Structure based design of novel inhibitors for histidinol dehydrogenase from Geotrichum candidum

Article abstract of DOI:10.1016/j.bmcl.2010.04.116

Histidinol dehydrogenase, the product of the HisD gene, mediates the final step in the histidine biosynthetic pathway. This enzyme has captured attention for drug discovery studies in past few years. Recently, our group cloned and expressed Geotrichum candidum histidinol dehydrogenase and successful screening of substrate analog inhibitors of histidinol dehydrogenase led to some antifungal compounds with IC₅₀ values in micromolar range. In this study, we have done docking analysis of these antifungal agents in G. candidum. Two new compounds were designed based on the docking results and these compounds turned out to be potent inhibitors of G. candidum histidinol dehydrogenase, showing IC₅₀ values as low as 3.17 μM.

Full text of DOI:10.1016/j.bmcl.2010.04.116

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3972–3976
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure based design of novel inhibitors for histidinol dehydrogenase from
Geotrichum candidum
Sonia Pahwa a, Simranjeet Kaur a, Rahul Jain b, Nilanjan Roy a,
*
a Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India
b Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Histidinol dehydrogenase, the product of the HisD gene, mediates the final step in the histidine biosyn-
thetic pathway. This enzyme has captured attention for drug discovery studies in past few years.
Recently, our group cloned and expressed Geotrichum candidum histidinol dehydrogenase and successful
screening of substrate analog inhibitors of histidinol dehydrogenase led to some antifungal compounds
with IC50 values in micromolar range. In this study, we have done docking analysis of these antifungal
agents in G. candidum. Two new compounds were designed based on the docking results and these com-
pounds turned out to be potent inhibitors of G. candidum histidinol dehydrogenase, showing IC50 values
Received 7 December 2009
Revised 25 March 2010
Accepted 27 April 2010
Available online 18 May 2010
Keywords:
Geotrichum candidum
Histidinol dehydrogenase
Homology modeling
Docking analysis
as low as 3.17 lM.
Ó 2010 Elsevier Ltd. All rights reserved.
Antifungal compound
The histidine biosynthesis pathway is an important pathway
found in bacteria, fungi and plants.1 One of the enzymes of this
histidine can inhibit Brucella growth in minimal medium and abol-
ish multiplication in human macrophages.10
pathway, histidinol dehydrogenase
(
L
-histidinol-NAD+ oxido-
Geotrichum candidum is an extremely common fungus with a
world-wide distribution and is the causative agent of geotricho-
sis.11,12 Pulmonary involvement is the most frequently reported
form of the disease, but bronchial, oral, vaginal, cutaneous, and ali-
mentary infections have also been reported.13–15 In our previous
studies, we have used a hybrid approach of screening substrate
analog inhibitors of G. candidum histidinol dehydrogenase
(GcHDH).16 GcHDH has been cloned, expressed, an assay method
developed for screening compounds and essential structures of
antifungal compounds have been identified by quantitative struc-
ture–activity relationship (QSAR) analysis.17 Based on our previ-
ously reported 3D QSAR studies, our aim was to design new
active compounds starting from the docking analysis of imidazole
derivatives reported in previous16 and in this paper. In this study,
docking of these substrate analog inhibitors of GcHDH was per-
formed and results analyzed. Two new inhibitors were designed,
synthesized and biologically evaluated as novel inhibitors of
G. candidum histidinol dehydrogenase.
reductase) is of particular interest as a target of antimicrobial
agents.2 Moreover, it is essential for virulence in various pathogens
and plays an essential role in maintenance of infection.3 Histidinol
dehydrogenase (EC 1.1.1.23) (HDH) catalyses the terminal step in
the biosynthesis of histidine, the four-electron oxidation of
dinol to histidine. HDH catalyzes the formation of -histidine in
two steps; NAD-dependent oxidation of -histidinol to -histindal-
dehyde, followed by another NAD-dependent oxidation to form
-histidine. This enzyme is a homodimeric zinc metalloenzyme
L-histi-
L
L
L
L
with one Zn2+ binding site per monomer.4,5 In bacteria HDH is a
single chain polypeptide; in fungi it is the C-terminal domain of
a multifunctional enzyme which catalyzes three different steps of
histidine biosynthesis; and in plants it is expressed as nuclear en-
coded protein precursor which is exported to the chloroplast.6
Very few inhibitors of histidinol dehydrogenase been reported
till date.7–9 Dancer et al. reported the development of potential
herbicides targeted against cabbage histidinol dehydrogenase.9
Further, Abdo et al. reported the synthesis and biological activity
of potential antibacterial compounds tested against histidinol
dehydrogenase of Brucella suis cloned and expressed in Escherichia
coli.8 These substituted benzylic ketone derivatives derived from
The molecular modeling studies of GcHDH were carried out on
Windows XP platform using MOE (Molecular Operating Environ-
ment)18 and SYBYL7.319 molecular modeling packages. Homology
modeling module of the MOE was used for comparative protein
modeling and molecular docking studies were performed using
the FlexX program implemented in SYBYL7.3.
