De novo fragment design: A medicinal chemistry approach to fragment-based lead generation

Article abstract of DOI:10.1021/jm4002605

The use of fragments with low binding affinity for their targets as starting points has received much attention recently. Screening of fragment libraries has been the most common method to find attractive starting points. Herein, we describe a unique, alt

Full text of DOI:10.1021/jm4002605

Brief Article
pubs.acs.org/jmc
De Novo Fragment Design: A Medicinal Chemistry Approach to
Fragment-Based Lead Generation
Francisco X. Talamas,* Gloria Ao-Ieong, Ken A. Brameld, Elbert Chin, Javier de Vicente, James P. Dunn,
Manjiri Ghate, Anthony M. Giannetti, Seth F. Harris, Sharada S. Labadie, Vincent Leveque, Jim Li,
Alfred S-T. Lui, Kristen L. McCaleb, Isabel Najera, Ryan C. Schoenfeld, Beihan Wang, and April Wong
́
Hoffmann-La Roche Inc., Pharma Research & Early Development, 340 Kingsland Street, Nutley, New Jersey 07110, United States
S
* Supporting Information
ABSTRACT: The use of fragments with low binding affinity for their targets as starting points has received much attention
recently. Screening of fragment libraries has been the most common method to find attractive starting points. Herein, we
describe a unique, alternative approach to generating fragment leads. A binding model was developed and a set of guidelines were
then selected to use this model to design fragments, enabling our discovery of a novel fragment with high LE.
INTRODUCTION
RESULTS AND DISCUSSION
■
■
A library of ∼2700 fragments was screened against the HCV
NS5B polymerase using surface plasmon resonance (SPR) and
confirmed 163 hits. Using different parameters, 29 fragments
were selected for crystallography but only fragment 1
In the last two decades, fragment screening has been embraced
by the pharmaceutical and biotechnology industry, becoming
an integral part of drug discovery. Much of this can be
attributed to the early success of several companies that
developed fragments into clinical candidates1,2 or a marketed
drug.3
cocrystallized with NS5B (NS5B BK KD = 78 μM and IC50
=
130 μM). Attempts to directly improve potency, and some of
the physicochemical and ADME properties of the scaffold
provided by 1 were unsuccessful.
At the same time that the screening activities were ongoing, a
de novo fragment design approach was also taken to come up
with leads that could then be moved into optimization as an
alternative to library screening. Of the four allosteric sites in
NS5B known,9 the palm I site was selected for targeting using
this approach.
Our first step was to build a model containing key
interactions in the palm I allosteric site. We used the
crystallographic data available from our internal efforts (2 and
310,11) and the relevant structures in the public domain (4−
612−14) available at the time (early 2007).
As mentioned above, we were not able to use 1 as an explicit
starting point for our medicinal chemistry efforts, however, 1
played an important role in helping us develop our model.
Careful analysis of the crystallographic data revealed that the
pyrazolopyrimidine N-2 makes a hydrogen bond accepting
interaction with the backbone NH of Tyr448, and the NH
hydrogen on the N-1 was within hydrogen bond distance (2.4
Å) to the backbone carbonyl of Gln446 (Figure 1). This
interaction with Gln446 was unexpected and to our knowledge
the first time that a small molecule binding in the palm I site
exhibited it.
Fragments as starting points for lead identification efforts are
typically found by using two complementary approaches: (1)
screening a library of fragments or (2) by de novo fragment
design using computer-assisted methods. The use of fragment
libraries is the most common approach. A summary of the
process and successes of using libraries for fragment-based drug
discovery has been documented by Erlanson.4 Two decades
ago, computer-assisted de novo design methods such as
LEGEND5 attracted the attention of researchers to use
computational tools to create novel structures that could
become drugs. Since then, a large number of new programs
have been described in the literature that addresses the
approach of de novo design. Two excellent reviews by Loving6
and Rognan7 describe the methods and approaches used by the
different programs. Even with the success obtained by using
software to do de novo fragment design, some drawbacks have
been identified and are difficult to overcome.7
Herein, we describe our approach to de novo fragment
design targeting the hepatitis C virus (HCV) nonstructural 5B
(NS5B) polymerase that plays an important role in the life
cycle of the virus by replicating the viral genome.8 We
approached de novo fragment design by implementing some of
the same principles utilized by computer programs. Key
receptor interactions were identified, and design constraints
were defined. The generation of the novel compounds was
done differently. The fragments were created by medicinal
chemists that applied their own experience to design novel
molecules that addressed all the recognized disadvantages.
