Minimal pharmacophoric elements and fragment hopping, an approach directed at molecular diversity and isozyme selectivity. Design of selective neuronal nitric oxide synthase inhibitors

Article abstract of DOI:10.1021/ja0772041

Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early "ADME/Tox" considerations and provides a basic platform for medicinal chemistry-driven efforts.

Full text of DOI:10.1021/ja0772041

Published on Web 03/06/2008
Minimal Pharmacophoric Elements and Fragment Hopping, an
Approach Directed at Molecular Diversity and Isozyme
Selectivity. Design of Selective Neuronal Nitric Oxide
Synthase Inhibitors
Haitao Ji,† Benjamin Z. Stanton,† Jotaro Igarashi,‡,# Huiying Li,‡ Pavel Marta´sek,§,
Linda J. Roman,§ Thomas L. Poulos,‡ and Richard B. Silverman*,†
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology,
and Center for Drug DiscoVery and Chemical Biology, Northwestern UniVersity,
EVanston, Illinois 60208-3113, Departments of Molecular Biology and Biochemistry,
Pharmaceutical Sciences, and Chemistry, UniVersity of California,
IrVine, California 92697-3900, Department of Biochemistry, The UniVersity of Texas Health
Science Center, San Antonio, Texas 78384-7760, and Department of Pediatrics, First Faculty of
Medicine, Charles UniVersity, Prague, Czech Republic
Received September 17, 2007; E-mail: Agman@chem.northwestern.edu
Abstract: Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on
ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the
minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme
selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries
are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the
toxicophore library, and the side chain library. These libraries are employed to generate focused fragment
libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the
fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase
(nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-
containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different
chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-
fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a
constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore,
a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules
that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de
novo design, has been established as a new type of fragment-based inhibitor design. As an open system,
the newly established approach efficiently incorporates the concept of early “ADME/Tox” considerations
and provides a basic platform for medicinal chemistry-driven efforts.
chemical starting points for inhibitor optimization.2 The good
hits identified from historical compound collections usually have
Introduction
Lead generation is a critical first step in the drug discovery
process. Over the past decade, high-throughput screening (HTS)
of corporate compound collections has emerged as the paradigm
for hit or lead discovery. Despite the fact that this approach
has often been successful, it has some inherent challenges and
limitations. Typically, targets are interrogated with 106 to 107
discrete compounds in parallel, which fall far short of potential
chemical diversity space, estimated to be upward of 1060
molecules containing up to 30 non-hydrogen atoms.1 For some
target classes the HTS hit rate is low and results in few good
moderate biological activity (Ki: 1-10 µM) but with relatively
high molecular weights (the average molecular weight is 400
Da) and excessive lipophilicities,3 which are frequently not
amenable for lead optimization to generate compounds with
drug-like properties.4 Stimulated by the introduction of the “rule
of five”,5 many research programs profile compound collections
for lead identification with low average molecular weights. This
trend leads to the generation of fragment-based screening
(1) Bohacek, R. S.; McMartin, C.; Guida, W. C. Med. Res. ReV. 1996, 16,
3-50.
† Northwestern University.
(2) Posner, B. A. Curr. Opin. Drug DiscoVery DeV. 2005, 8, 487-494.
(3) Teague, S. J.; Davis, A. M.; Leeson, P. D.; Oprea, T. Angew. Chem., Int.
Ed. 1999, 38, 3743-3748.
‡ University of California, Irvine.
§ The University of Texas Health Science Center.
Charles University.
(4) Oprea, T. I.; Davis, A. M.; Teague, S. J.; Leeson, P. D. J. Chem. Inf.
Comput. Sci. 2001, 41, 1308-1315.
(5) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. AdV. Drug
DeliVery ReV. 1997, 23, 3-25.
# Current address: Institute of Multidisciplinary Research for Advanced
Materials, Tohoku University, 2-1-1 Katahira, Sendai, Miyagi 9808577,
Japan.
9
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Minimal Pharmacophoric Elements and Fragment Hopping
A R T I C L E S
approaches.6 Low molecular weight chemical fragments (usually
120-250 Da) are initially selected on the basis of their ability
to bind to the target of interest or to inhibit/promote its biological
function. Following the binding event, various affinity-based
techniques7 have been proposed to accurately and efficiently
identify the weak binding fragment (typically the binding
affinities of fragments are in the 1 mM to 30 µM range), such
as nuclear magnetic resonance-based screening8 (SAR by
NMR9), mass spectroscopy-based identification10 (especially
tethering11), X-ray crystallography-based approaches,12 or sur-
face plasmon resonance spectroscopy-based screening.13 Alter-
natively, substrate activity screening highlights the roles of
bioassay-based techniques in the identification of effective
fragments.14 These fragments, which can be considered as the
building blocks of a more complex lead structure, are then
evolved or combined/merged into compounds. The exploration
of the approaches to construct molecules from fragments leads
to the generation of in situ fragment assembly techniques, such
as click chemistry,15 dynamic combinatorial library design,16
and tethering with extenders.17
Fragment-based screening offers a number of attractive
features compared to HTS. First, whereas compounds from HTS
libraries are more restricted in their rotational degrees of
freedom, and thus less able to adapt to a given target site, a
high proportion of the atoms of a fragment hit are directly
involved in the desired receptor-ligand interaction, which
allows for optimal positioning within the receptor pocket.
Therefore, a fragment is a more efficient binder (high binding
energies per unit molecular mass). Second, a fragment-based
strategy provides a combinatorial advantage. The number of
fragments screened is in the range of only hundreds to a few
thousands, but they can explore a larger chemical space than a
preassembled large compound library. On the other hand,
developing and maintaining a small set of fragments with
simpler structures is easier than maintaining a massive HTS
library. Third, when the binding of a fragment is identified, the
subsequent structural optimization can benefit from extensive
design and can lead to a higher success rate and greater
flexibility for generating novel chemical entities. And last,
starting with a low-molecular-mass fragment is likely to produce
leads with rather small and simple structures, which allow for
enhancement during the lead optimization process.
However, there are some internal problems and challenges
in current fragment-based approaches. First, a fragment-based
strategy can provide a combinatorial advantage relative to
preassembled large chemical libraries, that is, a collection of
103 fragments can typically probe the chemical diversity space
of 109 molecules, a tremendous increase relative to HTS;
however, it is still a small fraction of the total diversity space.18
Second, because most fragments have low binding affinities as
a result of limited interactions with the target, the identification
of relevant fragments and determination of how to link them
productively in three-dimensional space are still quite intractable
problems in some cases,14,18,19 although many affinity-based
assay techniques have been developed. Third, ligand specificity
for its targets is a particularly important goal of drug discovery
in the postgenomic era because a myriad of functional proteins
have been characterized, and the enzymatic pockets within a
target family/or superfamily, which execute the same/similar
metabolic reactions and functions, are often quite similar. An
important challenge in modern medicine is how to design
compounds that can modulate a specific enzyme while leaving
related isozymes unaffected. Known fragment-based approaches,
however, are only able to identify and characterize fragment
binding sites on the target protein (often called “hot spots”, that
is, the regions of a protein surface that are major contributors
to the ligand binding free energy19,20). In fact, many binding
sites in the active site that are responsible for target specificity
and/or selectivity are not included in these “hot spots”.
Utilizing the basic tenets of fragment-based inhibitor design
in our earlier structure-based design of inhibitors of neuronal
nitric oxide synthase (nNOS), this pharmacophore-driven ap-
proach was proposed that attempted to search for selective
inhibitors of nNOS over the other two isozymes.21 Nitric oxide
synthase (NOS, EC 1.14.13.39) is a multidomain enzyme
consisting of an N-terminal catalytic oxygenase domain, a
central linker region, and a C-terminal electron-supplying
reductase domain that catalyzes the five-electron, two-step
oxidation of L-arginine (L-Arg) to produce L-citrulline and nitric
oxide (NO). NO is an important signaling molecule involved
in a wide range of physiological functions as well as patho-
physiological states mainly through the soluble guanylate
cyclase/cGMP pathway.22 Three distinct NOS isoforms, neu-
ronal (nNOS), endothelial (eNOS), and inducible (iNOS), have
been identified in mammals; these isozymes have 50-60%
sequence identity and share identical overall architecture.23 The
N-terminal catalytic oxygenase domain binds heme (Fe-proto-
(6) (a) Erlanson, D. A.; McDowell, R. S.; O’Brien, T. J. Med. Chem. 2004,
47, 3463-3482. (b) Rees, D. C.; Congreve, M.; Murray, C. W.; Carr, R.
Nat. ReV. Drug DiscoVery 2004, 3, 660-672.
(7) (a) Makara, G. M.; Athanasopoulos, J. Curr. Opin. Biotech. 2005, 16, 666-
673. (b) Comess, K. M.; Schurdak, M. E. Curr. Opin. Drug DiscoVery
DeV. 2004, 7, 411-416.
(8) Lepre, C. A.; Moore, J. M.; Peng, J. W. Chem. ReV. 2004, 104, 3641-
3675.
(9) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996,
274, 1531-1534.
(10) (a) Swayze, E. E.; Jefferson, E. A.; Sannes-Lowery, K. A.; Blyn, L. B.;
Risen, L. M.; Arakawa, S.; Osgood, S. A.; Hofstadler, S. A.; Griffey, R.
