower concentra-
tion than our standard concentration (250 μM rather than
500 μM). It is well documented that poor solubility has
negative ramifications in terms of drug discovery and clinical
candidacy.30,31 We plan to address this problem by adding
anionic functional groups that can interact with the anion
binding pocket, and the addition of a carboxylate group at
the 6 position is a first step in this process. Second, we would

174 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Hevener et al.
Figure 9. DHPS pterin site binding interactions of compound 10. (a) Fo - Fc electron density map contoured at 3σ using Pymol.52 Map was
calculated from model refined after removing the compound to avoid bias. (b) Details of the interaction using MolScript.53 (c) LigPlot18
diagram of binding interactions.
prefer our hit compounds to have more diversity in chemical
structure and to include novel scaffolds. It is now recognized
that “scaffold hopping” is an important part of the drug
discovery process .32 The use of a ligand-based filter may
explain why so many pterin-like compounds were detected
by virtual screening, and a receptor-based filter is planned
for future studies in an attempt to identify alternate scaf-
folds.
compounds to include in our future screening efforts based
upon this scaffold. Our current compounds and future
libraries all target key active site residues and, unlike the
sulfonamide drugs, avoid the flexible loops that typically
accrue resistance mutations.
Methods
Compound Procurement. The majority of compounds used in
this study were procured from the following commercial ven-
dors and compound repositories: 3, Toronto Research Chemi-
cals, Inc.; 5, Ryan Scientific, Inc.; 6, 7, 10, 12, 16, 17, 19, 21, and
22, National Cancer Institute’s Drug Testing Program;34 8 and
11, Specs, Inc.; 9, 13, 18, and 20, Sigma Aldrich, Inc.; 14,
ChemDiv, Inc.; 15, ChemBridge, Inc. The remaining com-
pounds that were not commercially available were synthesized
according to the following published procedures: 1,12,16 2,35 and
4.17 Details of the preparation of 2 and 4 are provided in the
Supporting Information (Scheme S1 and synthesis methods).
Compound Purity Testing. Proof of purity for all compounds
purchased or synthesized was determined by analytical reverse-
HPLC was conducted on a Shimadzu HPLC system using a
One compound with a novel scaffold, 10, did result from
the virtual screening studies. Although non-pterin-like, the
compound is able to make many of the key binding inter-
actions defined in the pharmacophore model. Parts a-c of
Figure 9 show how 10 accesses the pocket, and it is satisfy-
ing in terms of our general approach that docking success-
fully recapitulated the crystal structure. The N1 nitrogen
forms an H-bonding interaction with Lys220 and a key
nitrogen triad interacts with the conserved and crucial
Asn120 and Asp184. Finally, due to its planar nature, 10
is also able to engage Arg254 in the characteristic stacking
interaction seen with all of the pterin-like hit compounds.
The positions of the carboxylate group of Asp101 and the
N6 ring nitrogen of 10 indicate the presence of an electro-
static interaction at this position which facilitates binding.
Microspecies and pKa calculations performed with 10 re-
veal that the nitrogen is weakly basic (pKa 6.13) and has a
predicted microspecies population of only 2.62% at pH
7.4.33 However, the charge on the adjacent Asp101 is likely
to raise the pKa and the bound species of 10 may actually
carry a significant positive charge centered at the N6
position. One key binding feature that is absent in 10 is a
negatively charged group that can engage the anionic
pocket. 10 has a molecular weight of 150.1 and is a bona
fide “fragment” molecule with many opportunities for
elaboration. Future studies are planned with 10 analogues
to explore the SAR of substitutions or modifications at the
7 position to take advantage of these opportunities to
improve binding affinity.
˚
Phenomenex Luna C18 column (100 A, 3 μm, 4.6 mm ꢀ 50 mm),
flow rate 1.0 mL/min, and a gradient of solvent A (water with
0.1% TFA) and solvent B (acetonitrile): 0-2.00 min 100% A;
2.00-8.00 min 0-100% B (linear gradient) and UV detection at
254 nm/215 nm. Compounds were determined to be of g95%
purity.
Enzyme Assay. Enzyme Preparation. The E. coli HPPK-GST
fusion gene was provided by Dr. Honggao Yan and transformed
into competent BL21 (DE3) E. coli cells (Novagen cat.
