Structure-based library design and the discovery of a potent and selective mast cell β-tryptase inhibitor as an oral therapeutic agent

Article abstract of DOI:10.1016/j.bmcl.2011.11.119

A solid phase combinatorial library was designed based on X-ray structures and in-silico models to explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible S4+ pocket is unique to β-tryptase and does not exist for other trypsin-like serine proteases of interest. Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other proteases in the same family. A member of this library was found to be a potent and selective β-tryptase inhibitor with a suitable pharmacokinetic profile for further clinical evaluation.

Full text of DOI:10.1016/j.bmcl.2011.11.119

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1049–1054
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based library design and the discovery of a potent and selective
mast cell b-tryptase inhibitor as an oral therapeutic agent
Guyan Liang a, , Suzanne Aldous b, Gregory Merriman a, Julian Levell g, James Pribish c, Jennifer Cairns d,
⇑
Xin Chen a, Sebastien Maignan e, Magali Mathieu e, Joseph Tsay b, Keith Sides b, Sam Rebello g,
Brian Whitely h, Isabelle Morize a, Henry W. Pauls f
a Molecular Innovative Therapeutics, Sanofi Pharmaceuticals, USA
b Fibrosis and Wound Repair, Sanofi Pharmaceuticals, USA
c Oncology, Sanofi Pharmaceuticals, USA
d Immunology and Inflammation, Sanofi Pharmaceuticals, USA
e Structure Design and Informatics, Sanofi Pharmaceuticals, France
f Campbell Family Institute for Breast Cancer Research, Canada
g Novartis Institutes for BioMedical Research, USA
h Bristol Myers Squibb, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A solid phase combinatorial library was designed based on X-ray structures and in-silico models to
explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible
S4+ pocket is unique to b-tryptase and does not exist for other trypsin-like serine proteases of interest.
Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other pro-
teases in the same family. A member of this library was found to be a potent and selective b-tryptase
inhibitor with a suitable pharmacokinetic profile for further clinical evaluation.
Received 29 October 2011
Revised 25 November 2011
Accepted 28 November 2011
Available online 7 December 2011
Keywords:
Serine protease
Asthma
Ó 2011 Elsevier Ltd. All rights reserved.
COPD
Tryptase
Inhibitor
Molecular modeling
Structure-based drug design
X-ray
Benzylamine
The physiological role of human b-tryptase for airway inflam-
matory and allergic disorders, such as asthma and chronic obstruc-
tive pulmonary disease (COPD), has been well documented.1–7
Following allergen stimulation, mast cells degranulate and secrete
various inflammatory mediators, including b-tryptase, a trypsin-
like serine protease. By cleaving and activating endogenous prote-
ase-activated receptor 2 (PAR-2), which subsequently triggers the
activation of various cell types, including fibroblasts, b-tryptase is
implicated in both bronchoconstriction and airway smooth muscle
remodeling.8 In a mouse asthma model, a b-tryptase inhibitor
administered either intraperitoneally or intranasally reduced air-
way inflammation and inhibited the release of IL-4 and IL-13 in
bronchoalveolar lavage fluid.9 As a major mediator of allergic
inflammation and airway tissue remodeling, b-tryptase has
emerged as an attractive target in pharmaceutical development.6,8
A variety of small molecular inhibitors of b-tryptase have been
developed for pharmaceutical purposes, as shown in Figure. 1. In
1999, Ono and co-workers reported several homo- and hetero-
dimeric inhibitors, such as a in Figure. 1, exploring the tetrameric
nature of the b-tryptase bioactive folding.10 A similar strategy
was also used by Rice and co-worker from Axys Pharmaceuticals
in the development of APC 2059 for ulcerative colitis (compound
b).11,12 A series of 4-carboxyazetidinone derivatives was discov-
ered by scientists from Bristol Myers Squibb (BMS), where com-
pounds, such as c, exhibited efficacy in a guinea pig asthma
model through intratracheal dosing.13 Compounds a–c incorporate
very basic functions in the P1 groups (pKa > 11), which results in
poor oral bioavailability.
To improve oral exposure, a series of inhibitors (compounds
d–i) with a less basic benzylamine P1 group (pKa ꢀ 9) have been
explored and exhibited significantly improved pharmacokinetic
⇑
Corresponding author. Address: 1041 Route 202/206, Mailstop 203A, Bridge-
water, NJ 08807, USA. Tel.: +1 908 231 4573.
E-mail address: guyan.liang@sanofi.com (G. Liang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.119

