Structural analysis of the active sites of dihydrofolate reductase from two species of Candida uncovers ligand-induced conformational changes shared among species

Article abstract of DOI:10.1016/j.bmcl.2013.01.008

A novel strategy for targeting the pathogenic organisms Candida albicans and Candida glabrata focuses on the development of potent and selective antifolates effective against dihydrofolate reductase. Crystal structure analysis suggested that an essential loop at the active site (Thr 58-Phe 66) differs from the analogous residues in the human enzyme, potentially providing a mechanism for achieving selectivity. In order to probe the role of this loop, we employed chemical synthesis, crystal structure determination and molecular dynamics simulations. The results of these analyses show that the loop residues undergo ligand-induced conformational changes that are similar among the fungal and human species.

Full text of DOI:10.1016/j.bmcl.2013.01.008

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1279–1284
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural analysis of the active sites of dihydrofolate reductase from
two species of Candida uncovers ligand-induced conformational
changes shared among species
⇑
⇑
Janet L. Paulsen, Kishore Viswanathan, Dennis L. Wright , Amy C. Anderson
Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Rd., Storrs, CT 06269, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel strategy for targeting the pathogenic organisms Candida albicans and Candida glabrata focuses on
the development of potent and selective antifolates effective against dihydrofolate reductase. Crystal
structure analysis suggested that an essential loop at the active site (Thr 58-Phe 66) differs from the anal-
ogous residues in the human enzyme, potentially providing a mechanism for achieving selectivity. In
order to probe the role of this loop, we employed chemical synthesis, crystal structure determination
and molecular dynamics simulations. The results of these analyses show that the loop residues undergo
ligand-induced conformational changes that are similar among the fungal and human species.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 4 October 2012
Revised 20 December 2012
Accepted 2 January 2013
Available online 11 January 2013
Keywords:
Antifolate
DHFR
Protein flexibility
X-ray crystallography
Molecular dynamics
The severe health risks presented by systemic fungal infections
continue to produce significant increases in mortality and morbid-
ity. Candida spp. are primary pathogenic organisms in systemic
infections with Candida albicans accounting for approximately
50–70% of infections.1,2 However, Candida glabrata, associated with
a high rate of mortality, now accounts for an increasing proportion
of infections.3,4 Additionally, C. glabrata is less sensitive to com-
monly used antifungal agents such as fluconazole and other
azoles.1,5 The development of new classes of antifungal agents with
good potency against these Candida species is a high priority.
The vast majority of the current antifungal agents target the
fungal cell wall or its biosynthesis, leaving many of the essential
metabolic functions unexplored as therapeutic targets. One such
essential enzyme, dihydrofolate reductase (DHFR), has long been
appreciated as an effective target for antimicrobial therapy. As
DHFR is also essential to human cells, an effective antifungal antif-
olate design must take advantage of differences in the pathogenic
enzyme relative to the human enzyme. Over the past several years
we have focused on the development of a novel class of antifolates
characterized by a conserved diaminopyrimidine moiety linked
through a propargylic spacer to a variable hydrophobic domain.6–
11 Leads such as compound 1 (shown in Table 1) have been shown
to function as inhibitors of C. glabrata (CgDHFR) and C. albicans
DHFR (CaDHFR) as well as exhibit antifungal activity (MIC values
of 1.5 lg/mL) and thus are interesting as potential antifungal ther-
apeutics.7,8,11 We have reported several crystal structures of
CgDHFR and CaDHFR bound to NADPH and several members of this
lead series.7,8,11 Analysis of these crystal structures has strongly
suggested that residues in a loop at the fungal active site (Thr
58-Phe 66) may be displaced further from the active site relative
to the analogous residues in the human structure (Thr 56-Asn
64), a property that may be advantageous for gaining selectivity
in inhibitor design.
In order to investigate whether this critical loop truly plays an
active role in determining the affinity of the propargyl-linked
antifolates for the fungal and human enzymes, here we present
an analysis of existing crystal structures of CgDHFR and CaDHFR
bound to the propargyl-linked antifolates as well as four new ter-
nary crystal structures of CgDHFR and CaDHFR bound to a recent
generation of inhibitors possessing a heterocyclic moiety in-
tended to improve solubility.12 We then designed and synthe-
sized four additional propargyl-linked antifolates with a bulkier,
fused ring system intended to especially probe the flexibility of
the loop. Crystal structures of CgDHFR and CaDHFR with one of
these compounds emphasize the ligand-induced conformational
changes observed with these more sterically demanding inhibi-
tors. Finally, we carried out a detailed molecular dynamics study
that focused on the loop regions of both the fungal and human
enzymes. These MD results show that this loop in the active site
is particularly flexible and while the loop undergoes ligand-in-
duced conformational changes, these changes are similar across
the three species.
⇑
Corresponding authors. Tel.: +1 860 486 6145/9451; fax: +1 860 486 6857.
E-mail addresses: dennis.wright@uconn.edu (D.L. Wright), amy.anderson@ucon-
n.edu (A.C. Anderson).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.008