As a first step towards 3D structure prediction using homology
modeling, the amino acid sequence of GcHDH (GenBank Accession
* Corresponding author. Tel.: +91 172 2214 687; fax: +91 172 2214 692.
E-mail address: nilanjanroy@nipeer.ac.in (N. Roy).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.116

S. Pahwa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3972–3976
3973
No. ABE73158) was obtained from the NCBI protein database. The
HIS4 gene in Saccharomyces cerevisiae encodes a trifunctional pro-
tein,20 which comprises the enzyme activities for AMP 1,6-cyclohy-
drolase (HIS4A), AMP-pyrophosphohydrolase (HIS4B), and
histidinol dehydrogenase (HIS4C) and catalyzes three steps in the
pathway of histidine biosynthesis. G. candidum HIS4 protein was
also found to have HIS4A, HIS4B, and HIS4C subregions.17 Amino
acid sequence encoding HIS4C region (358–842) was selected
based on sequence alignment with S. cerevisiae (Uniprot ID:
P00815) and Pichia pastoris HIS4 protein (Uniprot ID: P45353).
NCBI-BLAST (http://www.ncbi.nlm.nih.gov/BLAST)21 was used to
identify homologous structure for GcHDH by searching the struc-
tural database of protein sequences in the Protein Data Bank
(PDB).22 The BLAST search result picked up the crystal structure
deviation of the GcHDH model chain A and chain B with chain A
and chain B of the template E. coli histidinol dehydrogenase
(1KAR), at the C-a region was calculated to be 0.867 and 0.827 Å,
respectively, and in the backbone region 0.879 and 0.835 Å, respec-
tively, and in the side chain region 1.976 and 1.803 Å, respectively.
The final model was refined by energy minimization to remove
any steric clashes of the side chains with each other and/or with
backbone atoms. The model was first subjected to a highly tethered
of E. coli L
-histidinol dehydrogenase homodimer23 (PDB ID: 1KAR)
determined by Barbosa et al. with the sequence identity of 48%
for GcHDH. 1KAR was then used as template for the homology
modeling of both A and B chains of GcHDH protein. The final align-
ment of template with GcHDH was performed in needle program
in EMBOSS.24 The identity and similarity between the target and
template was 43.5% and 55.5%, respectively (Fig. 1).
After the development of final best packing quality model for
GcHDH, coordinates of Zn2+ and water molecule were added using
MOE and optimized using an MMFF94 force field and PM3, a gradi-
ent 0.01 kcal/mol Å Hamiltonian, in MOE. For the reported homol-
ogy model, coordinates of Zn2+ and water molecules in the active
site were mimicked from the crystal structure of E. coli histidinol
dehydrogenase. The catalytic Zn2+ cation in E. coli histidinol dehy-
drogenase is located at the bottom of the cavity occupied by the
substrate histidinol and is octahedrally coordinated by Gln-
259OE1, His-262NE2, Asp-360OD2, His-419BNE2, and two ligands
from L
-histidinol, ND1 and N.23 The distances between the Zn2+ and
Figure 2. Structural topology of GcHDH homodimer.
are colored cyan, magenta, and salmon, respectively, for chain A and in red, green
and yellow, respectively, for chain B. The N- and C-terminal regions of chain A are
denoted by N, C. For chain B, N- and C-terminal regions are denoted by N and C ,
respectively.
a Helices, b-strands and loops
residue atoms involved (Gln-311OE1, His-314NE2, Asp-414OD2,
and His-473B-NE2) in zinc coordination in GcHDH are within the
acceptable limits (Supplementary file 1 and Fig. 1). The RMS
*
*
Figure 1. Sequence alignment of the GcHDH with E. coli
are denoted by double dots and colored cyan. Gray color depicts less conserved residues denoted by single dot. For GcHDH sequence, HIS4C region (358–844) was selected for
alignment.