Once we had selected the relevant molecules (1−6) from
which to develop the model, it was decided to focus mainly on
the hydrophobic area of the allosteric site (for additional
descriptions of all the interactions in this site see ref 10). First,
Received: February 19, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm4002605 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
Figure 2. Model depicting key interactions. Displayed are the
identified amino acids important for the binding of the known
inhibitors. The dashed circles indicate possible modes of hydrogen
bond donor−acceptor interactions with the backbone, and the gray
areas represent volumes where hydrophobic interactions were
identified. The red sphere represents a conserved molecule of water.
It was preferred at this point of the program to focus on
molecules with no acidic functionality to avoid potential issues
with permeability. Because fragment 1 was the only known
binder to palm I without an acidic moiety, we focused on
incorporating the unique NH Gln446 interaction into our
design. The first step was to identify ring systems containing a
hydrogen donor (NH) and an acceptor (N or O) separated by
two, three, or four bonds.
A comprehensive set of heterocycles satisfying these criteria
was derived from the CSD and a ring system identification
algorithm.15 All docking, energy minimization, and visual
analyses were performed using the Molecular Operating
Environment (MOE).16 The initial pose in the binding pocket
of each heterocycle was generated using a three-point
pharmacophore defining the ideal location of the hydrogen
bond donor, hydrogen bond acceptor, and a ring center, each
with a radius of 1 Å. These initial poses were then subjected to
energy minimization with the MMFF94s forcefield using the
“R-field” solvent model. The RMSD of the pharmacophore
points for the energy minimized coordinates was used to rank
order the heterocycles for visual analysis. Each heterocycle was
visually studied both for the ability to satisfy the desired
hydrogen bonds and to provide a vector toward the other key
interaction points. In general, the ring systems capable of 1,2-
and 1,3-interactions modeled the best, some of them shown in
Figure 3.
Figure 1. Co-crystal structure of fragment 1 with HCV NS5B
polymerase. Simplified view of the binding of 1 in the palm I allosteric
site. Dashed lines indicate the hydrogen bond interactions of the
ligand with the backbone residues.
it was recognized that all the inhibitors made a primary
interaction not only with the backbone NH of Tyr448 but also
with either the carbonyl of Gln446, as described for 1, or with
the NH of Gly449 through a conserved molecule of water (2
and 3) or directly (5 and 6). It was also observed that the
hydrophobic pocket can expand and has a relatively wide entry
point that accommodates hydrophobic groups of different sizes
from different directions. The largest pocket observed was with
4. The importance of the aryl side chain of Tyr448 was also
recognized, as all of 1−6 make aromatic edge-to-face
interactions. Hydrophobic interactions with Gly410 and/or
Met414 side chain were observed with all the inhibitors. An
idealized design model was constructed utilizing all of these
important interactions for the purpose of de novo design
(Figure 2).
With the model in place, the following set of guidelines were
proposed for the design of de novo fragments: (a) satisfy
backbone carbonyl of Gln446 and NH of Tyr448, optionally
displacing or engaging the conserved molecule of water, (b)
occupy large hydrophobic pocket, probing with variable groups
to satisfy pocket size, (c) position an aromatic group to make
edge-to-face interaction with the aryl side chain group of
Tyr448, and (d) include at least one hydrophobic interaction
with Gly410 and/or Met414. The following steps were
followed for the design and selection of targets: the medicinal
chemist discussed his ideas with the computational chemist and
modeled them. In a routine manner, the ideas were discussed
and brainstormed within the team and collectively selected the
most promising proposals for synthesis.