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Table 1
Enzyme inhibition of CaDHFR, CgDHFR and human DHFR with propargyl-linked antifolates
ID
Structure
CaDHFR IC50 (nM) (selectivity)
CgDHFR IC50 (nM) (selectivity)
0.60 0.27 (2350)
huDHFR IC50 (nM)
1
22 1 (64)
1410 15
2
3
4
21 4 (19)
23 4 (11)
61 3 (25)
20 3 (20)
22 2 (11)
97 9 (15)
400 40
250
4
1500 80
5
60 2 (22)
89 8 (15)
16 1 (3.8)
11 1 (4.1)
1300 10
12
13
71 20 (0.84)
60
45
6
3
17 5 (2.6)
18
19
78 2 (1.8)
19 4 (7.4)
140
9
23 2 (11)
18 3 (14)
260 20
20
21
100 7 (13)
33 14 (9)
8.2 1.3 (156)
11 2 (26)
1280 15
290 17
Over the past several years, we have reported eight CgDHFR and
three CaDHFR crystal structures bound to NADPH and antifolates
possessing a pyrimidinyl ring linked through the propargyl bridge
to substituted monophenyl or biphenyl systems.7,8,11 Analysis of
these structures has been critical to improving the potency and
selectivity of the inhibitors for the fungal enzymes. While there

J. L. Paulsen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1279–1284
1281
are a few residue substitutions between the human and fungal en-
zymes, including Phe 31/Met 33[Ile 33], Val 115/Ile 122 and Asn
64/Phe 66 (Fig. 1A), a broad study of these fungal structures in
comparison to structures of the human DHFR enzyme13 has sug-
gested the intriguing possibility that selective inhibitors could be
designed by taking advantage of conformational differences in a
key active site loop found in both fungal pathogens (residues 63–
66) as well as human DHFR (residues 61–64). Figure 1 shows a
structural alignment of CaDHFR, CgDHFR and human DHFR (huD-
HFR) as well as a superposition of the two fungal enzymes bound
to propargyl-linked antifolates with monophenyl moieties and
the human enzyme bound to a pyridopyrimidine inhibitor.13 The
structural comparison suggests that the loop in human DHFR
(61–64) restricts the active site more profoundly than the corre-
sponding loop in fungal DHFR (58–66) and that selective inhibitors
may be designed with increased bulk at the distal ring to sterically
interfere with the loop in human DHFR.
As we had observed that a third generation of compounds pos-
sessing heterocyclic ring systems at the distal position (ring C in
structure 1) showed improved solubility,12 we also evaluated these
compounds as inhibitors of the fungal enzymes (Table 1). Overall,
conversion of the phenyl ring in 1 to either aromatic or alicyclic
heterocycles (2–5, 12, 13, 18, 19) results in an attenuation of en-
zyme inhibition (3- to 20-fold loss) and selectivity ratios. In order
to understand the structural basis of the reduced potency, we
determined four ternary crystal structures: CgDHFR bound to 3
(PDB ID: 3ROA), CgDHFR bound to 5 (PDB ID: 3RO9), CaDHFR
bound to 5 (PDB ID: 4H95) and CaDHFR bound to 1 (PDB ID:
4H97), as a comparator (crystallographic details in Supplementary
data). Analysis of these structures shows that compounds 3 and 5,
especially as bound to CgDHFR, reorient to bring the polar distal
ring away from loop residues Pro 63 and Phe 66 and toward the
solvent accessible surface, thus decreasing hydrophobic contacts
(Fig. 2A). As previously noted, the loop residues appear to be flex-
ible in CgDHFR with the position of the C
a atoms of Pro 63 varying
between 0.5 and 1.1 Å. Similarly, in the structures with CaDHFR
bound to 1 and 5, the distance between Pro 63 C
a atoms is 0.8 Å
(Fig. 2B).
Our prior structural analysis suggested that increasing bulk at
the distal C-ring position of the inhibitors may improve potency
by increasing contacts with the fungal enzymes (specifically resi-
dues Pro 63 and Phe 66) while simultaneously improving selectiv-
ity by inducing steric interference with the human residues Pro 61
and Asn 64, which appeared to be closer to the inhibitors. This
strategy relies on species-specific conformations of the loop in
both enzymes. In order to evaluate this hypothesis, we first de-
signed a focused docking library for evaluation in silico. The design
involved the annulation of the distal C ring with the specification of
Figure 1. (A) Structural alignment of CaDHFR, CgDHFR and huDHFR with the loop residues boxed, (B) structural differences in the loop region of CaDHFR (green; PDB ID:
3QLR11), CgDHFR (pink; PDB ID: 3QLX11) and human DHFR (purple; PDB ID: 1PD813).