N69450).36 The initial culture was grown overnight at 26 ꢀC in
100 mL of LB containing 100 mg/L ampicillin, and 13 mL of this
culture was used to inoculate 1 L of the same medium. This was
grown at 37 ꢀC until the OD reached 0.8, at which point
isopropyl-B-D-thiogalactopyranoside (IPTG) was added to a
final concentration of 0.4 mM. Cells were then grown overnight
in IPTG at 28 ꢀC and harvested by centrifugation at 4 ꢀC, 4000
rpm, for 15 min. The cell pellet was suspended in 100 mL of PBS
with 10 mg of lysozyme and lysed by a microfluidizer
(Microfluidics M-110), and the cell debris was cleared by
centrifugation at 30000 rpm for 30 min. The supernatant was
filtered through a 0.45 mm syringe filter and applied to a 5 mL
GSTrap FF column (Amersham Biosciences), and the column
was washed with 70 mL of PBS binding buffer and then step-
eluted using 50 mM Tris-HCl, 10 mM reduced glutathione, pH
8.0. The eluted protein was essentially pure and adjusted to a
concentration of 5 mg/mL, dialyzed in 100 mM NaCl, 20 mM
Tris pH 8.0, and finally stored at -80 ꢀC. The B. anthracis
DHPS enzyme was expressed in E. coli and purified as pre-
viously described.12 Protein concentrations were measured by
the Bradford protein assay (Bio-Rad Laboratories) using bo-
vine serum albumin as standard.
Conclusions
In these studies, we have thoroughly characterized the
pterin binding pocket of DHPS and generated a detailed
structure-activity based pharmacophore map that will facil-
itate the development of novel DHPS inhibitors that specifi-
cally target the pterin site. We have identified the optimal
binding features of the pterin scaffold and will apply these
insights to the production of pterin-based libraries for further
screening efforts. We have also identified a nonpterin scaffold
that engages the pocket and we will create a second library of

Article
DHPS Substrates. 6-Hydroxymethyl-7,8-dihydropterin
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 175
using F-score, PMF-score, and ChemScore and selected on the
basis of their consensus score as well as visual inspection and
comparison with the DHPP substrate.
hydrochloride was purchased from Schircks Laboratories,
Switzerland. [ring-14C]para-aminobenzoic acid (14C pABA,
55 mCi/mmol) was obtained from Moravek Biochemicals,
USA. 6-Hydroxymethyl-7,8-dihydropterin diphosphate is un-
stable and was prepared enzymatically using HPPK-GST.37,38
6-Hydroxymethyl-7,8-dihydropterin was incubated at 37 ꢀC for
30 min in 5 mM ATP, 10 mM magnesium chloride, 3% dimethyl
sulfoxide, 20 μg/mL GST-HPPK, and 50 mM HEPES pH 7.6.
The reaction solution was passed through the GST•Bind Resin
(Novagen) to remove the GST-HPPK enzyme and stored in
aliquots at -80 ꢀC.
Enzyme Assay. DHPS activity was measured in a 30 μL
reaction containing 5 μM 14C pABA, 10 μM 6-hydroxy-
methyl-7,8-dihydropterin diphosphate, 10 mM magnesium
chloride, 2% DMSO, 50 mM HEPES pH 7.6, and 10 ng
DHPS.29,39 After 30 min incubation at 37 ꢀC, the reactions were
stopped by addition of 1 μL of 50% acetic acid in an ice bath.
The labeled product of the reaction, 14C dihydropteroate, was
separated from 14C pABA by thin layer chromatography.
Aliquots (15 μL) of the reaction mixture were spotted onto
Polygram TLC plates (CEL 300 PEI) purchased from Macher-
ey-Nagel and developed with ascending chromatography in 100
mM phosphate buffer pH 7.0. The plates were scanned using a
Typhoon (GE Healthcare) and analyzed with ImageQuant TL.
Inhibitor compounds were dissolved in DMSO, and inhibition
was tested at 500 or 250 μM depending on solubility. The final
concentration of DMSO in the reaction mixture was 2%. To
determine the 50% inhibitory concentration (IC50) values,
DHPS activities were measured in the presence of various
concentrations of the compounds using the conditions described
above but with 5 ng DHPS. Data were analyzed by using Prism
GraphPad software.40
Crystallography. The structures of all DHPS-inhibitor com-
plexes were obtained by soaking the small molecules into the
P6222 B. anthracis DHPS crystals described earlier.12 The small
molecules were first dissolved in crystal mother liquor (1.3 M
Li2SO4, 0.1 M Bis-Tris propane, pH 9.0) to the saturation
allowed by their typically limited solubility (approximately 1
mM), and the crystals were transferred into these solutions for
24-48 h soaking periods. Crystals were then cryoprotected by
brief immersion in 50% Paratone-N and 50% mineral oil, and
flash frozen in liquid nitrogen. All data were collected at the
SER-CAT beamlines 22-ID and 22-BM at the Advanced
Photon Source and processed using HKL2000.41 Structures
were directly refined using REFMAC42,43 and the deposited
coordinates of B. anthracis DHPS bound with pteroic acid (PDB
code 1TX0). Model building was performed using the COOT
program.44 Relevant data collection and refinement statistics
are presented in Supporting Information Tables S1 and S2.