H. J. Med. Chem. 2002, 45, 3816-3819. (b) Erlanson, D. A.; Wells, J. A.;
Braisted, A. C. Annu. ReV. Biophys. Biomol. Struct. 2004, 33, 199-223.
(11) Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud, R.
M.; Gordon, E. M.; Wells, J. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
9367-9372.
(12) (a) Hartshorn, M. J.; Murray, C. W.; Cleasby, A.; Frederickson, M.; Tickle,
I. J.; Jhoti, H. J. Med. Chem. 2005, 48, 403-413. (b) Nienaber, V. L.;
Richardson, P. L.; Klighofer, V.; Bouska, J. J.; Giranda, V. L.; Greer, J.
Nat. Biotechnol. 2000, 18, 1105-1108.
(13) Dickopf, S.; Frank, M.; Junker, H. D.; Maier, S.; Metz, G.; Ottleben, H.;
Rau, H.; Schellhaas, N.; Schmidt, K.; Sekul, R.; Vanier, C.; Vetter, D.;
Czech, J.; Lorenz, M.; Matter, H.; Schudok, M.; Schreuder, H.; Will, D.
W.; Nestler, H. P. Anal. Biochem. 2004, 335, 50-57.
(18) Erlanson, D. A.; Hansen, S. K. Curr. Opin. Chem. Biol. 2004, 8, 399-
406.
(14) Wood, W. J.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman, J. A. J.
Am. Chem. Soc. 2005, 127, 15521-15527.
(19) Murray, C. W.; Verdonk, M. L. J. Comput.-Aided Mol. Des. 2002, 16,
741-753.
(15) (a) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.;
Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41,
1053-1057. (b) Whiting, M.; Muldoon, J.; Lin, Y. C.; Silverman, S. M.;
Lindstrom, W.; Olson, A. J.; Kolb, H. C.; Finn, M. G.; Sharpless, K. B.;
Elder, J. H.; Fokin, V. V. Angew. Chem., Int. Ed. 2006, 45, 1435-1439.
(16) Ramstrom, O.; Lehn, J. M. Nat. ReV. Drug DiscoVery 2002, 1, 26-36.
(17) Erlanson, D. A.; Lam, J. W.; Wiesmann, C.; Luong, T. N.; Simmons, R.
L.; DeLano, W. L.; Choong, I. C.; Burdett, M. T.; Flanagan, W. M.; Lee,
D.; Gordon, E. M.; O’Brien, T. Nat. Biotechnol. 2003, 21, 308-314.
(20) (a) Vajda, S.; Guarnieri, F. Curr. Opin. Drug DiscoVery DeV. 2006, 9,
354-362. (b) Verdonk, M. L.; Hartshorn, M. J. Curr. Opin. Drug DiscoVery
DeV. 2004, 7, 404-410.
(21) Ji, H.; Tan, S.; Igarashi, J.; Li, H.; Derrick, M.; Marta´sek, P.; Roman, L.
J.; Va´squez-Vivar, J.; Poulos, T. L.; Silverman, R. B. Submitted.
(22) Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001, 357,
593-615.
(23) Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. ReV. Pharmacol. Toxicol. 1999,
39, 191-220.
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Figure 1. Chemical structures and NOS inhibitory activities of L-nitroarginine-containing dipeptide/peptidomimetic inhibitors.
porphyrin IX), substrate L-Arg, and (6R)-5,6,7,8-tetrahydro-
biopterin (H4B). The central linker region binds calmodulin
(CaM), and the C-terminal reductase domain has the binding
sites for flavin mononucleotide (FMN), flavin adenine dinucle-
otide (FAD), and nicotinamide adenine dinucleotide phosphate
(NADPH). All three NOS isozymes are functional only as tight
homodimers, and CaM binding to the central linker region
mediates electron transfer from the reductase domain of one
subunit trans to the oxygenase domain of the other subunit of
the dimer.24 The NOS isozymes differ in cellular distribution,
regulation, and activity. Under normal physiological conditions,
both constitutively expressed isozymes (nNOS and eNOS) are
regulated by intracellular Ca2+/CaM and generate trace amounts
of NO as an intercellular messenger. The eNOS-derived NO is
a vasodilator essential for vascular homeostasis and also inhibits
platelet aggregation and leukocyte adhesion, while NO generated
by nNOS participates in neurotransmission in both the central
and peripheral nervous systems. iNOS binds CaM irreversibly
and is instead regulated mainly by transcriptional control of
enzyme expression in response to cytokines.
In line with the central biological role of NO, there are a
number of pathological processes associated with its over- or
underproduction.25 The impaired NO production by eNOS is
associated with hypertension, atherosclerosis, and arterial throm-
bosis.26 Excess formation of NO from nNOS has been implicated
in stroke and neurodegenerative diseases.27 iNOS-produced NO
appears to be involved in a broad range of inflammatory
pathologies, such as septic shock, rheumatoid arthritis, and
multiple sclerosis.28 Owing to these many pathological condi-
tions, in addition to the basic physiological functions related to
NOS, indiscriminate inhibition of NOS would be detrimental.29
Therefore, selective inhibition of one isozyme over the others
is essential. In particular, compounds that control the overpro-
duction of NO by nNOS or iNOS, while leaving undisturbed
the vasoprotective function of eNOS, are desired.30
points out of the substrate-binding site.21 Previously, we
synthesized and evaluated nitroarginine-containing dipeptide or
peptidomimetic inhibitors and identified a family of compounds
that had high potency and selectivity for inhibition of nNOS
over eNOS and iNOS. The most potent nNOS inhibitors among
these compounds were L-Nω-nitroarginine-2,4-L-diaminobutyra-
mide (1),32 (4S)-N-[4-amino-5-(aminoethyl)aminopentyl]-N′-
nitroguanidine (2),33,34 and L-Nω-nitroarginine-(4R)-amino-L-
proline amide (3)35,36 (Figure 1). The selectivity of these
dipeptide/peptidomimetic inhibitors for nNOS over eNOS and/
or iNOS was investigated by crystallographic analysis37 and by
GRID/CPCA in the earlier studies.38
In this paper the concept of minimal pharmacophoric elements
is proposed, and focused (or targeted) fragment libraries that
match the requirements of the minimal pharmacophoric elements
are subsequently generated. On the basis of these focused
fragment libraries a pharmacophore-driven strategy for fragment-
(24) Siddhanta, U.; Presta, A.; Fan, B.; Wolan, D.; Rousseau, D. L.; Stuehr, D.
J. J. Biol. Chem. 1998, 273, 18950-18958.
(25) Vallance, P.; Leiper, J. Nat. ReV. Drug DiscoVery 2002, 1, 939-950.
(26) Forstermann, U.; Munzel, T. Circulation 2006, 113, 1708-1714.
(27) Reif, D. W.; McCarthy, D. J.; Cregan, E.; Macdonald, J. E. Free Radic.
Biol. Med. 2000, 28, 1470-1477.
(28) Tinker, A. C.; Wallace, A. V. Curr. Top. Med. Chem. 2006, 6, 77-92.
(29) Babu, B. R.; Griffith, O. W. Curr. Opin. Chem. Biol. 1998, 2, 491-500.
(30) (a) Salerno, L.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.; Siracusa, M. A.
Curr. Pharm. Des. 2002, 8, 177-200. (b) Erdal, E. P.; Litzinger, E. A.;
Seo, J.; Zhu, Y.; Ji, H.; Silverman, R. B. Curr. Top. Med. Chem. 2005, 5,
603-624. (c) Tafi, A.; Angeli, L.; Venturini, G.; Travagli, M.; Corelli, F.;
Botta, M. Curr. Med. Chem. 2006, 13, 1929-1946.
(31) (a) Crane, B. R.; Arvai, A. S.; Ghosh, D. K.; Wu, C.; Getzoff, E. D.; Stuehr,
D. J.; Tainer, J. A. Science 1998, 279, 2121-2126. (b) Raman, C. S.; Li,
H.; Marta´sek, P.; Kral, V.; Masters, B. S.; Poulos, T. L. Cell 1998, 95,
939-950. (c) Fischmann, T. O.; Hruza, A.; Niu, X. D.; Fossetta, J. D.;
Lunn, C. A.; Dolphin, E.; Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula,
S. K.; Weber, P. C. Nat. Struct. Biol. 1999, 6, 233-242. (d) Li, H.; Shimizu,
H.; Flinspach, M.; Jamal, J.; Yang, W.; Xian, M.; Cai, T.; Wen, E. Z.; Jia,
Q.; Wang, P. G.; Poulos, T. L. Biochemistry 2002, 41, 13868-13875.
(32) Huang, H.; Marta´sek, P.; Roman, L. J.; Masters, B. S.; Silverman, R. B. J.
Med. Chem. 1999, 42, 3147-3153.
(33) Hah, J.-M.; Roman, L. J.; Marta´sek, P.; Silverman, R. B. J. Med. Chem.
2001, 44, 2667-2670.
(34) Hah, J.-M.; Marta´sek, P.; Roman, L. J.; Silverman, R. B. J. Med. Chem.
2003, 46, 1661-1669.