L
-histidinol (1KAR). Identical residues are highlighted in red and denoted by asterisk (Ã). Highly conserved residues

3974
S. Pahwa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3972–3976
series of conjugate gradient minimization steps. The medium min-
imization options using Truncated Newton Optimization algo-
rithm, with RMS gradient tests of 1 Å was used for GcHDH
protein homodimer. For further refinement of the BsHDH and
GcHDH model, the outlier residues were determined using Protein
Geometry tool in MOE. Energy minimization of the selected outlier
residues was carried out using AMBER94 force field with the RMS
gradient of 0.05 Å. The stereochemistry and accuracy of the model
was further gauged using Procheck25 and Verify 3D26,27 program in
Structural Analysis and Verification Server (SAVES) (http://
www.nihserver.mbi.ucla.edu/SAVES). The Ramachandran plot of
GcHDH model obtained through Procheck showed 99.4% residues
in the allowed region, 0.4% in the generously allowed region, and
only 0.1% in the disallowed region (Supplementary file 1 and
Fig. 2). This indicates that the backbone dihedral angles u and
w
in the model were reasonably accurate. Verify 3D calculates 3D
to 1D compatibility score and the graphical representation por-
trays the properly folded and misfolded regions in the protein
structure by performing an Eisenberg analysis of the model (Sup-
plementary file 1 and Fig. 3). From the above observations, the
model was found sufficiently accurate for further structure-based
analysis (Fig. 2).
To study the ligand–protein interactions in the active site of
GcHDH, molecular docking was performed on the final refined
homology model. Docking experiments were carried out using
the FlexX module in SYBYL7.3 which utilizes the incremental con-
struction algorithm. The binding site of substrate histidinol has
already been defined for E. coli histidinol dehydrogenase.23 Most
of the active site residues of E. coli HDH superimposed well on
GcHDH active site residues as observable from Figure 3. In order
to validate our docking protocol, crystal structure of histidinol
was extracted from E. coli histidinol dehydrogenase (PDB ID:
1KAE) and docked into the active site of GcHDH. A set of 30 confor-
mations for histidinol was generated, from which one with the
highest consensus score was selected. Once the docking protocol
was validated, 6 imidazole derivative compounds were docked into
the active site of GcHDH using same protocol. Compounds showing
good activity from Class I and II were selected and docked into the
Figure 3. Active site residues of the template (purple) and GcHDH (cyan). Asterisk
(Ã) denotes residues from B chain of respective enzymes.
Table 1
Interaction of GcHDH active site residues
Mol. ID
Structure
Interacting residues
Total No. of H bonds
IC50 (lM)
Chain A
Chain B
Class I
79
Ser646, His738, Leu780
–
9
29.3
10.6
5.2
92
Thr545, Ser646, Glu737, His738, Gln668, Leu780
Gln668, His738, Ser646
–
11
8
344
Glu825
Class II
334
Ser646, Gln668, His671, His738, Glu737, Leu780
His830
10
122.2
333
339
Thr545, Ser646, His738, Gln668, Glu767, Asp771
–
–
13
4
58.7
24.2
Leu780, His778

S. Pahwa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3972–3976
3975
M)
Table 2
Interaction and activity data for new designed compounds.
Mol. ID
Structure
Interacting residues
Total No. of H bonds
IC50 (l
Chain A
Chain B
COOH
COOH
S-3
Gln668, Ser646, His738, His671, Asp771
His830, Glu825
13
3.17
HN
N
N
N
NH
RJ-278
His671, Ser646, Gln668, His738, Glu737, Leu780
His830
15
3.56
O
active site of GcHDH homology model (Table 1). The activity of
these compounds has been determined experimentally by our pre-
vious studies (reference). Based on the docking conformations and
activity of these inhibitors, two novel inhibitors S-3 and RJ-278
were designed (Table 2) and docked in to the active site of GcHDH
using same docking protocol and tested in vitro for their inhibitory
activity against GcHDH.