1050
G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1049–1054
O
O
NH
O
H
O
N
O
O
NH2
NH
H
N
H2N
N
N
H
O
NH
O
N
N
N
N
H
O
O
N
N
NH2
N
H
H2N
O
O
N
O
H
O
HN
O
O
a
b
NH2
HO
O
O
H
O
O
N
O
NH2
N
H
H
O
N
N
N
N
NH2
N
O
NH2
N
N
N
O
O
O
Cl
HN
O
N
c
d
e
f
O
O
Cl
N
H
N
NH2
N
Cl
H2N
O
O
S
O
H
N
O
N
O
N
N
NH2
N
N
H
O
H2N
N
H
N
O
H
O
NH2
g
h
i
j
Figure 1. Representative b-tryptase inhibitors reported in the literature.
properties.14–20 Celera Genomics reported a series of inhibitors
with an aliphatic amine as P1 group, such as compound j. While
excellent potency for b-tryptase and exquisite selectivity against
other serine proteases were demonstrated for those inhibitors, oral
bioavailability was only moderate. In the present study, we contin-
ued the optimization of b-tryptase inhibitors with a benzylamine
P1 and a piperidine amide scaffold by focusing on the diversity
of aromatic P4 groups and their extended binding in an inducible
S4+ pocket.
X-ray structures of b-tryptase were first resolved by Bode and
co-workers in 1998,21,22 providing a structure-based insight for
early inhibitor designs. Based on their findings, the bioactive
b-tryptase was determined to be a homo-tetramer with four inde-
pendent catalytic sites in a pair-wise distribution. While the S1
pocket of each catalytic site is located exclusively within an inde-
pendent monomer, the S4 pocket is situated on the interface be-
tween two adjacent monomers. The most prominent binding
pocket of each catalytic site is the S1 pocket featured by Asp189,
Ser190, and Tyr228. The S1 pocket is also the most conserved bind-
ing pocket in the trypsin-like serine protease family. While all tryp-
sin-like serine proteases prefer a basic P1 group in the S1 pocket by
forming an H-bonding interaction with Asp189, some members,
such as thrombin and fXa, can accommodate inhibitors with a non-
basic P1 group through a hydrophobic interaction with Tyr228. This
type of nonbasic P1 inhibitors, however, is limited to family mem-
bers with an alanine instead of a serine at the 190 position.23 Hav-
ing a serine at the 190 position, b-tryptase has followed this trend
with all its competitive nonmechanism-based inhibitors identified
so far comprising a basic P1 group H-bonding with Asp189.
Another important binding pocket for b-tryptase is the S4 pocket
Figure. 2(a) is a b-tryptase structure with inhibitor f bound in the
catalytic site, where residues in the S4 pocket retain their apopro-
tein-folding conformation. In this binding mode, the H-bonding
network between Tyr95 and Glu217 and between Glu217 and
Asp60B maintains the folding integrity of the S4 pocket back wall,
with which the terminal phenethyl group of the inhibitor interacts.
While this fold is the same as the one exhibited by the X-ray
structures with a vacant S4 pocket, it is not the only possible con-
formation that b-tryptase can adopt. Under certain circumstances,
an inducible S4+ pocket is revealed: upon binding inhibitors with
rigid phenylethynyl-aryl S-4 groups the S4 region can be induced
to a much deeper pocket, designated as S4+. Shown in Figure. 2(b)
is b-tryptase complexed with an analogous inhibitor f but with a
rigid acetylene joining the 2 aryl functions of the P4 group. As
shown in the figure, Glu217 shifts closer to Asp60B by forming a
stronger interlocking H-bonding network. The most significant
movement involves Tyr95, which rotates about 120ꢀ forming a
new H-bonding interaction with the side chain of Arg177 (not
shown in the figure). This rotation opens a gate to a tube-like pock-
et with a diameter of about 4.5 ÅA and a depth of about 6.5 ÅA. As
shown in Figure. 2, this induced S4+ pocket is situated on the inter-
face between two adjacent monomers and is unique to b-tryptase,
which offers an opportunity to design inhibitors selective to b-
tryptase. The present article describes a solid-phase combinatorial
library designed to explore this induced S4+ pocket for optimal
binding affinity and selectivity.
A combinatorial library was designed with a benzylamine P1
group, a piperidine amide scaffold linker, and various substituted
furan P4 groups to preserve the aforementioned binding mode
for the P1-scaffold component. As shown in Figure. 3, the benzyl-
amine group bound in the S1 pocket with the amino moiety
H-bonding with the carboxyl side chain of Asp189 and the back-
bone carbonyl oxygen of Gly219. The piperidine ring remained
almost perpendicular to the benzylamine ring with a torsion angle
0
0
featuring a
p-stacking interaction with Gln98 and a hydrophobic
interaction with Tyr95. Published X-ray structures have shown that
the catalytic site binds competitive b-tryptase inhibitors with little
conformational change vis-à-vis the apoprotein.21,22 Illustrated in