Ligand efficiency17 (LE) was tracked during this process to
drive the design toward efficient binders because it has been
shown that the initial LE for a lead is maintained or decreases as
the series evolve to a clinical candidate.18
This process can be exemplified with the imidazol-2-one
moiety (Figure 4). When this ring system was modeled, it was
apparent that good interactions with the backbone carbonyl
Gln446 and NH Tyr448 could be made. One of the ring
nitrogens was in a position to grow toward the hydrophobic
pocket (yellow arrow). When this model was overlapped with 1
(in gray), this assumption was confirmed and it was also noted
B
dx.doi.org/10.1021/jm4002605 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
a
Table 1. Evolution of Hydantoin and Pyridone Fragments
compd
R1
R2
R3
IC50 (μM) KD (μM)
LE
1
130
>1000
>1000
364
220
108
21
78
NA
NA
400
144
93
0.31
7
H
Cl
H
H
H
H
H
H
H
H
8
Cl
9
t-butyl
t-butyl
t-butyl
t-butyl
0.26
0.26
0.27
0.30
0.26
0.27
0.43
0.46
Figure 3. Ring systems with hydrogen bond donor−acceptor
functionalities modeled in the hydrophobic area of palm I site.
10
11
12
13
14
15
16
methyl
ethyl
i-propyl
18
431
80
NA
NA
5.8
4.0
0.44
0.57
a
IC50 were measured using GT-1b NS5B Con1 strain (7−16) and
GT-1b NS5B BK strain (1). KD was measured using GT-1b NS5B BK
strain. IC50 and KD values are the average of at least two experiments.
LE is calculated using the IC50 value in the formula reported by
Groom17.
Figure 4. Evolution of imidazol-2-one to a disubstituted hydantoin.
that N-5 nitrogen on the pyrimidine portion of 1 could be
mimicked by adding a carbonyl group to the five-membered
ring system, changing the imidazol-2-one to a hydantoin ring
system. In turn, the hydantoin could give us the opportunity to
add alkyl groups (R2) on C-5 to interact with Gly410 (small
hydrophobic site).
First, fragment 7 was prepared and screened but did not
show any measurable activity in the biochemical assay (Table
1). Adding 3,4-dichloro substitution to the aryl ring (8) did not
show any improvement, but 4-t-butyl substitution (9) started
showing some activity. Other substitutions on the aryl ring did
not improve the activity of this early fragment. After having
fulfilled three of the requirements in our model (carbonyl
Gln446/NH Tyr448 backbone interactions, filling of hydro-
phobic pocket and edge-to-face with Tyr448), we explored the
R3 substitution on the hydantoin to make interactions with the
hydrophobic site. Interacting with this part of the palm I site
proved to be important to gain extra affinity. As seen in Table 1,
as the size of the alkyl group (R3) increased from hydrogen (9,
IC50 = 364 μM) to i-propyl (12, IC50 = 21 μM), the potency
improved by more than 15-fold. Fragment 12 was cocrystallized
with NS5B and confirmed the expected binding mode as
described above (Figure 5). Up to this point, our model and
guidelines had helped us design a novel fragment for the NS5B
polymerase.
Another ring system that showed good interactions when
modeled into the hydrophobic pocket of the palm I site was the
2-pyridone moiety (Figure 3). The NH and carbonyl of the
pyridone looked to be able to provide good hydrogen bond
distances to the two relevant interactions on the backbone.
Analyzing the positions to grow toward the lipophilic pocket,
C-3 of the pyridone appeared to be a reasonable choice to add
substitutions. The pyridone with the 4-t-benzyl group (13) on
Figure 5. Co-crystal structure of fragment 12 with HCV NS5B
polymerase in palm I allosteric site.
C-3 was synthesized and screened in the enzyme assay against
NS5B, displaying similar activity as 9 (Table 1).