The crystal structures of the dimeric oxygenase domain for
all three NOS isoforms have been solved, which provide the
possibility for structure-based inhibitor design.31 However, this
has proven to be a challenging problem because the active sites
of NOS isozymes are highly conserved. Sixteen out of eighteen
residues within 6 Å of the substrate binding site are identical,
and the side chain of one of these two dissimilar amino acids
(35) Go´mez-Vidal, J. A.; Marta´sek, P.; Roman, L. J.; Silverman, R. B. J. Med.
Chem. 2004, 47, 703-710.
(36) Ji, H.; Go´mez-Vidal, J. A.; Marta´sek, P.; Roman, L. J.; Silverman, R. B.
J. Med. Chem. 2006, 49, 6254-6263.
(37) Flinspach, M. L.; Li, H.; Jamal, J.; Yang, W.; Huang, H.; Hah, J.-M.;
Go´mez-Vidal, J. A.; Litzinger, E. A.; Silverman, R. B.; Poulos, T. L. Nat.
Struct. Mol. Biol. 2004, 11, 54-59.
(38) Ji, H.; Li, H.; Flinspach, M.; Poulos, T. L.; Silverman, R. B. J. Med. Chem.
2003, 46, 5700-5711.
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A R T I C L E S
Figure 2. Schematic flow diagram for fragment hopping, the pharmacophore-driven strategy for fragment-based de noVo design.
based de noVo design, we term fragment hopping, is proposed
that utilizes the minute structural differences of the active sites
of the three NOS isozymes and leads to the design of a class of
non-peptide inhibitors that are highly selective for nNOS over
the other two isozymes.
determine the potential pharmacophores. The multiple solvent
crystal structures method (MSCS)39 and various affinity-based
biophysical techniques mentioned above are efficacious tools
for understanding how small molecules bind to the active site
of the enzymes. The energetic hot spots of enzymes for ligand
binding can be unraveled in combination with alanine scan-
ning.40 The computational methods for active site analysis are
useful when the receptor structure is known, or, if unknown,
the structure can be constructed by homology modeling.41 Two
of the most popular and venerable algorithms are GRID,42 which
Results and Discussion
Fragment Hopping, a Pharmacophore-Driven Strategy for
Fragment-Based de NoWo Design. Figure 2 summarizes the
pharmacophore-driven strategy for fragment-based inhibitor
design. The first step of the strategy is to determine the
pharmacophores of a specific drug target. If the target structure
can be determined by X-ray crystallography or NMR spec-
troscopy, various experimental approaches can be used to
(39) (a) Mattos, C.; Ringe, D. Nat. Biotechnol. 1996, 14, 595-599. (b) Mattos,
C.; Bellamacina, C. R.; Peisach, E.; Pereira, A.; Vitkup, D.; Petsko, G. A.;
Ringe, D. J. Mol. Biol. 2006, 357, 1471-1482.
(40) DeLano, W. L. Curr. Opin. Struct. Biol. 2002, 12, 14-20.
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calculates 3D energy maps around protein binding sites, thus
highlighting favorable sites for small functional groups, and
multiple copy simultaneous search43 (MCSS), which randomly
places thousands of copies of small functional groups into the
binding site, and the copies of small functional groups are
subject to energy minimization. The copies with the lowest
energies highlight hot spots of ligand binding. Many other
computational methods, such as the knowledge-based equiva-
lents of GRID (X-SITE44 and SuperStar45) and energy-based
approaches (PocketFinder,46 Q-SiteFinder47), also can be used
to explore sensitive and specific hot spots in the active site.
Computational solvent mapping48 and binding site determination
technology, based on grand canonical thermodynamics ensemble
Monte Carlo simulations (Lotus),49 can be regarded as an
important new breakthrough in this field. GRID/CPCA is an
excellent tool for understanding the selectivity of inhibitors for
a specific target over the other structure-related enzymes.50 If
the structure of the receptor is unknown, the pharmacophore
can be identified by structure-activity analysis of ligands or
by various computational methods, such as Catalyst, DISCO,
and GASP.51 Self-organizing maps (SOM) can be used as a
ligand-based approach to predict compound selectivity.52
Three-point or four-point pharmacophore models can be
generated from the above analyses.53 However, the key point
of the above pharmacophore investigation is to derive the
minimal pharmacophoric elements for each pharmacophore,
which means that a combinatorial application of different
pharmacophore identification methods is required to provide
as much information as possible. The minimal pharmacophoric
element can be an atom, a cluster of atoms, a virtual graph, or
vector(s). On the basis of the derived minimal pharmacophoric
elements, the second step of this approach is to query two main
general-purpose libraries: (1) A basic fragment library, which
is constructed on the basis of the fragments extracted directly
from known drugs and/or drug candidates. The fragments are
either from well-known libraries, such as the MDL compre-
hensive medicinal chemistry (CMC) database,54 the World Drug
Index (WDI),55 the Maccs Drug Data Report (MDDR),56 or from
the literature.57 These are summarized in Supporting Information
Figure 1. (2) A bioisostere library, which is constructed on the
basis of known bioisosteric principles reported in the literature
(Supporting Information Figure 2).58 The basic fragment library
is searched first to find all of the possible fragments that are
able to match the requirements of the minimal pharmacophoric
elements for each pharmacophore. Then the bioisostere library
is utilized to generate a focused fragment library with diverse
structures. The generated focused fragment library is then
interrogated with the rules for metabolic stability (see Supporting
Information Figure 3)59 and a toxicophore library (see Support-
ing Information Figure 4)60 to provide a focused library for a
specific pharmacophore. The focused library is then converted
into a LUDI fragment library, and the LUDI program is used
to search the optimal binding position for each fragment of each
pharmacophore.61
The third step of this approach is to link these fragments. A
constructed side chain library is used for this purpose, in which
the synthetic accessibility is considered.55b,c,62 This library,
shown in Supporting Information Figure 5, has been converted
into a LUDI linking library. SciFinder Scholar 2006,63 in
conjunction with the bioisostere library, also plays a key role
in securing the synthetic accessibility of the formed chemical
bond. The bioisostere library plays an assistant role in enhancing
the binding capabilities and optimizing the chemical properties
of the generated ligands. The generated ligand is interrogated
again with the rules for metabolism stability and the toxicophore
library.
The ligands generated by this iterative process are then docked
into the active site using AutoDock3.0,64 scored with consensus
(55) (a) Hajduk, P. J.; Bures, M.; Praestgaard, J.; Fesik, S. W. J. Med. Chem.
2000, 43, 3443-3447. (b) Ertl, P. J. Chem. Inf. Comput. Sci. 2003, 43,
374-380. (c) Lewell, X. Q.; Judd, D. B.; Watson, S. P.; Hann, M. M. J.
Chem. Inf. Comput. Sci. 1998, 38, 511-522.
(56) (a) Vieth, M.; Siegel, M. G.; Higgs, R. E.; Watson, I. A.; Robertson, D.
H.; Savin, K. A.; Durst, G. L.; Hipskind, P. A. J. Med. Chem. 2004, 47,
224-232. (b) Ertl, P.; Jelfs, S.; Muhlbacher, J.; Schuffenhauer, A.; Selzer,
P. J. Med. Chem. 2006, 49, 4568-4573. (c) Boda, K.; Johnson, A. P. J.
Med. Chem. 2006, 49, 5869-5879. (d) Sheridan, R. P. J. Chem. Inf.
Comput. Sci. 2002, 42, 103-108.
(57) (a) Boehm, H. J.; Boehringer, M.; Bur, D.; Gmuender, H.; Huber, W.; Klaus,
W.; Kostrewa, D.; Kuehne, H.; Luebbers, T.; Meunier-Keller, N.; Mueller,
F. J. Med. Chem. 2000, 43, 2664-2674. (b) Muller, G. Drug DiscoVery
Today 2003, 8, 681-691. (c) Carr, R.; Jhoti, H. Drug DiscoVery Today
2002, 7, 522-527. (d) Lewell, X. Q.; Jones, A. C.; Bruce, C. L.; Harper,
G.; Jones, M. M.; McLay, I. M.; Bradshaw, J. J. Med. Chem. 2003, 46,
3257-3274.
(58) (a) Patani, G. A.; LaVoie, E. J. Chem. ReV. 1996, 96, 3147-3176. (b)
Lima, L. M.; Barreiro, E. J. Curr. Med. Chem. 2005, 12, 23-49. (c)
Thornber, C. W. Chem. Soc. ReV. 1979, 8, 563-580. (d) Wermuth, C. G.
Molecular variations based on isosteric replacements. In The practice of
medicinal chemistry, 2nd ed.; Wermuth, C. G., Ed.; Academic Press: San
Diego, 2003; Chapter 13, pp 189-214. (e) Abraham, D. J. Analog design.
In Burger’s medicinal chemistry and drug discoVery, Vol. 1 drug discoVery,
6th ed.; Cannon, J. G., Ed.; John Wiley and Sons, Inc.: New York, 2003;
Chapter 16, pp 687-714.
(59) (a) van de Waterbeemd, H.; Smith, D. A.; Beaumont, K.; Walker, D. K. J.
Med. Chem. 2001, 44, 1313-1333. (b) Nassar, A.-E. F.; Kamel, A. M.;
Clarimont, C. Drug DiscoVery Today 2004, 9, 1020-1028. (c) Rishton,
G. M. Drug DiscoVery Today 1997, 2, 382-384. (d) Rishton, G. M. Drug
DiscoV. Today 2003, 8, 86-96.