G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1049–1054
1051
a
b
O
O
N
N
H2N
N
H2N
N
Tyr-95
Tyr-95
Glu-217
Gly-219
Glu-217
Gly-219
Asp-189
Asp-189
Cat. Triad
Cat. Triad
Figure 2. Catalytic binding site of b-tryptase. Carbon atoms of the inhibitor are colored in white, while those of b-tryptase are colored in green and magenta, respectively for
two adjacent monomers. Another copy of the inhibitor bound in the adjacent catalytic site is also visible, whereas most residues around the second inhibitor are not shown.
Figure 3. A stereo view of compound 2 modeled in the b-tryptase substrate-binding site. Carbon atoms of the two adjacent monomers are colored in green and magenta,
respectively, while carbon atoms of compound 2 are colored in white. This binding mode was confirmed by an in-house X-ray structure later deposited with the protein data
bank, 4A6L.
between 65ꢀand 90ꢀ. The amide nitrogen of the scaffold was situ-
ated above the carbonyl oxygen of the Gly216 backbone, while
the amide oxygen H-bonded with the backbone NH of Gly219.
The furan ring of the P4 group bound in the original S4 pocket,
sition of the furan ring to project the terminal P4 attachment into
the S4+ binding pocket, which is the target binding site of the pres-
ent library.
The S4+ pocket is a tube-shaped binding pocket surrounded by
residues from two adjacent monomers of b-tryptase, as shown in
Figure. 3, Tyr172 at the bottom, Pro60A and Thr96 on the top,
Ile175 and Tyr95 at the right, and Asp60B and Glu217 at the left.
p-stacking with the side chain amide of Gln98. This p-stacking
interaction and the conjugation between the furan ring and the
piperidine amide scaffold formed a binding vector from the 5-po-

1052
G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1049–1054
Residues lining the surface of the S4+ pocket are mostly hydropho-
bic except Asp60B and Glu217 on the left side. In general, aspartic
acid and glutamic acid are deprotonated under physiological con-
dition. Asp60B and Glu217 of b-tryptase, however, adopted a
head-to-head folding motif, which is highly similar to the one
exhibited by the catalytic dyad of aspartic proteases. While the
protonation state of the two aspartic acids in the dyad is not di-
rectly observable from X-ray structures, numerous studies have
been published on the topic using various approaches including
quantum mechanical calculations, molecular dynamics simula-
tions, thermodynamic and NMR studies.24–31 According to those
publications, the protonation state of the dyad can be affected by
many factors including the neighboring residues, the pH, and the
substrate or inhibitors bound in the catalytic site. Several studies
concluded that a mono-protonated state (only one of the two
aspartic acids was protonated) is likely with a substrate or certain
types of inhibitor bound in the catalytic site.26,27,30 It was also sug-
gested that both aspartic acids in the dyad could be protonated if
no substrate was in the binding site or the binding site was occu-
pied by other types of inhibitors.26,27,30
The present scaffold includes a benzylamine P1 group, which is
positively charged due to protonation and is required by the H-
bonding interaction with Asp189. Therefore, it is preferred not to
introduce another positively charged group at the P4 position in
the library for consideration of pharmacokinetics properties. The
possibility that Glu217 and Asp60B can be both protonated under
a proper binding environment offered a good opportunity to en-
gage in a strong S4+ interaction without introducing a positively
charged moiety. Therefore, in our binding models, both Glu217
and Asp60B were protonated in order to select possible hydropho-
bic S4+ binders.
plate, as shown in Scheme 1. N-Teoc-protected 4-piperidinone was
converted to the triflate and coupled utilizing Suzuki type method-
ology as previously described15 to give the N-Teoc-protected 4-(3-
cyanophenyl)-1,2,36-tetrahydropyridine. Tandem reduction of the
nitrile and endocyclic double bond was followed by loading this
material onto Wang resin (via the p-nitrophenyl carbonate Wang
resin) and removal of the Teoc group to give s2. The resin bound
template underwent standard amide coupling with the carboxylic
acid s3 to give s4. The key step of the target synthesis was accom-
plished by Sonogashira type reaction on the resin bound furan-al-
kyne s4. After optimizing the reaction conditions it was found that
the reaction proceeded in 10 min at 80 °C, using microwave irradi-
ation. The preferred catalyst system was Pd2(dba)3/PtBu3, in the ab-
sence of copper, using tetramethylguanidine as base. The final
products were cleaved from the solid phase with TFA and purified
by standard reverse phase prep-HPLC. This method allowed for the
rapid preparation of the selected compounds.
A search of commercially available chemical databases resulted
in 1502 suitable reagents. A virtual combinatorial library was con-
structed by merging the core scaffold and each reagent. The library
was then docked into the catalytic site of b-tryptase by constrain-
ing the binding mode of the core scaffold to the one identified by
previous X-ray structures. Docking poses were generated using
GlideÒ from SchrodingerÒ. Van de Waals radii of all residues in
the S4+ pocket were reduced by 40% during the initial binding pose
generation to accommodate flexibility of the binding pocket. An in-
house customized scoring function was used with the electrostatic
component of Glu217 and Asp60B eliminated to reflect their ‘dyad-
like’ nature and to favor hydrophobic groups purposely. Out of the
list of 1502 virtually enumerated compounds, 59 compounds sur-
vived the virtual screening and 25 highly scored members were
synthesized and tested for their b-tryptase activity.
It was envisioned that the desired alkyne target compounds
could be synthesized utilizing the Sonagashira reaction and that
the library synthesis could be accomplished on a resin bound tem-
The compound inhibition for b-tryptase was measured in a
chromogenic assay with human recombinant b-tryptase and
N
O
NH2 HCl
N
H
O
OTf
O
(i)
(ii)
(iv)
(v)
(iii)
N
N
N
Teoc
Teoc
N
N
H
Teoc
Teoc
s1
s2
O
NH2. TFA
N
H
O
(vii)
(viii)
(vi)
HO
s2
+
O
H
O
N
N
O
s3
O
O
O
s4
Ar
H
(i) LiHMDS, PhN(Tf)2, THF, -78oC; (ii) m-cyanophenyl boronic acid, Na2CO3, Pd(Ph3)4, MeCN;
(iii) H2, Pd/C, conc HCl/EtOH; (iv) p-nitrophenylcarbonate Wang resin, DIEA, DMAP, DMF;
(v) TBAF, THF; (vi) DIC, HOBt, DMF;
(vii) ArI, Pd2(dba)3, P(tBu)3, THF, tetramethylguanidine,10 min microwave 80°C; (viii) TFA/CH2Cl2
Scheme 1. Target synthesis by resin bound template.