Another way to fill the lower pocket, based on modeling, was
by directly attaching on the C-3 position of the pyridone an aryl
ring with a substituent on the meta position. We started with
one of the groups initially reported by Pfizer.12 Fragment 14
was prepared and presented an increase in potency with respect
to the previous fragment, however, the ligand efficiency stayed
the same. The use of LE at this point of fragment design was
very useful because it was an excellent metric to indicate if the
changes improved the binding affinity of our fragments.
Because of the null gain in LE by attempting to fill the large
hydrophobic pocket, we explored again the use of the t-butyl
group. Fragment 15, with a t-butyl group in the meta position,
was prepared and screened in the enzyme assay to give an IC50
= 4.0 μM, over 100-fold increase in potency with respect to 13.
The properties of 15 stayed within the desired range
(calculated pKa 10.4 and PSA 26) and had an excellent LE of
C
dx.doi.org/10.1021/jm4002605 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
0.43. Co-crystallization of 15 with NS5B confirmed that the
fragment was binding in the palm I site (Figure 6) as expected.
and information for cocrystals of NS5B with 1, 12, and 15. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
The coordinates and structure factor files have been deposited
in the Protein Data Bank under the accession codes 4IH5 (1),
4IH6 (12), and 4IH7 (15).
AUTHOR INFORMATION
Corresponding Author
*Phone: (973) 235-3626. E-mail: franciscotalamas@gmail.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge all members of the Analytical
Department in Roche Palo Alto and the Physical Chemistry
Department at Hoffmann-La Roche, Nutley, for the character-
ization of the compounds. We thank Romyr Dominique for
helpful discussions. The Advanced Light Source is supported by
the Director, Office of Science, Office of Basic Energy Sciences,
of the U.S. Department of Energy under contract no. DE-
AC02-05CH11231.
Figure 6. Co-crystal structure of fragments 15 with HCV NS5B
polymerase in palm I allosteric site. A figure overlaying the cocrystal
structures of the protein bound to fragment 1 and fragment 15 can be
found in the Supporting Information.
ABBREVIATIONS USED
HCV, hepatitis C virus; NS5B, nonstructural 5B
It is important to notice that the de novo designed fragment
15 exhibits significantly more potent enzymatic activity and
binding affinity than 1, found in our library, while both have the
same number of heavy atoms (17) and thus 15 has a much
higher LE than 1 (0.43 vs 0.31).
■
REFERENCES
■
(1) Wendt, M. D. Discovery of ABT-263, a Bcl-Family Protein
Inhibitor: Observations on Targeting a Large Protein−Protein
Interaction. Expert Opin. Drug Discovery 2008, 3, 1123−1143.
(2) Wyatt, P. G.; Woodhead, A. J.; Berdini, V.; Boulstridge, J. A.;
Carr, M. G.; Cross, D. M.; Davis, D. J.; Devine, L. A.; Early, T. R.;
Feltell, R. E.; Lewis, E. J.; McMenamin, R. L.; Navarro, E. F.; O’Brien,
M. A.; O’Reilly, M.; Reule, M.; Saxty, G.; Seavers, L. C. A.; Smith, D.
M.; Squires, M. S.; Trewartha, G.; Walker, M. T.; Woolford, A. J. A.
Identification of N-(4-Piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-
pyrazole-3-carboxamide (AT7519), A Novel Cyclin Dependent Kinase
Inhibitor Using Fragment-Based X-Ray Crystallography and Structure
Based Drug Design. J. Med. Chem. 2008, 51, 4986−4999.
(3) Bollag, G.; Tsai, J.; Zhang, J.; Zhang, Ch.; Ibrahim, P.; Nolop, K.;
Hirth, P. Vemurafenib: The First Drug Approved for BRAF-Mutant
Cancer. Nature Rev. Drug Discovery 2012, 11, 873−886.
Introduction of the methoxy group on the 4-position of the
aryl ring of 15 gave a 10-fold increase in potency and improved
the LE to 0.46 (fragment 16). A similar effect was observed by
the GSK group on their template.14 Fragment 16 was further
optimized to become a clinical candidate that entered phase I.