(60) (a) Kazius, J.; McGuire, R.; Bursi, R. J. Med. Chem. 2005, 48, 312-320.
(b) Nassar, A.-E. F.; Kamel, A. M.; Clarimont, C. Drug DiscoVery Today
2004, 9, 1055-1064. (c) Williams, D. P.; Naisbitt, D. J. Curr. Opin. Drug
DiscoVery DeV. 2002, 5, 104-115. (d) Llorens, O.; Perez, J. J.; Villar, H.
O. J. Med. Chem. 2001, 44, 2793-2804.
(61) (a) Bo¨hm, H.-J. J. Comput.-Aided Mol. Des. 1992, 6, 61-78. (b) Bo¨hm,
H.-J.; Banner, D. W.; Weber, L. J. Comput.-Aided Mol. Des. 1999, 13,
51-56.
(62) Bemis, G. W.; Murcko, M. A. J. Med. Chem. 1999, 42, 5095-5099.
(63) Wagner, A. B. J. Chem. Inf. Model. 2006, 46, 767-774.
(64) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.;
Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 1639-1662.
(41) Ji, H.; Zhang, W.; Zhang, M.; Kudo, M.; Aoyama, Y.; Yoshida, Y.; Sheng,
C.; Song, Y.; Yang, S.; Zhou, Y.; Lu, J.; Zhu, J. J. Med. Chem. 2003, 46,
474-485.
(42) (a) Goodford, P. J. J. Med. Chem. 1985, 28, 849-857. (b) von Itzstein,
M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Van Phan,
T.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman, P. M.; Varghese,
J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham, V. J.; Cameron,
J. M.; Penn, C. R. Nature 1993, 363, 418-423.
(43) (a) Miranker, A.; Karplus, M. Proteins 1991, 11, 29-34. (b) Zoete, V.;
Meuwly, M.; Karplus, M. Proteins 2005, 61, 79-93.
(44) Laskowski, R. A.; Thornton, J. M.; Humblet, C.; Singh, J. J. Mol. Biol.
1996, 259, 175-201.
(45) Boer, D. R.; Kroon, J.; Cole, J. C.; Smith, B.; Verdonk, M. L. J. Mol.
Biol. 2001, 312, 275-287.
(46) An, J.; Totrov, M.; Abagyan, R. Mol. Cell. Proteomics 2005, 4, 752-761.
(47) Laurie, A. T.; Jackson, R. M. Bioinformatics 2005, 21, 1908-1916.
(48) (a) Dennis, S.; Kortvelyesi, T.; Vajda, S. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 4290-4295. (b) Silberstein, M.; Dennis, S.; Brown, L.;
Kortvelyesi, T.; Clodfelter, K.; Vajda, S. J. Mol. Biol. 2003, 332, 1095-
1113.
(49) (a) Vajda, S.; Guarnieri, F. Curr. Opin. Drug DiscoVery DeV. 2006, 9,
354-362. (b) Moore, W. R., Jr. Curr. Opin. Drug DiscoVery DeV. 2005,
8, 355-364. (c) Clark, M.; Guarnieri, F.; Shkurko, I.; Wiseman, J. J. Chem.
Inf. Model. 2006, 46, 231-242.
(50) Kastenholz, M. A.; Pastor, M.; Cruciani, G.; Haaksma, E. E. J.; Fox, T. J.
Med. Chem. 2000, 43, 3033-3044.
(51) Patel, Y.; Gillet, V. J.; Bravi, G.; Leach, A. R. J. Comput.-Aided Mol.
Des. 2002, 16, 653-681.
(52) Noeske, T.; Sasse, B. C.; Stark, H.; Parsons, C. G.; Weil, T.; Schneider,
G. ChemMedChem 2006, 1, 1066-1068.
(53) Mason, J. S.; Good, A. C.; Martin, E. J. Curr. Pharm. Des. 2001, 7, 567-
597.
(54) (a) Fejzo, J.; Lepre, C. A.; Peng, J. W.; Bemis, G. W.; Ajay, Murcko, M.
A.; Moore, J. M. Chem. Biol. 1999, 6, 755-769. (b) Bemis, G. W.; Murcko,
M. A. J. Med. Chem. 1996, 39, 2887-2893.
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Minimal Pharmacophoric Elements and Fragment Hopping
A R T I C L E S
in the following discussion because these are the sources of
the NOS X-ray crystal structure data. In the GRID analysis,
hydrophobic interactions are calculated with the DRY probe
(see Table 1 for a compilation of the probes used). The C3 and
NM3 probes describe the steric interactions. The polar probes
consist of N1, NHd, O, and O1. The COO- probe is negatively
charged, while the probes N3+, NM3, N1+ are the positively
charged single-atom probes. Since the natural ligand for all three
isozymes of NOS is L-Arg, a polar and basic residue that needs
a polar and acidic environment to stabilize it in the active site,
more polar and positive probes were used in the active site
analysis, including two multiatom probes Aramidine and Ami-
dine. There are two significant molecular interaction fields
(MIFs) in the active site of nNOS for steric effect probes C3
and NM3, as indicated in Figure 3A for the C3 probe. One is
located in the S pocket, which is encompassed by residues P565,
A566, V567, F584, S585, G586, W587, and the heme cofactor.
The maximal interaction energy is -5.00 kcal/mol for C3 and
-12.50 kcal/mol for NM3, respectively (Table 1). The second
region is located in the M pocket, which is defined by D597
and the heme propionate of the pyrrole A ring. The correspond-
ing maximal interaction energy is 4.00 kcal/mol for C3 and
-10.00 kcal/mol for NM3. The MIFs for the polar hydrogen
bond (H-bond) donor probes N1 and O1 are distributed almost
everywhere in the active site. This is understandable because
the substrate L-Arg is very polar and hydrogen donor-rich.
Among these MIFs, there are two main regions for the N1
probe: One is in the S pocket determined by the backbone amide
of W587 and the side chain carboxylate of E592. This is where
the guanidine group of L-Arg is located. The second one is in
the M pocket determined by the side chains of D597, R603,
and Y588. The main MIF region for the O1 probe is determined
by the M pocket enclosed by D495, Y562, R481, D597, R603,
Q478, and Y588. The MIF of the polar H-bond acceptor probes
NHd and O is much clearer compared to those of the H-bond
donor probes. It is mainly located in the M pocket also
determined by D495, Y562, R481, D597, R603, Q478, and
Y588. The negatively charged probe COO- is only located in
the S pocket, which is mainly driven by the presence of the
heme Fe cation.
Figure 3. Results of GRID analysis of the substrate binding site of nNOS
(PDB: 1p6i). The residues and cofactors (heme and H4B) are represented
in an atom-type style. The S and M pockets are indicated. A: GRID contours
of the C3 probe at an energy level of -3.50 kcal/mol. B: GRID contours
of the N3+ probe at an energy level of -9.60 kcal/mol; “×” represents the
position of the heme iron atom.
As shown in Figure 3B, there are three main MIFs for the
positively charged single-atom probes N3+ and N1+ in the
active site of nNOS. In the M pocket one region is determined
by D597 and Y588 (the maximal interaction energy is -13.50
kcal/mol for N3+ and -11.00 kcal/mol for N1+). The second
one is determined by the two heme propionates (the maximal
interaction energy is -11.00 kcal/mol and -8.50 kcal/mol for
N3+ and N1+, respectively). The last MIF is located in the S
pocket, which is determined by E592, the same position where
the guanidine group of substrate L-Arg is located in the active
site. There are two main MIFs for the multiatom probes
Aramidine and Amidine, determined by D597 and E592,
respectively. The hydrophobic interaction is mainly determined
by the hydrophobic residues in the C1 pocket (Table 1).
scoring functions,65 and filtered with absorption, distribution,
metabolism, excretion, and toxicity (ADME/Tox) consider-
ations.5,66 If the ligands generated are not satisfactory, the
molecule is reconstructed using the generated focused fragment
libraries, the side chain library, and the bioisostere library
(Figure 2).
Application of Fragment Hopping: The de NoWo Design
of Selective Neuronal Nitric Oxide Synthase Inhibitors. 1.
Active Site Analysis. The active site of NOS was investigated
prior to the design of inhibitors by two different methods, GRID
and MCSS. The active site of NOS has been divided into 4
pockets (S, M, C1, and C2) as described in the earlier study.38
The residue numbering for rat nNOS and bovine eNOS are used
The nine aromatic, aliphatic, polar, or charged functional
groups have been mapped into the active site of NOS by MCSS
calculations (Table 2). Similar to what was obtained from the
GRID analysis, the minima with the most favorable interaction
energies for the apolar and bulky groups, such as benzene
(minima no. 1-no. 3: -24.16 kcal/mol ∼ -21.88 kcal/mol),
(65) Charifson, P. S.; Corkery, J. J.; Murcko, M. A.; Walters, W. P. J. Med.
Chem. 1999, 42, 5100-5109.
(66) (a) Norinder, U.; Haeberlein, M. AdV. Drug DeliVery ReV. 2002, 54, 291-
313. (b) Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie,
C.; Howe, T.; Vianello, R. J. Med. Chem. 2005, 48, 6970-6979.
9
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A R T I C L E S
Ji et al.