G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1049–1054
1053
Table 1
Table 2
Representative members of the designed library and their Ki values for b-tryptase
inhibition
Selectivity profile of most potent compounds against other trypsin-like serine
proteases (IC50 M).
, l
N
Compounds
fXa (IC50 M)
Thrombin (IC50
Trypsin (IC50
Elastase (IC50
Chymase (IC50
1
2
15
R
,
l
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
,
l
l
l
l
M)
M)
M)
M)
>100
>100
>100
>100
O
,
N
,
,
O
Compound#
R
Ki (nM)
Table 3
1
X
31
Pharmacokinetics (PK) profile of compounds 1, 2, and 15 (rat, 2 mg/kg i.v. dosing and
10 mg/kg p.o. dosing)
F
Compounds
1
2
15
2
10
X
X
Bioavailability
CL (L/h/kg)
i.v. T1/2 (h)
Vss (L/kg))
p.o. Cmax (ng/ml)
p.o. Tmax (h)
32%
5.1
2.5
14.1
68
77%
8.4
2.2
18.5
121
3.7
0%
18.6
0.46
3
4
112
324
3.3
X
X
F
were used to generate IC50 curves. For each concentration, the
compound was mixed with 50 ng/mL b-tryptase, followed by addi-
tion of 500 lM substrate S-2366. The reaction mixture was incu-
5
6
742
67
F
bated at room temperature for 30 min before the absorbance of
the released product, p-nitroaniline, was measured at a wave-
length of 405 nm with Wallac Victor2 VÒ spectrophotometer. The
IC50 values were determined by fitting the dose–response data into
a sigmoid curve and the Ki values were calculated using a compet-
itive inhibition equation: Ki = IC50/(1 + [S]/Km).
Ki values of representative members of the library are shown in
Table 1 with an SAR supporting the original design hypothesis. This
entire library of compounds, dominated by largely hydrophobic P4
tails, is well accepted by b-tryptase with exceptional potency. The
activity of this library confirms that a polar or H-bonding group,
more specifically a positively charged group, is not absolutely re-
quired to interact with Glu217 and Asp60B. With a head-to-head
folding motif, the two carboxyl side chains could serve as H-bond-
ing donors and acceptors to each other without deprotonation and
requiring H-bonding donors from the inhibitor bound in the S4+
pocket.
However, the S4+ pocket is not symmetric in nature with Glu/
Asp on the left and Ile/Tyr on the right. Even if the interlocked
Glu217 and Asp60B satisfied the H-bonding requirement, their side
of the S4+ pocket is still less hydrophobic than the other side.
Hence, almost all compounds with an asymmetric charge/dipole
distribution are more potent than their symmetric counterpart,
especially if the charge is brought on at the ortho position. For
example, compounds 2 and 10 are more potent than compound
1, compound 6 is more potent than compound 8, and compound
15 is more potent than compounds 1 and 18. Additionally, the size
of the S4+ pocket is rather limited with space only capable of
accommodating a single 5- or 6-member aromatic ring. A substitu-
tion on the ring, even as small as a methyl or chloro group, may not
be able to fit into the pocket without lowering the potency for b-
tryptase inhibition.
F
X
X
X
F
7
8
Cl
F
166
279
F
9
98
X
X
Cl
10
11
22
N
101
X
X
Cl
S
12
13
236
327
S
X
N
14
15
84
5
X
X
X
N
N
16
27
F
N
17
18
X
X
2769
157
Compounds in this library are highly selective against other
trypsin-like serine proteases, such as factor Xa (fXa), thrombin,
trypsin, elastase, and chymase. Most compounds exhibit no inhibi-
tion against those enzymes within the detectable limit of 100
except Compound 15 having an IC50 against fXa of 9 M, as shown
N
N
N
lM,
l
in Table 2. This exquisite selectivity was expected in light of the
fact that the S4+ pocket occupied by the diversity point of this li-
brary was exclusive to b-tryptase and not available to other tryp-
sin-like serine proteases on the selectivity panel.
substrate S-2366 (from DiaPharmaÒ),
arginine-p-Nitroaniline hydrochloride. Eight concentrations of the
compound, 0.0046, 0.0137, 0.04, 0.12, 0.37, 1.11, 3.3, and 10 M,
L
-Pyroglutamyl-
L
-prolyl-L-
l