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building blocks were docked to the fungal and human structures.
Compounds with substituted isoquinoline and benzodioxane ring
systems emerged as potential leads. The parent compounds (12,
13, 18, and 19 in Table 1) were chosen for synthesis in order to
make a preliminary evaluation of this new design.
Compounds 12, 13, 18, and 19 were prepared based on analogy
to prior routes12 and involved a key Suzuki coupling between the
isoquinoline or benzodioxane boronic acids and the brominated
benzaldehyde (6) or acetophenone (7) to give 8 and 9 in excellent
yields (Scheme 1). After completion of the coupling, the aldehyde
or methyl ketone was homologated to the corresponding terminal
alkynes 10 and 11 through sequential Wittig condensation, hydro-
lysis and Ohira–Bestmann reaction. The inhibitors (12, 13, 18, and
19) were ultimately prepared in high yield by a final Sonogashira
coupling with an iodo-pyrimidine.
Evaluation of enzyme inhibition (Table 1) shows that 12, 13, 18
and 19 are potent inhibitors of CgDHFR and additionally that 13
and 19 are potent inhibitors of CaDHFR. Thus, the expanded ring
system enhanced affinity for the fungal enzymes, as designed.
Unfortunately, the compounds also potently inhibit the human
form of the enzyme and in fact, lowered the selectivity index rela-
tive to the biphenyl compounds. The fact that the docking scores
predicted better selectivity than was observed experimentally
may point to the difficulty of negative design (selectivity) using
algorithms developed for positive design (potency). Although these
results indicate that this strategy would not be useful for improv-
ing selectivity, it also revealed that the understanding of this re-
gion of the active site is incomplete and that these molecules
could serve as useful probes to better characterize the dynamic as-
pects of the active site residues.
In an effort to understand the basis of potency and the dimin-
ished selectivity of the compounds with fused ring heterocycles,
crystal structures of CgDHFR and CaDHFR bound to NADPH and
18 were determined (PDB IDs: 4H98 and 4H96). Again, the loop
residues in the fungal DHFR enzymes show ligand-induced confor-
mational changes. In CgDHFR with benzodioxane 18, the loop con-
taining Pro63-Phe 66 shows modest displacement by 0.75 Å to
more optimally interact with the dioxane moiety as compared to
the structure of CgDHFR bound to the biphenyl 1 (Fig. 3A). In CaD-
HFR, although Pro 63 remains in the same position in the struc-
tures with both 1 and 18, the remainder of the residues in the
Figure 2. (A) CgDHFR in complex with 3 (cyan), 5 (pink) and 1 (purple), (B) CaDHFR
in complex with 5 (green) and 1 (purple).
incorporated heteroatom(s). Using Surflex-Dock,14 approximately
250 compounds derived from commercially available boronic acid
R
R
O
O
O
NH2
N
O
O
R
O
b-d
e
a
R
N
B(OH)2
H2N
Br
N
N
N
N
R
R= H
6
R= H 8, 80%
R= Me 9, 86%
R= H 10
R= Me 11
R= H 12, 76%
R= Me 7
R= Me 13, 81%
O
R
R
O
O
O
NH2
N
O
O
a
R
b-d
e
N
B(OH)2
H2N
Br
O
O
O
O
O
O
O
O
R= H
R= Me
6
7
R= H 14, 85%
R= Me 15, 88%
R= H 16
R= Me 17
R= H 18, 78%
R= Me 19, 84%
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, Cs2CO3, dioxane, 80 °C; (b) Ph3P@CHOMe, THF; (c) Hg(OAc)2, KI, THF/H2O; (d) dimethyl(1-diazo-2 oxopropyl)phos-
phonate, K2CO3, MeOH; (e) 5-iodo-6-ethyl pyrimidine, Pd(PPh3)2Cl2, CuI, Et3N, DMF.