Figure 4. Representative MCSS-minimized positions of the functional groups in the active site of rat nNOS (PDB: 1p6i). Cofactors heme and H4B are
shown in cyan and magenta, respectively. The S and M pockets are indicated. A. Benzene (red, minimum no. 1 is in the S pocket, while minimum no. 8 is
in the M pocket). Cyclohexane (yellow, minimum no. 1 is in the S pocket, while minimum no. 3 is in the M pocket) and isobutene (green, minimum no.
1 is in the S pocket, while minimum no. 3 is in the M pocket). B. Methylammonium (red, 4 minima are shown in the M pocket. Minima no. 1 and no. 7:
-79.74 kcal/mol bind to the heme propionate, minimum no. 4 binds to E592 and the heme propionate, and minimum no. 6 is close to D597. Minimum no.
9 is shown in the S pocket). Trimethylammonium cation (yellow, 2 minima are shown in the M pocket. Minimum no. 1 is close to D597 and E592, and
minimum no. 2 binds to the heme propionate. Minimum no. 22 is shown in the S pocket). The labels of the minima denote their ranking. C. The relative
positions of minima no. 4 and no. 6 of methylammonium, minimum no. 1 of trimethylammonium cation, and the R-amino group of compound 2 in Figure
2. The labels of the minima denote their ranking.
cyclohexane (minima no. 1-no. 2: -9.80 kcal/mol ∼ -9.65
kcal/mol), and _sobutene (minima no. 1-no. 2: -11.29 kcal/
mol ∼ -11.22 kcal/mol), are located in the hydrophobic cavity
of the S pocket (Figure 4A). Among them, the heavy atom-
only models of cyclohexane and isobutene are appropriate to
explore steric effects in the active site. The region displaying
the second most favorable interaction for these two functional
groups is located in the M pocket (Figure 4A), which is defined
by D597 and the heme propionate of the pyrrole A ring.
(cyclohexane, minima no.3-no.4: -9.02 kcal/mol to -8.90
kcal/mol and isobutene, minima no. 3-no. 11: -7.33 kcal/
mol to -6.24 kcal/mol). The polar functional groups (N-
methylacetamide and methanol), containing H-bond donors,
generated more MCSS minima than the H-bond acceptor (ether).
The lowest energy minima of all three polar functional groups
were located in the S pocket and bound to the heme iron atom.
The minima of the H-bond donors displaying the second most
favorable interaction energies formed H-bonds with the heme
propionate groups (N-methylacetamide minimum no. 8 and
methanol minimum no. 3 in Table 2), while the minimum of
ether displaying the second most favorable interaction energies
(minimum no. 2 in Table 2) formed H-bonds with Q478 and
R603 of the M pocket. The minima with the most favorable
interaction energies for the positively charged functional groups
were located in the M pocket (Table 2). Minima no. 1-no. 3
of methylammonium (-121.90 kcal/mol to -120.62 kcal/mol)
and minimum no. 2 of trimethylamine cation (-82.79 kcal/mol)
bound to both heme propionates (Figure 4B). Minima no. 4 and
no. 5 of methylammonium (-115.83 kcal/mol and -115.09
kcal/mol, respectively) bound to E592 and the heme propionate
of the pyrrole A ring in the same way that the R-amino group
of Nω-nitro-L-arginine (L-NNA) acts in its crystal structure
complexed with NOS.38 Minimum no. 6 of methylammonium
bound to D597 only. On the other hand, minimum no. 1 of the
trimethylamine cation was located in a position close to E592
and D597. This position is where the R-amino group of
9
3906 J. AM. CHEM. SOC. VOL. 130, NO. 12, 2008

Minimal Pharmacophoric Elements and Fragment Hopping
A R T I C L E S
A focused fragment library was generated based on the
minimal pharmacophoric elements for each pharmacophore. The
fragments were then docked into the active site of nNOS where
the corresponding pharmacophore is located. It is noteworthy
that the amidino group and the nitrogen atoms are directional
and require rather rigorous positioning for optimal ligand-
receptor interactions. Thus, the fragments that are able to match
their requirements are limited. However, the options for
fragments for hydrophobic and steric interactions are rather
broad when the basic fragment library and the bioisostere library
are queried. That is, targeting hydrophobic and steric interactions
would offer diverse fragments for each pharmacophore initially.
A small subset of this focused fragment library is described
here. To match the requirements of the amidino group and
hydrophobic/steric effects, the 2-aminopyridine group was
selected as a basic fragment. One advantage of the 2-aminopy-
ridine fragment is that the pKa value of 2-amino-6-methylpy-
ridine is 6.69.67 This fragment could act as a charge switch: in
the small intestine the fragment could be in its neutral form,
which is favorable for absorption; in the NOS active site, the
local acidic environment could convert it into the positively
charged form, which is favorable for binding. Starting with the
nitrogen atom close to D597 in Figure 5, the pyrrolidino
fragment was generated as a substitute for the R-amino group
of 1-3. The pyrrolidino group is not only able to meet the
charge-charge interaction requirement for nNOS selectivity but
also to match the steric effect requirement for NOS binding.
Another advantage of using the pyrrolidine ring is that the
secondary amino group is more lipophilic and has less polar
surface area (PSA) compared to the primary amino group of
1-3, which is better for in ViVo inhibitor delivery.68 The
ethylenediamine fragment was chosen to form a charge-charge
interaction and H-bonds with the two heme propionate groups.
After the linking of these fragments, compound 4 in Figure 6
emerged as the desired molecule. The mode of action of this
molecule with the active site of nNOS was confirmed by
AutoDock docking analysis. Some analogues (5-9) and their
corresponding trans isomers (10-15) were designed to verify
the derived pharmacophores and to provide preliminary structure-
activity relationships.
Figure 5. Minimal pharmacophoric elements for selective nNOS inhibitor
design. An aminidino group is positioned close to E592. A yellow nitrogen
atom is close to D597. The regions where hydrophobic and/or steric
interactions play important roles are indicated by circles. Three blue nitrogen
atoms are placed close to the heme propionate.
compounds 1-3 is located in the crystal structure of nNOS. It
is this site that is mainly responsible for the nNOS/eNOS
selectivity of the nitroarginine-containing dipeptide/peptidomi-
metic inhibitors (Figure 4C).37,38 It is interesting that the MCSS
interaction energies between the positively charged functional
groups and E592 in the S pocket were much higher than those
between the positively charged functional groups and D597 and/
or the heme propionate. As indicated in Table 2 and Figure
4B, the minima bound to E592 are minimum no. 9 of
methylammonium (-73.97 kcal/mol) and no. 22 of the trim-
ethylammonium cation (-32.36 kcal/mol).
2. Minimal Pharmacophoric Elements and Lead Structure
Design. Our earlier research found that a single-residue differ-
ence in the active site, rat nNOS D597 versus bovine eNOS
N368, is mostly responsible for the favored selectivity of nNOS
over eNOS; this high selectivity is determined by the R-amino
groups of 1-3.37,38 The maximum electrostatic stabilization
arising from residues D597 and E592 and the R-amino group
of inhibitors 1-3 forces inhibitors to adopt a curled conforma-
tion. Such stabilization is rather weak in eNOS because N368
does not bear a negative charge. Inhibitors 1-3 in eNOS adopt
an extended conformation, and the R-amino group is shifted
away from the corresponding selective region defined in
nNOS.37,38 The minimal pharmacophoric elements are proposed
in Figure 5 on the basis of the above active site analyses and
many structure-activity relationship studies conducted by us
and others.30 An amidino group is positioned in the same place
as the guanidino group of substrate L-Arg. This group is the
minimal binding element to form a charge-charge interaction
and also H-bonds with the carboxylate side chain of E592 and
the backbone amide of W587. One sp3-hybridized nitrogen
cation is placed in the selective region defined by D597 of nNOS
and N368 of eNOS. The other three nitrogen atoms are placed
close to the heme propionate to form a charge-charge interac-
tion and H-bonds. In the S pocket steric and hydrophobic effects
play important roles in ligand binding. The steric effect is
prominent at the position close to D597 and the heme propi-
onate, as indicated by the circles in Figure 5.
3. Chemistry. Compounds 8 and 9 and their corresponding
trans isomers 14 and 15 were synthesized by the route shown
in Scheme 1a. The epoxidation of 1-benzyl-3-pyrroline using
3-chloroperoxybenzoic acid (m-CPBA) generated 17 in a 77%
yield. Sulfuric acid was used to protonate the tertiary amine to
avoid an N-oxidation side reaction. Boc-protected 2-amino-6-
picoline (16) was treated with 2 equiv of n-butyllithium (n-
BuLi) and allowed to react with epoxide 17 to form alcohol 18
in a 90% yield. The alcohol was then converted to ketone 19
by a standard Swern oxidation. The amine compounds were
prepared by a reductive amination reaction. Cis isomer 20 and
trans isomer 21 were separated by silica gel column chroma-
tography; the more nonpolar fraction corresponded to the cis
structure. The structures of the isomers were characterized by
mass spectrometry, 1H NMR, 13C NMR, 13C NMR-DEPT, 1H-
1H COSY, 1H-13C HMQC, and 1H-1H NOESY spectra
(Supporting Information Figure 6). The NOE of the hydrogens
communicating between carbon 6 and carbon 10 was only
(67) Paudler, W. W.; Blewitt, H. L. J. Org. Chem. 1966, 31, 1295-1298.
(68) Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714-3717.