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Rat pharmacokinetic (PK) studies were carried out for represen-
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Dupuy, A.; Davidson, J.; Harrison, T.; Morley, A.; Watson, S.; Fenton, G.;
McCarthy, C.; Romano, J.; Mathew, R.; Engers, D.; Gardyan, M.; Sides, K.;
Kwong, J.; Tsay, J.; Rebello, S.; Shen, L.; Wang, J.; Luo, Y.; Giardino, O.; Lim, H.-
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tative compounds of the library with 2 mg/kg intravenous (i.v.) dos-
ing and 10 mg/kg oral (p.o.) dosing. As shown in Table 3, compound
15 exhibited extremely low oral bioavailability, presumably due to
the terminal pyridyl group, and was eliminated from further profil-
ing. Both compounds 1 and 2 have a Volume of Distribution (Vss)
greater than the total body water for rat (0.67 L/kg), indicating a sig-
nificant tissue distribution. Since b-tryptase is not a plasma target, a
significant tissue distribution may not necessarily be a negative
parameter for our purpose. The plasma clearance (CL) values for
both compounds are higher than the average rat hepatic blood flow
(3.3 L/h/kg), which indicates that other clearance mechanisms may
exist in addition to the hepatic route. Notwithstanding the possibil-
ity of those additional clearance mechanisms, both compounds
have a satisfactory plasma half-life, as shown in the table. With a
combination of higher potency against b-tryptase and overall supe-
rior PK profile, Compound 2 was accepted for further clinical
evaluation.
In conclusion, Tyr95 of b-tryptase can rotate away from its apo-
protein folding position, which presents an inducible S4+ pocket.
This S4+ pocket is largely hydrophobic in nature even though
Asp60B and Glu217 form one side of the pocket. Based on this
working hypothesis, a combinatorial library was designed to ex-
plore the hydrophobic interaction within the S4+ pocket, which re-
sulted in the discovery of a potent, selective, and orally bioavailable
b-tryptase inhibitor suitable for further clinical evaluation.
19. Liang, G.; Choi-Sledeski, Y. M.; Poli, G.; Chen, X.; Shum, P.; Minnich, A.; Wang,
Q.; Tsay, J.; Sides, K.; Cairns, J.; Stoklosa, G.; Nieduzak, T.; Zhao, Z.; Wang, J.;
Vaz, R. J. Bioorg. Med. Chem. Lett. 2010, 20, 6721.
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Authors would like to thank Dr. Sukanthini Thurairatnam
and Dr. Yong-Mi Choi-Sledeski for support and constructive
suggestions.
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