The new compounds S-3 and RJ-238 were prepared as described
in Supplementary file 2 and tested in enzyme and cell based assays
according to previously reported procedures.16
in its activity. Based on the above observations, a new compound
S-3 was designed in which the alkyl carbamate (–NHCOOR) substi-
tution was detached and methyl group from the side chain was re-
placed by free –OH group. Docking of S-3 into the active site of
GcHDH was performed, which showed imidazole ring situated
deep inside the cavity and total 13 hydrogen bonding interactions
with the receptor (Fig. 4D). The imidazole ring formed three hydro-
gen bonds inside the cavity. The compound S-3 was then tested for
its inhibitory activity against GcHDH which showed an IC50 value
of 3.17 lM (Table 2). This new compound resulted in a significant
The most active Class I compound 344 showed favorable con-
formations into GcHDH active site. The imidazole ring of 344
docked deep inside the cavity (Fig. 4A) and formed hydrogen bond-
ing interaction with Glu825 and His738. Substitution of bulkier
improvement in the inhibition of GcHDH.
Class II compounds docked in the outer cavity of the GcHDH ac-
tive site ( Fig. 5A–C). 339 compound formed hydrogen bonding
interaction with Leu780 and His738. In 333 and 334, substitution
of electronegative atom on the imidazole ring decreased the effi-
ciency of imidazole ring for making hydrogen bonding interaction
inside the cavity. Total 13 and 10 hydrogen bonds were observed
for 333 and 334, respectively. Methyl substitution on side chain
–COOH group reduces the hydrogen bonding interactions; there-
fore it can be assumed that free –OH of side chain carboxyl group
is required for better activity. Based on these observations, another
molecule RJ-278 was designed in which imidazole ring was kept
unsubstituted and has a free carboxyl group on the side chain of
group at the
a carbon decreases the inhibitory activity of the Class
I molecules. Alkyl carbamate (–NHCOOR) substitution in com-
pound 92 and 79 also decreases their activity. Compound 92
docked outside the cavity and its imidazole ring formed hydrogen
bonding with Thr545 whereas, compound 79 formed no hydrogen
bonding interaction with imidazole ring (Fig. 4B and C). Therefore
it was assumed that imidazole ring should be less substituted and
docked deep inside the cavity for better activity as steric hin-
drances due to N-methyl substituted imidazole causes a decrease
Figure 4. Selectively docked Class I compounds in GcHDH active site; 344(4A), 92(4B), 79(4C) and S-3(4D).

3976
S. Pahwa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3972–3976
Figure 5. Selectively docked Class II compounds in GcHDH active site; 339(5A), 333(5B), 334(5C) and RJ-278(5D).
the molecule. As illustrated in Fig. 5D, RJ-278 docked well in the
outer cavity of GcHDH active site and formed total fifteen hydro-
gen bonds. RJ-278 was then tested for biological activity, which
References and notes
1. Alifano, P.; Fani, R.; Lio, P.; Lazcano, A.; Bazzicalupo, M.; Carlomagno, M. S.;
Bruni, C. B. Microbiol. Rev. 1996, 60, 44.
2. Black, M. T.; Hodgson, J. E.; Knowles, D. J. C.; Riechard, R. W.; Nicholas, R. O.;
Burnham, M. K. R.; Pratt, J. M.; Rosenberg, M.; Ward, J. M.; Lonetto, M. A. Google
Patents, 2001.
3. Kishore, G. M.; Shah, D. M. Annu. Rev. Biochem. 1988, 57, 627.
4. Nagai, A.; Ohta, D. J. Biochem. 1994, 115, 22.
5. Grubmeyer, C.; Skiadopoulos, M.; Senior, A. E. Arch. Biochem. Biophys. 1989, 272,
311.
showed IC50 value of 3.56 lM.
Our structural data are in agreement with the overall mecha-
nism of action for HisD as proposed by Barbosa et al. where the res-
idues Glu326 and His327 participate in acid–base catalysis.23 From
the crystal structure of E. coli histidinol dehydrogenase complexed
with substrate, zinc and imidazole (PDB ID: 1KAE), it was observed
6. Nagai, A.; Ward, E.; Beck, J.; Tada, S.; Chang, J. Y.; Scheidegger, A.; Ryals, J. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 4133.
that the hydroxyl group of substrate (L-histidinol) formed hydrogen
bonds to the His367 and His327. The imidazole ring provided coor-
dination to Zn2+ and also formed hydrogen bond to Glu414*. Similar
interactions with corresponding residues in GcHDH were detected
in S-3 and RJ-278 from our docking results. S-3 and RJ-278 made
hydrogen bonds with above mentioned catalytically important res-
idues, that is, His738, His778, Glu737, and Glu825 (His327, His367,
Glu326, and Glu414 in E. coli HDH, respectively) (Fig. 3 and Table 2).