9
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A R T I C L E S
Ji et al.
Table 1. Summary of the Main Probe Interactions Observed in the Active Site of Rat nNOS. The Relevant Residues and Cofactors and the
Maximal Interaction Energy Involved in a Particular Interaction Are Given
probe
chemical group
pocket
maximal energy (kcal/mol)
main residues and cofactors
C3
methyl group
S
M
S
M
S
M
M
M
M
S
S
M
S
M
M
S
M
M
S
M
S
C1
-5.00
-4.00
P565, A566, V567, F584, S585, G586, W587, heme
D597, heme propionate
P565, A566, V567, F584, S585, G586, W587, heme
D597, heme propionate
W587, E592
Y588, D597, R603
Q478, R481, D495, Y562, Y588, D597, R603
Q478, R481, D495, Y562, Y588, D597, R603
Q478, R481, D495, Y562, Y588, D597, R603
backbone of A566 and V567
heme Fe cation
D597, Y588
E592
heme propionate
D597, Y588
E592
NM3
N1
trimethylammonium cation
neutral flat NH (e.g., amide)
-12.50
-10.00
-9.50
-7.30
O1
NHd
O
alkyl hydroxyl OH group
sp2 NH with lone pair
sp2 carbonyl oxygen
-12.00
-12.00
-8.50
-7.00
COO-
N3+
carboxylic acid anion
sp3 amine NH3 cation
-17.00
-13.50
-11.40
-11.00
-11.00
-9.50
N1+
sp3 amine NH cation
-8.50
heme propionate
D597
E592
D597
ARamidine
amidine
DRY
aromatic cationic amidine group
aliphatic cationic amidine group
hydrophobic probe
-20.00
-12.00
-21.00
-15.00
-1.85
E592
Y706, L337, M336, W306 (the other NOS monomer)
Table 2. Minima Found by MCSS in the Active Site of Rat nNOS
range of Ubinda for
minima (kcal/mol)
selected minima
initial no.
of copies
no. of
MCSS functional groups
minima
from
to
Ubind; pocket
residues/cofactors involved
benzene
2500
5000
5000
5000
27
98
22
85
-1.75
-0.09
-5.75
-2.44
-24.16
-9.80
no. 1: -24.16; S
no. 8: -12.99; M
no. 1: -9.80; S
V567, F584, heme
D597, heme
V567, F584, heme
D597, heme
V567, F584, heme
D597, heme
heme
heme, R603
Q478, E592
heme
D597, Q478
E592
Q478, R603
R481
heme Fe
Q478, R481
heme
cyclohexane
no. 3: -9.02; M
no. 1: -11.29; S
no. 3: -7.33; M
no. 8: -43.97; M
no. 12: -42.15; M
no. 13: -41.91; M
no. 3: -30.65; M
no. 5: -29.52; M
no. 7: -25.26; S
no. 2: -19.91; M
no. 1: -79.51; C1
no. 2: -77.73; S
no. 5: -42.93; M
no. 1: -121.90;M
no. 4: -115.83; M
no. 6: -85.32; M
no. 9: -73.97; S
no. 1: -87.43; M
no. 2: -82.79; M
no. 22: -32.36; S
isobutane
-11.29
-54.77
N-methylacetamide
methanol
5000
64
-3.19
-32.66
ether
acetate ion
5000
5000
27
42
-3.21
-5.93
-25.20
-79.51
methylammonium
trimethylamine ion
5000
5000
16
28
-0.75
-2.18
-121.90
-87.43
E592, heme
D597
E592
D597, E592
heme
E592
a Ubind, the binding energy for a given functional group in each minimized replica obtained from the MCSS calculation, is defined as Ubind ) Uprotein-group
+ Ugroup0, where Uprotein-group represents the nonbonded interactions between nNOS and the given functional group. Ugroup represents the internal energy of
0
the functional group within the complex, and Ugroup represents the internal energy of the isolated functional group in vacuum.
observed in the trans compound as indicated in Supporting
Information Figure 7B. No NOE was observed for the cis isomer
(Supporting Information Figure 7A). When sodium cyanoboro-
hydride was used as the reducing reagent, 3 Å molecular sieves
were added as a water trap, dry methanol was used as the
solvent, and 1 equiv of acetic acid was added as a proton source.
The ratio of the cis to trans isomers was generally 45:55.
Deprotection of the Boc group of compounds 20 or 21 afforded
that the 13C chemical shift of the carbon atom attached to the
pyridine ring (carbon 6 in Supporting Information Figure 7) is
29.5 ppm for the cis compound, but 34.1 ppm for the trans
isomer, which can be used for structural characterization in
future inhibitor optimizations.
Compounds 4-6 and their trans isomers 10-12 (Figure 6)
were synthesized by the route in Scheme 1b. There was only
one reaction different from that of Scheme 1a. Alcohol
intermediate 23 was oxidized to ketone 24 by a Dess-Martin
oxidation in a yield of 96%, while the Swern oxidation reaction
was rather inefficient and only afforded a 54% yield. The two
isomers (25 and 26) also can be separated cleanly by silica gel
1
final products 8, 9, 14, and 15. It is noteworthy that the H
NMR and 13C NMR spectra of the cis and trans isomers of the
final products are quite different from each other, as shown in
Supporting Information Figure 8. One prominent difference is
9
3908 J. AM. CHEM. SOC. VOL. 130, NO. 12, 2008

Minimal Pharmacophoric Elements and Fragment Hopping
A R T I C L E S
Figure 6. Chemical structures of the initially designed molecules.
Scheme 1
column chromatography after the reductive amination reaction.
The more nonpolar isomers, which were believed to be the cis
isomers (4 and 6) based on the above multidimensional NMR
analyses, were cocrystallized with rat nNOS. The X-ray crystal-
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A R T I C L E S
Ji et al.
Figure 7. Superimposition of the binding conformation (blue) and predicted
bioactive conformation (yellow) of 4 (A) and 6 (B) in the active site of rat
nNOS. The heme (orange), H4B (violet), and structural water (green)
involved in the binding of 4 and 6 to nNOS are shown. The distances of
some important H-bonds between the residues, structural water, cofactors,
and inhibitors are given in angstroms (Å).
Figure 8. A. Binding conformation of 2 in complex with rat nNOS. B.
Superimposition of the binding conformations of 4 (blue) and 2 (yellow)
in rat nNOS. Heme (orange), H4B (violet), and the structural water molecules
(green) involved in the binding of the inhibitors to nNOS are shown. The
distances of some important H-bonds between the residues, structural water,
cofactors, and inhibitors are given in angstroms (Å).
lographic analysis described below confirms the cis stereo-
chemistry for 4 and 6.
conversion of 29 to 30. A combination of tetrapropylammonium
perruthenate (TPAP)/4-methylmorpholine N-oxide (NMO) oxi-
dation in 10% acetonitrile in dichloromethane successfully
generated 30 in a 67% yield,71 while neither the Swern oxidation
nor Dess-Martin oxidation was effective. Reductive amination
provided amines 31 and 32, which can be separated cleanly by
silica gel column chromatography; the ratio of the cis to trans
isomer was 60:40.
4. NOS Inhibition and Structure-Activity Relationships.
The racemic mixtures of 4 and 6 were used for the inhibitor
complex crystal structure determination with the rat nNOS heme
domain. Crystallographic analysis of new NOS inhibitors
generated from the proposed fragment hopping method is
described in detail elsewhere.72 To briefly summarize, the
Compound 7 and its trans isomer 13 were prepared according
to the synthetic route in Scheme 2. 3-Cyclopenten-1-ol was
converted to its N-Boc amine derivative 27 by a Mitsunobu
reaction and a hydrolysis reaction.69 The epoxidation of 27 using
3-chloroperoxybenzoic acid (m-CPBA) generated 28 and its
trans isomer 28a, which were separated by silica gel column
chromatography. The structural characterization of 28 and its
trans isomer has been elucidated by Barrett et al.70 The ratio of
the cis to trans isomers was 75:25. Three equivalents of n-BuLi
were essential to successfully generate alcohol intermediate 29
in a 50% yield. Several methods were attempted for the
(69) Berre´e, F.; Michelot, G.; Le Corre, M. Tetrahedron Lett. 1998, 39, 8275-
8276.
(70) Barret, S.; O’Brien, P.; Steffens, H. C.; Towers, T. D. Voith, M.
Tetrahedron. 2000, 56, 9633-9640.
(71) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P. Synthesis 1994,
639-666.