Isothermal Titration Calorimetry. The purified B. anthracis
DHPS protein was dialyzed against 50 mM HEPES, 5 mM MgCl2,
pH 7.6. ITC titrations were performed in 40 mM HEPES, 4 mM
MgCl2 at pH 7.6 and 25 ꢀC. 5% DMSO was added to the ITC
buffer for the titration experiment of Compound 6. Nineteen
injections of 2 μL each (except 0.5 μL for the first injection) of
500 μM ligand solution were added to 200 μL of 20 μM protein
solution. ITC titrations were performed on an ITC200 Microca-
lorimeter (MicroCal), and data was analyzed using MicroCal
Origin 7.0 software using a one-site binding model.
For the second stage of virtual screening (large scale screen-
ing), the screening compounds used were downloaded in sdf for-
mat from the vendors subsets of ZINC database (version 6).46
The sdf files were converted to UNITY databases for pharma-
cophore screening using the UNITY tool available with the
Sybyl 7.3 molecular modeling suite of Tripos, Inc.19,20,45 2D and
Macro fingerprints were created using default settings. Concord
was used to generate 3D coordinates, when necessary.47 Default
values were accepted for all other UNITY database preparation
settings. The UNITY pharmacophore filter consisted of 1 donor
and 4 acceptor positions based upon the H-bonding patterns
observed in the crystal structure of 2 (Figure 4) as discussed in
˚
the Results and Discussion section. A spatial tolerance of 0.3 A
was used for each macro and 2 partial match constraints were
applied. The UNITY databases were screened using a 3D Flex
search with modified “Rule of Three” search options as dis-
cussed in the Results and Discussion section.48 The flex ring
search option was also enabled. All other settings retained their
default values.
Hit lists from the pharmacophore filtering were merged to
eliminate duplicate compounds and then the converted to a
multi-mol2 file for docking. Charges were loaded to the com-
pounds using the Gasteiger-Huckel method.49 Surflex docking
utilized the multi-mol2 file and a protomol generated using a
threshold of 0.50 and bloat of zero (default values).22 These
settings are the same as those used in our previously reported
docking validation study.26 An active site water was retained for
all docking runs. The ring flexibility function was enabled; all
other docking settings retained their default values. Compounds
docked with Surflex were scored with the native Surflex scoring
function; the Cscore option was disabled. The top 2% of the
Surflex scored compounds were selected for procurement and
testing in the enzyme assay described above.
The final stage of compound selection was used to maximize
our emerging structure activity relationship results obtained in
the previous stages. This screen was performed using simple 2D
scaffold similarity search using SciFinder against all commer-
cially available compounds in the Chemical Abstracts Services
(CAS) registry.50,51 The scaffold search criteria are shown in
Figure 6. The search criteria were identified by visual SAR
analysis of compounds 1 through 15, which were identified in
the earlier stages of screening or were known pterin site binders
from previous studies. Key binding features were identified in
these earlier compounds using their measured % inhibition of
enzyme activity. Compounds matching the scaffold search
constraints were procured and tested as described above.
Acknowledgment. Funding for this research was provided
by National Institutes of Health grants AI060953 (to SWW
and REL) and AI070721 (to SWW and REL), Cancer Center
core grant CA21765, and the American Lebanese Syrian
Associated Charities (ALSAC). SER-CAT supporting insti-
tutions may be found at www.ser.anl.gov/new/index.html.
Use of the Advanced Photon Source was supported by the
U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences, under contract no. W-31-109-Eng-38. We
acknowledge the technical assistance of D. Ball and J. Scar-
borough at the University of Tennessee, Health Science
Center, and of M. Frank in the Department of Infectious
Diseases at St. Jude Children’s Research Hospital. Support of
this research by the American Foundation for Pharmaceutical
Education is also gratefully acknowledged.
Computational and Experimental Methods. The first stage of
preliminary virtual screening utilized a simple 2D pharmaco-
phore search with imposed distance constraints between speci-
fied donor and acceptor groups followed by flexible docking of
the hit compounds (Supporting Information Figures S2 and S3).
The UNITY program implemented in the Sybyl v6.9 molecular
modeling package was used to perform the 2D search and the
FlexX implementation in Sybyl v6.9 was used to perform the
docking.19,20,45 The Maybridge and NCI databases supplied
with the Sybyl package were screened. Compounds were scored
Supporting Information Available: Pharmacophore screening
methods, organic synthesis scheme and methods, statistics of

176 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Hevener et al.
X-ray crystallography data collection, and refinement. This
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