The clinical candidate, which has a LE of 0.35, contains all the
atoms present in this fragment.
Figures were drawn with PyMOL.19
CONCLUSION
■
In this paper, we described a novel approach for de novo
fragment design that relies on the design by medicinal chemists.
The approach allowed us to incorporate all the relevant
characteristics during the design of the fragments (synthetic
route, uniqueness of the fragments with respect to prior art, and
drug-like properties) to minimize the efforts and time to find an
excellent starting point for the program. This is the first case, to
our knowledge, in which medicinal and computational chemists
applied structure-based methodologies to the de novo design of
a complete fragment by following a predefined set of
interaction criteria.
(4) Erlanson, D. A. Introduction to Fragment-Based Drug Discovery.
Top. Curr. Chem. 2012, 317, 1−32.
(5) Nishibata, Y.; Itai, A. Automatic Creation of Drug Candidate
Structures Based on Receptor Structure. Starting Point for Artificial
Lead Generation. Tetrahedron 1991, 47, 8985−8990.
(6) Loving, K.; Alberts, I.; Sherman, W. Computational Approaches
for Fragment-Based and de Novo Design. Curr. Top. Med. Chem. 2010,
10, 14−32.
(7) Rognan, D. Fragment-Based Approaches and Computer-Aided
Drug Discovery. Top. Curr. Chem. 2012, 317, 201−222.
(8) Sofia, M. J.; Chang, W.; Furman, P. A.; Mosley, R. T.; Ross, B. S.
Nucleoside, Nucleotide, and Non-Nucleoside Inhibitors of Hepatitis C
Virus NS5B RNA-Dependent RNA-Polymerase. J. Med. Chem. 2012,
55, 2481−2531.
This approach offers several advantages compared to virtual
screening or de novo fragment design using only computational
methods. We believe that the unique process described in this
paper can also be successfully applied to other targets for which
it may be difficult to otherwise find high quality starting leads.
(9) Le Pogam, S.; Seshaadri, A.; Kosaka, A.; Chiu, S.; Kang, H.-S.;
Hu, S.; Rajyaguru, S.; Symons, J.; Cammack, N.; Najera, I. Existence of
Hepatitis C Virus NS5B Variants Naturally Resistant to Non-
Nucleoside, but not to Nucleoside, Polymerase Inhibitors Among
Untreated Patients. J. Antimicrob. Chemother. 2008, 61, 1205−1216.
(10) de Vicente, J.; Hendricks, R. T.; Smith, D. B.; Fell, J. B.; Fischer,
J.; Spencer, S. R.; Stengel, P. J.; Mohr, P.; Robinson, J. E.; Blake, J. F.;
Hilgenkamp, R. K.; Yee, C.; Adjabeng, G.; Elworthy, T. R.; Tracy, J.;
ASSOCIATED CONTENT
* Supporting Information
Experimental procedure for the preparation of 7−16,
spectroscopic data of intermediates and target compounds,
description of the HCV NS5B polymerase biochemical and
surface plasma resonance assays, and crystallographic protocols
■
S
D
dx.doi.org/10.1021/jm4002605 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
Chin, E.; Li, J.; Wang, B.; Bamberg, J. T.; Stephenson, R.; Oshiro, C.;
Harris, S. F.; Ghate, M.; Leveque, V.; Najera, I.; Le Pogam, S.;
Rajyaguru, S.; Ao-Ieong, G.; Alexandrova, L.; Larrabee, S.; Brandl, M.;
Briggs, A.; Sukhtankar, S.; Farrell, R.; Xu, B. Non-Nucleoside
Inhibitors of HCV Polymerase NS5B. Part 2: Synthesis and
Structure−Activity Relationships of Benzothiazine-Substituted Quino-
linediones. Bioorg. Med. Chem. Lett. 2009, 19, 3642−3646.