9
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Minimal Pharmacophoric Elements and Fragment Hopping
A R T I C L E S
Scheme 2
Table 3. NOS Enzyme Assay Results for the Lead Compounds
structure of nNOS complexed with 4 (PDB accession 3B3N)
was solved to 1.98 Å resolution with R/Rfree ) 0.23/0.27, and
the complex with 6 (PDB accession 3B3M) was solved to 1.95
Å with R/Rfree ) 0.20/0.23). Only one of the two cis enanti-
omers, the (3′S,4′S)-isomer, was bound to the active site. The
enantiomer binding preference for 4 and 6 in the crystal
structures is the same as that predicted by the above pharma-
cophore-driven strategy for fragment-based de noVo design
(fragment hopping). Figure 7 is the superimposition of the
binding conformation (blue) and the predicted bioactive con-
formation (yellow) for 4 or 6 in the active site of nNOS. The
2-aminopyridino group of 4 and 6 binds to residue E592 in the
active site. The nitrogen atom of the pyrrolidine ring is located
in the selective region defined by residues nNOS D597/eNOS
N368. The nitrogen atom next to the pyrrolidine ring forms a
H-bond with one of the heme propionate groups. There is some
difference concerning the location of the terminal amino group
of 4. As indicated in Figure 7A, in the crystal structure it forms
H-bonds and charge-charge interactions with the heme propi-
onate of the pyrrole D ring and one structural water, while in
the predicted bioactive conformation it is located in the middle
of two heme propionates to form H-bonds and charge-charge
interactions with both heme propionates. The location of the
terminal hydroxyl group of 6 in the crystal structure is also
different from that of the predicted bioactive conformation. The
bioactive conformation was predicted to form a H-bond directly
Ki (
µM)
selectivity
compd
nNOS
eNOS
iNOS
n/e
n/i
(()-4
0.388
3.17
9.44
434.5
88.5
58.4
1114
28
39
254
3
3
99
6
150
53
15
33
13
8
9
11
8
(()-5
166.2
143.1
51.9
609.4
2523
77.4
143.8
233.8
71.5
60.1
1750
(()-6
366.6
403.5
121.6
849.2
866.2
85.2
683.7
242.3
44.7
(()-7
1.59
(()-8
47.94
305.0
8.76
13.28
28.54
2.34
(()-9
(()-10
(()-11
(()-12
(()-13
(()-14
(()-15
24
104
3
31
4
9
13.98
190.3
677.7
4
with the heme propionate of the pyrrole A ring. However, the
binding conformation from the crystal structure in complex with
nNOS exhibits a H-bonding interaction with the heme propi-
onate through the medium of a structural water molecule (Figure
7B). Since the interaction of the terminal hydroxyl group of 6
with the enzyme is rather weak, the root-mean-square (rms)
deviation of the side chain heavy atoms of 6 between the actual
binding conformation and the predicted bioactive conformation
is larger than that for 4.
Table 3 shows the results of the NOS enzyme assays. The
nNOS inhibitory activity (Ki) for 4 is 388 nM with high
selectivity for nNOS over eNOS (1100 fold). Compound 4 is a
racemic mixture; theoretically only the active (3′S,4′S)-enanti-
omer binds to the active site, which means that the single
(72) Igarashi, J.; Li, H.; Jamal, J.; Ji, H.; Fang, J.; Silverman, R. B.; Poulos, T.
L. To be submitted.
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A R T I C L E S
Ji et al.
enantiomer should generate enhanced inhibitory activity. The
selectivity of 4 between nNOS and iNOS is 150-fold. These
selectivities are comparable to those attained with the nitroargi-
the selective region in the active site of NOS being defined by
nNOS D597/eNOS N368 was verified by inhibitor design.
Furthermore, the flexibility of dipeptide inhibitors in Figure 1
was constrained in 4 by the introduction of a pyrrolidine ring.
Peptide or peptidomimetic inhibitors are often very potent and
exhibit high specificity for their targets. However, poor oral
bioavailability, metabolic instability, and difficulty in passage
through biomembranes are serious disadvantages. The meth-
odology presented here offers a way to design small biologically
active peptidomimetic molecules.
nine dipeptide amide analogues made previously.32-36
A
comparison of the NOS assay results between 4 and 10 reveals
that the cis isomer is more potent and more selective than the
corresponding trans isomer, as was predicted by fragment
hopping. This indicates that the nitrogen atom attached to the
pyrrolidine ring is a pharmacophore for nNOS inhibition and
also is important for nNOS selectivity because it binds to the
heme propionate and maintains the nitrogen atom of the
pyrrolidine ring in the selective region defined by nNOS D597/
eNOS N368.37,38 A comparison of the results for compounds
4, 5, and 6 indicates that the terminal amino group in the side
chain of 4 also is an important pharmacophore for nNOS
binding. The nNOS inhibitory activity for 5 is better than that
for 6, which means that the phenyl group probably is located
in a hydrophobic pocket in the active site of nNOS defined by
M336 and L337. The residue corresponding to L336 in murine
iNOS is N115. The nNOS/iNOS selectivity for 5 is better than
that for 6, which further confirms that the phenyl group of 5
binds to the above hydrophobic pocket. The NOS inhibitory
activities of the cis compounds, 8 and 9, and their trans isomers,
14 and 15, are very weak, which suggests that the benzyl group
is too large to be accommodated by the M pocket. Derivative
7 is one atom longer than 4, but its nNOS inhibitory activity is
4-fold lower than that of 4. The bond length between the
exocyclic primary amino nitrogen atom of 7 and the attached
cyclopentane carbon atom (1.49 Å) is not long enough to allow
the exocyclic amino group to form a direct H-bond interaction
between the bridging water molecule and D597 (Figure 7). The
selectivity between nNOS and eNOS also is decreased in 7,
despite the exocyclic primary amino group of 7 being closer to
nNOS D597 compared to the pyrrolidine nitrogen of 4. This
suggests that maximal nNOS/eNOS selectivity requires that not
only must a positively charged functional group be placed into
the selective region defined by nNOS D597/eNOS N368, but
it also has to be placed in an appropriate position to form strong
interactions with the enzyme. It is interesting that the nNOS
potency and the nNOS/eNOS selectivity of 13 are similar to
those of its cis isomer 7, which means that the nitrogen atom
of the ethylene diamine fragment which is attached to the five-
membered ring is less important in the cyclopentane derivatives,
compared to the pyrrolidine-type lead structure, although the
overall trend of structure-activity/selectivity relationship be-
tween the cis and trans isomers is still the same.
5. Comparison of Fragment Hopping with Known Ex-
perimental and Computational Approaches. The fragments
used in conventional fragment-based approaches are usually in
the molecular weight range of 120-250 Da, containing 8-18
non-hydrogen atoms.6b This molecular size is essential because
of the detection limit of biophysics-guided fragment screening;7
however, the essential structural requirement for a specific
binding pocket is much smaller than these fragments in most
cases. To elucidate the minimal pharmacophoric elements for
each binding pocket of a druggable target and to derive various
fragments with different chemotypes, a much wider chemical
space can be explored. On the other hand, with the use of
fragment hopping, key fragments can be identified and linked
more efficiently in three-dimensional space. The selective region
in the active site responsible for ligand selectivity is rather
delicate in many cases. As discussed above, the chemical
structure of compound 7 is very similar to that of 4. The primary
amino group attached to the cyclopentane ring is also located
in the selective region defined by nNOS D597/eNOS N368,
but its selectivity is much lower than that of 4. As noted in our
previous study,38 L-NNA also contains an R-amino group as in
the case of compounds in Figure 1, yet L-NNA exhibits no
selectivity between nNOS and eNOS. This is because the
carboxylate group of L-NNA is shielding the R-amino group
from the influence of the selective residues nNOS D597/eNOS
N368. It is difficult for fragment-based approaches to differenti-
ate this kind of small difference. Although conventional
fragment-based approaches are able to identify and characterize
those fragments located in the “hot spot” of the active site,73
the fragments that are responsible for isozyme selectivity are
generally not located in the “hot spots”. As indicated in Figure
9, thirteen MCSS energy minima were obtained when the
pyrrolidinium group was subjected to a MCSS calculation. These
thirteen MCSS minima can be clustered into five groups. Group
I: minima no. 1-no. 4 (-132.65 kcal/mol to -129.74 kcal/
mol) are in the middle of the two heme propionates. Group II:
minima no. 5 (-129.30 kcal/mol) and no. 6 (-127.60 kcal/
mol) are located between E592 and the heme propionate of the
pyrrole A ring. Group III: minima no. 7 (-111.82 kcal/mol)
and no. 8 (-107.15 kcal/mol) form charge-charge interactions
only with the heme propionate of the pyrrole A ring. Group
IV: minima no. 9 (-104.75 kcal/mol) and no. 10 (-101.80
kcal/mol) form charge-charge interactions only with the heme
propionate of the pyrrole D ring. Group V: minima no. 11-
no. 13 (-99.25 kcal/mol to -97.86 kcal/mol) bind to D597
directly. None of the MCSS-minimized pyrrolidinium cations
are placed in the position of the pyrrolidine ring found in the
crystal structure of nNOS in complex with 4 (Figure 9).
Fragment hopping determines the minimal pharmacophoric
Designed lead compound 4 forms interactions with selective
residue D597 Via two structural water molecules. A similar
interaction was observed in the crystal structure of nNOS in
complex with the nitroarginine-containing dipeptide or pepti-
domimetic inhibitors 1-3.37 Figure 8A shows the binding mode
of 2 in the crystal structure of rat nNOS. A superimposition of
the binding conformations of 4 and 2 indicates that they have
similar binding modes with nNOS (Figure 8B). The NOS
inhibition and selectivity profile of 4 (Table 3) are very similar
to those of compounds 1-3 (Figure 1). This suggests that the
small organic molecule 4 derived from fragment hopping mimics
the mode of action of the peptide or peptidomimetic inhibitors
with the enzyme. The new peptidomimetic inhibitor was
generated by the approach we proposed, and the prediction about
(73) Bo¨hm, M.; Klebe, G. J. Med. Chem. 2002, 45, 1585-1597.