These observations further validate our results and strengthen the
proposed inhibition of GcHDH by S-3 and RJ-278.
In conclusion, histidinol dehydrogenase from G. candidum con-
stitutes a suitable target for reported antifungal compounds, which
represent valuable candidates for the potential development of no-
vel inhibitors. 3D structure of GcHDH was determined using homol-
ogy modeling technique. The docking studies show an extensive in
silico evaluation of the substrate analog inhibitors of GcHDH for
their potential antifungal activity. Moreover, the study of the inter-
actions of the inhibitors with the active site of the HDH enzyme of-
fered a better understanding of inhibitor action, allowing the design
of new potential drug leads with improved inhibitory activity.
7. Kanaori, K.; Uodome, N.; Nagai, A.; Ohta, D.; Ogawa, A.; Iwasaki, G.; Nosaka, A.
Y. Biochemistry 1996, 35, 5949.
8. Abdo, M. R.; Joseph, P.; Boigegrain, R. A.; Liautard, J. P.; Montero, J. L.; Köhler, S.;
Winum, J. Y. Bioorg. Med. Chem. 2007, 15, 4427.
9. Dancer, J. E.; Ford, M. J.; Hamilton, K.; Kilkelly, M.; Lindell, S. D.; O’Mahony, M.
J.; Saville-Stones, E. A. Bioorg. Med. Chem. Lett. 1996, 6, 2131.
10. Joseph, P.; Abdo, M. R.; Boigegrain, R. A.; Montero, J. L.; Winum, J. Y.; Kohler, S.
Antimicrob. Agents Chemother. 2007, 51, 3752.
11. Buchta, V.; Otcenasek, M. Mycoses 1988, 31, 363.
12. Sfakianakis, A.; Krasagakis, K.; Stefanidou, M.; Maraki, S.; Koutsopoulos, A.;
Kofteridis, D.; Samonis, G.; Tosca, A. Med. Mycol. 2007, 45, 81.
13. Jagirdar, J.; Geller, S. A.; Bottone, E. J. Hum. Pathol. 1981, 12, 668.
14. Kassamali, H.; Anaissie, E.; Ro, J.; Rolston, K.; Kantarjian, H.; Fainstein, V.;
Bodey, G. P. J. Clin. Microbiol. 1987, 25, 1782.
15. Kantardjiev, T.; Kuzmanova, A.; Baikushev, R.; Zisova, L.; Velinov, T. Folia Med.
1998, 40, 42.
16. Pahwa, S.; Chavan, A. G.; Jain, R.; Roy, N. Chem. Biol. Drug Des. 2008, 72, 229.
17. Pahwa, S.; Roy, N. Int. J. Integr. Biol. 2008, 3, 1.
18. Chemical computing groups Inc.: Montreal, Canada.
19. Tripos Associates Inc., 1699. S. Hanley Rd., St. Louis, MO 631444, USA.
20. Keesey, J. K., Jr.; Bigelis, R.; Fink, G. R. J. Biol. Chem. 1979, 254, 7427.
21. Altschul, S. F. G. W.; Miller, W.; Myers, E. W.; Lipman, D. J. J. Mol. Biol. 1990, 215,
403.
22. Berman, H. M. W. J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I.
N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
23. Barbosa, J. A.; Sivaraman, J.; Li, Y.; Larocque, R.; Matte, A.; Schrag, J. D.; Cygler,
M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1859.
24. Needleman, S. B.; Wunsch, C. D. J. Mol. Biol. 1970, 48, 443.
25. Laskowski, R. M. M.; Moss, D.; Thornton, J. J. Appl. Crystallogr. 1993, 26, 283.
26. Bowie, J. U.; Luthy, R.; Eisenberg, D. Science 1991, 253, 164.
27. Luthy, R.; Bowie, J. U.; Eisenberg, D. Nature 1992, 356, 83.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.116.