(11) de Vicente, J.; Hendricks, R. T.; Smith, D. B.; Fell, J. B.; Fischer,
J.; Spencer, S. R.; Stengel, P. J.; Mohr, P.; Robinson, J. E.; Blake, J. F.;
Hilgenkamp, R. K.; Yee, C.; Zhao, J.; Elworthy, T. R.; Tracy, J.; Chin,
E.; Li, J.; Lui, A.; Wang, B.; Oshiro, C.; Harris, S. F.; Ghate, M.;
Leveque, V. J. P.; Najera, I.; Le Pogam, S.; Rajyaguru, S.; Ao-Ieong, G.;
Alexandrova, L.; Fitch, B.; Brandl, M.; Masjedizadeh, M.; Wu, S.; de
Keczer, S.; Voronin, T. Non-Nucleoside Inhibitors of HCV Polymer-
ase NS5B. Part 3: Synthesis and Optimization Studies of
Benzothiazine-Substituted Tetramic Acids. Bioorg. Med. Chem. Lett.
2009, 19, 5648−5651.
(12) Pfefferkorn, J. A.; Greene, M. L.; Nugent, R. A.; Gross, R. J.;
Mitchell, M. A.; Finzel, B. C.; Harris, M. S.; Wells, P. A.; Shelly, J. A.;
Anstadt, R. A.; Kilkuskie, R. E.; Koptab, L. A.; Schwendea, F. J.
Inhibitors of HCV NS5B Polymerase. Part 1: Evaluation of the
Southern Region of (2Z)-2-(Benzoylamino)-3-(5-phenyl-2-furyl)-
acrylic Acid. Bioorg. Med. Chem. Lett. 2005, 15, 2481−2486.
(13) Gopalsamy, A.; Chopra, R.; Lim, K.; Ciszewski, G.; Shi, M.;
Curran, K. J.; Sukits, S. F.; Svenson, K.; Bard, J.; Ellingboe, J. W.;
Agarwal, A.; Krishnamurthy, G.; Howe, A. Y. M.; Orlowski, M.; Feld,
B.; O′Connell, J.; Mansour, T. S. Discovery of Proline Sulfonamides as
Potent and Selective Hepatitis C Virus NS5b Polymerase Inhibitors.
Evidence for a New NS5b Polymerase Binding Site. J. Med. Chem.
2006, 49, 3052−3055.
(14) Slater, M. J.; Amphlett, E. M.; Andrews, D. M.; Bravi, G.;
Burton, G.; Cheasty, A. G.; Corfield, J. A.; Ellis, M. R.; Fenwick, R. H.;
Fernandes, S.; Guidetti, R.; Haigh, D.; Hartley, C. D.; Howes, P. D.;
Jackson, D. L.; Jarvest, R. L.; Lovegrove, V. L. H.; Medhurst, K. J.;
Parry, N. P.; Price, H.; Shah, P.; Singh, O. M. P.; Stocker, R.;
Thommes, P.; Wilkinson, C.; Wonacott, A. Optimization of Novel
Acyl Pyrrolidine Inhibitors of Hepatitis C Virus RNA-Dependent RNA
Polymerase Leading to a Development Candidate. J. Med. Chem. 2007,
50, 897−900.
(15) Brameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small
Molecule Conformational Preferences Derived from Crystal Structure
Data. A Medicinal Chemistry Focused Analysis. J. Chem. Inf. Model.
2008, 48, 1−24.
(16) Molecular Operating Environment (MOE); Chemical Computing
Group Inc.: 1010 Sherbooke Street West, Suite #910, Montreal, QC,
Canada, H3A 2R7, 2008.
(17) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand Efficiency: A
Useful Metric for Lead Selection. Drug Discovery Today 2004, 9, 430−
431.
(18) Hajduk, P. J. Fragment-Based Drug Design: How Big is Too
Big? J. Med. Chem. 2006, 49, 6972−6976.
(19) The PyMOL Molecular Graphics System, version 1.5.0.4;
Schrodinger, LLC.
̈
E
dx.doi.org/10.1021/jm4002605 | J. Med. Chem. XXXX, XXX, XXX−XXX