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3912 J. AM. CHEM. SOC. VOL. 130, NO. 12, 2008

Minimal Pharmacophoric Elements and Fragment Hopping
A R T I C L E S
reported.76,77 In conventional computation-based de noVo design
strategies, the output structures obtained from the computer
program can be problematic with regard to synthetic accessibil-
ity76 and binding affinity prediction.78 Analyses of conventional
computation-based de noVo design techniques indicate that it
is rare to generate novel lead structures with nanomolar activity
initially.76 Recently de noVo design methods, such as graph
framework-based inhibitor design approaches, (e.g., scaffold
hopping79) and privileged substructure-based design,80 have been
proposed. The starting point for these types of methods is the
selection of a template structure. Then isofunctional but
structurally dissimilar substructures (scaffolds) are hopped into
the different parts of the template structure. These methods can
decrease the risks of molecular construction or synthetic
accessibility, increase the hit rate for lead generation, and offer
certain structural diversity. However, the skeleton of the newly
designed molecules is confined to the basic architecture of the
template structure, which usually comes from a known drug or
drug candidate. Moreover, mimicking the different parts of the
template structure with scaffolds often does not optimize the
interaction between the ligand and the receptor to the maximal
extent because the scaffold is quite large, and sometimes rigidity
of the template structure does not allow an optimal match
between ligand and receptor. To overcome the problem arising
from binding affinity predictions in de noVo design, the concept
of minimal pharmacophoric elements proposed here can map
an important interaction pattern between a ligand and a receptor
based on a priori knowledge and experience. As noted in Figure
2, experiment-based methods, computation-based methods,
receptor-based methods, or ligand-based methods can find their
roles in deriving an accurate pharmacophore model. The
approach proposed here is an open system that provides a basic
platform for medicinal chemistry-driven efforts. To solve the
problem arising from synthetic accessibility, common chemical
bonds in drugs or drug candidates are considered preferentially
in this approach (Supporting Information Figure 5).55b,c,62 The
bioisostere principles and SciFinder search engine are two
effective tools to design synthetically feasible molecules.
Figure 9. Representative MCSS-minimized positions of the pyrrolidinium
group in the active site of rat nNOS in complex with 4. Cofactors heme
and H4B are shown in cyan and magenta, respectively. The S pocket and
the M pocket are noted. The label of the minima denotes their ranking.
Hydrogen atoms attached to the nitrogen atom of the pyrrolidine ring are
shown to indicate the spatial orientation of the pyrrolidinium cation.
elements for each pharmacophore that are important for ligand
selectivity. Then fragments are generated to match the require-
ment of minimal pharmacophoric elements based on the basic
fragment and bioisostere libraries. After the focused fragment
library is generated for each pharmacophore, conventional
fragment-based approaches, such as NMR-based and/or X-
crystallography-based fragment screening techniques, click
chemistry, and dynamic combinatorial chemistry, can be utilized
to investigate the binding mode of the above-generated frag-
ments within the active site of the enzyme. The interaction of
the generated fragments with the selective regions of the active
site can be analyzed further by tethering or tethering with an
extender. Therefore, fragment hopping as a pharmacophore-
driven strategy is an open system that can incorporate other
techniques and provide a more efficient pathway to generate
more potent and more selective inhibitors. For example,
compound 4 obtained in the present study is one of the most
selective inhibitors of nNOS with nanomolar inhibitory potency.
In some cases, fragments identified by classical fragment-
based methods do not have the same binding site as the linked
molecule. Babaoglu and Shoichet parsed a typical, moderately
potent (Ki ) 1 µM) â-lactamase inhibitor into its component
fragments. Crystallographic analysis of less complex fragments
bound to the enzyme revealed that they do not recapitulate the
binding mode of the original inhibitor.74 Instead, the compounds
probe an entirely new region of the active site. That means that
less complex molecules (that is, fragments with fewer points
of interaction) should be able to bind in multiple ways to a given
receptor binding site.75 Fragment hopping proposed here
determines the positioning of the potential pharmacophores and
then places diverse fragments to match each minimal pharma-
cophoric element. After linking, the molecule shows the same
spatial orientation as the pharmacophores and is able to exhibit
the maximal desirable biological activities. To date there have
been about 38 computation-based de noVo design programs
Conclusion
To the best of our knowledge this is the first time that the
concept of minimal pharmacophore elements has been proposed,
and this concept has been combined with fragment-based
inhibitor design to generate inhibitors with novel structures. It
is also the first published example of the de noVo design of
potent and selective nNOS inhibitors. These small organic
molecules designed from the proposed approach mimic the
biological function of the earlier nitroarginine-containing peptide
inhibitors. Thus, a new peptidomimetic strategy, referred to as
fragment hopping, which creates small organic molecules that
mimic the biological function of peptides, has been established
as a new type of fragment-based inhibitor design.
(76) (a) Schneider, G.; Fechner, U. Nat. ReV. Drug DiscoVery 2005, 4, 649-
663. (b) Honma, T. Med. Res. ReV. 2003, 23, 606-632.
(77) (a) Bo¨hm, H.-J. Curr. Opin. Biotechnol. 1996, 7, 433-436. (b) Bohacek,
R. S.; McMartin, C. Curr. Opin. Chem. Biol. 1997, 1, 157-161.
(78) (a) Leach, A. R.; Shoichet, B. K.; Peishoff, C. E. J. Med. Chem. 2006, 49,
5851-5855. (b) Jorgensen, W. L. Science 2004, 303, 1813-1818. (c)
Shoichet, B. K. Nature 2004, 432, 862-865.
(79) (a) Schneider, G.; Neidhart, W.; Giller, T.; Schmid, G. Angew. Chem., Int.
Ed. 1999, 38, 2894-2896. (b) Barker, E. J.; Buttar, D.; Cosgrove, D. A.;
Gardiner, E. J.; Kitts, P.; Willett, P.; Gillet, V. J. J. Chem. Inf. Model.
2006, 46, 503-511.
(80) Schnur, D. M.; Hermsmeier, M. A.; Tebben, A. J. J. Med. Chem. 2006,
49, 2000-2009.
(74) Babaoglu, K.; Shoichet, B. K. Nat. Chem. Biol. 2006, 2, 720-723.
(75) Hajduk, P. J. Nat. Chem. Biol. 2006, 2, 658-659.
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A R T I C L E S
Ji et al.
This new de noVo design methodology is an open system; it
merges new discovery and development of related research fields
and provides a window for future modification. The final
decision regarding inhibitor design in this approach is made by
the medicinal chemist on the basis of his/her requirements and
an understanding of a specific research project. A minimal
pharmacophoric element is the core concept of the proposed
approach. It can be derived by receptor-based active site analyses
or ligand-based structure-activity relationship studies. In our
approach, the bioisoterism concept proved to be a research tool
of utmost importance in the generation of a focused fragment
library or for the linking of fragments to form effective and
accessible molecules with strong structural diversity. As an open
system, the approach established here also efficiently incorpo-
rates early “ADME/Tox” considerations. The functional groups
that potentially influence metabolic stability and toxicophores
are taken into account during the generation of the focused
fragment library and in the linking of fragments.
Using the established approaches, an inhibitor of nNOS,
compound 4 in Figure 6, was discovered with nanomolar
inhibitory potency and high selectivity for nNOS over eNOS
and iNOS. The structure-activity relationship analyses of the
derivatives are consistent with the model of minimal pharma-
cophoric elements. The crystal structure of rat nNOS in complex
with 4 confirms that the binding conformation of 4 is the same
as the predicted docking conformation. Compound 4, as a small
organic molecule with a constrained conformation, can exactly
mimic the mode of action of the dipeptide nNOS inhibitors.
Therefore, the proposed pharmacophore-driven strategy for
fragment-based de noVo design provides a new peptidomimetic
strategy. The molecular size of obtained compound 4 is rather
small (17 non-hydrogen atoms), indicating that the lead structure
designed by this newly established approach has high ligand
efficiency,81 which provides a good starting point for further
inhibitor optimization.
Acknowledgment. The authors are grateful for financial
support from the National Institutes of Health, GM 49725 to
R.B.S., GM57353 to T.L.P., and GM52419 to Dr. Bettie Sue
Masters, in whose lab P.M. and L.J.R. work, and Grant No.
AQ1192 from The Robert A. Welch Foundation to B.S.M. P.M.
is also supported by Grant KAN200200651 from Academy of
Science, Czech Republic.
Supporting Information Available: Complete refs 13, 42b;
full experimental details; a basic fragment library, which is
constructed on the basis of the fragments extracted directly from
known drugs and/or drug candidates; a bioisostere library, which
is constructed on the basis of known bioisosteric principles
reported in the literature; rules for metabolic stability; a
toxicophore library to provide a focused library for a specific
pharmacophore; a constructed side chain library to link frag-
ments, which is converted into a LUDI linking library; 1H NMR,
13C NMR, 13C NMR-DEPT, 1H-1H COSY, 1H-13C HMQC,
and 1H-1H NOESY spectra; the PDB registration numbers, data
collection, and refinement statistics of nNOS in complex with
4 and 6. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA0772041
(81) Hopkins, A. L.; Groom, C. R.; Alex, A. Drug DiscoVery Today 2004, 9,
430-431.
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3914 J. AM. CHEM. SOC. VOL. 130, NO. 12, 2008