J. L. Paulsen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1279–1284
1283
Figure 3. Superposition of structures of (A) CgDHFR bound to 18 (cyan) and 1 (purple) and (B) CaDHFR bound to 18 (green) and 1 (pink).
Figure 4. MD analysis by species A–C. (A) huDHFR, (B) CgDHFR and, (C) CaDHFR bound to 20 (pink), 21 (cyan), 1 (green) and in the binary state (purple). MD analysis by
ligand, (D–F) huDHFR (green), CgDHFR (cyan) and CaDHFR (pink) bound to (D) 20, (E) 21 and (F) 1.

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J. L. Paulsen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1279–1284
CaDHFR loop (64–66) are significantly displaced by up to 2 Å, dri-
ven largely by interactions with Phe 66 (Fig. 3B).
Targeting metabolic enzymes such as dihydrofolate reductase
in Candida spp. has the potential to lead to the development of no-
vel treatment strategies for fungal infections. The analysis of exist-
ing crystal structures of C. albicans and C. glabrata DHFR as well as
four new ternary structures presented here highlight that a key
loop at the active site undergoes ligand-induced conformational
changes. Based on docking results, four new compounds intended
to probe the flexibility of the loop were synthesized and found to
be potent inhibitors of the pathogenic and, somewhat surprisingly,
human enzymes. Crystal structures of one of these compounds
bound to the fungal enzymes again emphasized ligand-induced
conformational changes. Molecular dynamics simulations with
the fungal and human enzymes reveal that the loop undergoes li-
gand-induced conformational changes that are driven by the li-
gand, not the species. As the conformation of the loop may not
allow the design of selective inhibitors, greater emphasis should
be placed instead on residue substitutions.
Although it is clear that the loop residues in fungal DHFR show
ligand-induced conformational changes, it is not evident whether
the human DHFR loop residues show similar dynamics. Therefore,
it became essential to evaluate the differential dynamics of the
fungal and human loops in order to refine the model for future de-
sign work.
In order to assess the dynamics of the loop residues and the de-
gree to which they are influenced by the bound ligand in both fun-
gal and human DHFR enzymes, we carried out free MD simulations
inclusive of solvent using ternary and binary (only NADPH bound)
complexes. Three compounds (1, 20 and 21 in Table 1) represent-
ing a spectrum of potency and selectivity for human and fungal
DHFR were chosen to probe the loop conformation.
Crystal structures of CgDHFR and CaDHFR bound to compounds
1, 20 and 21 were used as starting structures; compounds 1, 20 and
21 were docked to human DHFR. Protein complexes were prepared
by assigning bond orders, adding hydrogens, capping termini,
deleting all waters, adding missing side chains and optimizing
hydrogen bonds.15 Desmond system builder was used to construct
the system for simulation. A periodic boundary box was built by
explicitly adding waters, counter ions and salt to simulate physio-
logical conditions. The resulting system was minimized using two
stages (restrained and unrestrained) followed by several stages of
MD runs to gradually remove any system restraints. The prepared
systems were transferred to the Extreme Science and Engineering
Digital Environment (www.xsede.org, formerly the TeraGrid). Sim-
ulations were run in Desmond with the OPLS force-field for 10 ns.
Visualization and analysis focused on the last 9 ns to ensure sys-
tem equilibrium was achieved prior to production.
Acknowledgements
The authors thank the NIH for funding this work (GM067542),
the Pittsburgh Supercomputing Center for time to perform the
molecular dynamics calculations and Melanie Allen for assistance
with enzyme assays.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.
008.
Analyzing each species independently reveals that the loop res-
idues appear to adopt ligand-induced conformations. Relative to
the loop conformation adopted with 20 in human DHFR (Fig. 4A),
References and notes
1. Pfaller, M.; Diekema, D. J. Clin. Microbiol. 2004, 42, 4419.
2. Pfaller, M.; Diekema, D. Clin. Microbiol. Rev. 2007, 20, 133.
3. Arendrup, M. Curr. Opin. Clin. Care 2010, 16, 445.
the loop is displaced 0.4 Å (as measured from C
a of Pro 61) in
the structure with 1, 1.4 Å in the structure with 21 and 1.6 Å in
the binary complex. For CgDHFR (Fig. 4B), relative to the loop con-
formation adopted with 20, the displacement is 1.2, 0.9, and 1.6 Å,
respectively. Lastly, in CaDHFR (Fig. 4C) the same comparison
yields displacements of 1.2, 2.3 and 4.3 Å. These results show that
the ligand induces significant displacement of the loop residues in
all three species.
4. Falagas, M.; Roussos, N.; Vardakas, K. Int. J. Infect. Dis. 2010, 14, e954.
5. Bennett, J. E.; Izumikawa, K.; Marr, K. Antimicrob. Agents Chemother. 2004, 48,
1773.
6. Bolstad, D.; Bolstad, E.; Frey, K.; Wright, D.; Anderson, A. J. Med. Chem. 2008, 51,
6839.
7. Liu, J.; Bolstad, D.; Smith, A.; Priestley, N.; Wright, D.; Anderson, A. Chem. Biol.
2008, 15, 990.
8. Liu, J.; Bolstad, D.; Smith, A.; Priestley, N.; Wright, D.; Anderson, A. Chem. Biol.
Drug Des. 2009, 73, 62.
We next investigated whether a given ligand would induce the
same loop conformation across the three different species. Super-
positions of the three species bound to the same ligand (Fig. 4D–
F) show that the loop residues show minimal displacements,
reflecting ligand-induced conformational changes are independent
of species. The fact that the ligand drives a specific conformation of
the loop, independent of species, implies that selectivity cannot be
easily achieved by attempting to induce species-specific steric
interference.
9. Pelphrey, P.; Popov, V.; Joska, T.; Beierlein, J.; Bolstad, E.; Fillingham, Y.; Wright,
D.; Anderson, A. J. Med. Chem. 2007, 50, 940.
10. Paulsen, J.; Liu, J.; Bolstad, D.; Smith, A.; Priestley, N.; Wright, D.; Anderson, A.
Bioorg. Med. Chem. 2009, 17, 4866.
11. Paulsen, J.; Bendel, S.; Anderson, A. Chem. Biol. Drug Des. 2011, 78, 505.
12. Viswanathan, K.; Frey, K.; Scocchera, E.; Martin, B.; Swain, P.; Alverson, J.;
Priestley, N.; Anderson, A.; Wright, D. PLoS One 2012, 7, e29434.
13. Cody, V.; Luft, J.; Pangborn, W.; Gangjee, A. Acta Crystallogr. 2003, D59, 1603.
14. Jain, A. J. Med. Chem. 2003, 46, 499.
15. Preparation performed with Maestro preparation wizard (Schrodinger, Inc.),
Desmond System builder is distributed through D. E. Shaw Research.