A general inhibitor scaffold for serine proteases with a (chymo)trypsin- like fold: Solution-phase construction and evaluation of the first series of libraries of mechanism-based inhibitors [13]

Full text of DOI:10.1021/ja990160e

8128
J. Am. Chem. Soc. 1999, 121, 8128-8129
A General Inhibitor Scaffold for Serine Proteases
with a (Chymo)trypsin-Like Fold: Solution-Phase
Construction and Evaluation of the First Series of
Libraries of Mechanism-Based Inhibitors
Rongze Kuang,† Jeffrey B. Epp,† Sumei Ruan,† Hongyi Yu,†
Peng Huang,† Shu He,† Juan Tu,† Norman M. Schechter,‡
Jane Turbov,§ Christopher J. Froelich,§ and
William C. Groutas*,†
Department of Chemistry, Wichita State UniVersity,
Wichita, Kansas 67260, Department of Dermatology &
Department of Biochemistry and Biophysics, UniVersity of
PennsylVania, Philadelphia, PA 19104, and Department of
Medicine, Northwestern UniVersity Medical School,
EVanston, IL 60201
ReceiVed January 19, 1999
Serine proteases are involved in various physiological as well
as pathological processes and, thus, are important therapeutic
targets. Proteolysis by serine proteases is a highly specific process,
which involves both sequential and conformational recognition
of protein/peptide substrates. Current approaches to the design
of specific inhibitors of serine proteases focus on the exploitation
of substrate sequential specificity of target proteases, while
conformational recognition has rarely been integrated into the
design process.1 Rigid inhibitor scaffolds resulting from integrated
sequential and conformational design not only can provide highly
specific inhibitors of target proteases but also can serve as an
excellent model system for addressing fundamental issues related
to protease-substrate recognition.
As part of an ongoing project related to the design of inhibitors
of neutrophil-derived serine proteases involved in a range of
inflammatory diseases,2 we have recently described the structure-
based design of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold
(I in Scheme 1) for the mechanism-based inhibition of serine
proteases.3 The design process began with the X-ray crystal
structure of the human leukocyte elastase-turkey ovomucoid
inhibitor (HLE-TOMI) complex.4 With the P1 backbone locked
with a sulfamide linkage, the central segment of the substrate
recognition loop (P2-P2′)5 was able to be conformationally frozen
in the rigid five-membered ring scaffold. As suggested by many
crystallographic studies of protein inhibitor-protease complexes,
the active backbone conformation of substrate recognition loop
P3-P3′ is conserved among most chymotrypsin-like serine pro-
Figure 1. Inhibitory activity of P1, R2, and L libraries toward human
leukocyte elastase, cathepsin G, and chymase: (a) each P1 sublibary is
derived from a single racemic amino acid (Ala, Leu, Phe), mixed
aldehydes (R2 is Me3CCH2-, (p-methoxy)benzaldehyde, (m-phenoxy)-
benzaldehyde), and selected L: Cl, sulfone (p-chloro)benzenesulfonyl),
and sulfonamide (MeSO2N(COOCH3); (b) each R2 sublibrary is derived
from a single aldehyde, mixed racemic amino acids and selected L; (c)
each L sublibrary is derived from p-anisaldehyde (R2), mixed racemic
amino acids and single carboxylic acid (acetate, benzoate, or phenyl-
acetate).
* To whom correspondence should be addressed. Tel: (316) 978 3120.
Fax: (316) 978 3431. E-mail: groutas@wsuhub.uc.twsu.edu.
† Wichita State University.
‡ University of Pennsylvania.
§ Northwestern University Medical School.
(1) Edward, P. D.; Bernstein, P. R. Med. Res. ReV. 1994, 14 (2), 127-
194. For an elegant example of integrated sequential and conformational design
that has emerged from the area of HIV protease inhibition, where the sequential
recognition elements (P2-P2′) were spatially presented on a rigid cyclic urea
that mimics the substrate conformation, see Lam, P. Y. S.; Jadhav, P. K.;
Eyermann, C. J.; Hodge, C. N.; Ru, Y.; Bacheler, L. T.; Meek, J. L.; Otto, M.
J.; Rayner, M. M.; Wong, Y. N.; Chang, C.-H.; Weber, P. C.; Jackson, D. A.;
Sharpe, T. R.; Erickson-Viitanen, S. Science 1994, 263, 380-384.
(2) Groutas, W. C.; Kuang, R.; Ruan, S.; Epp, J. B.; Venkataraman, R.;
Truong, T. M. Bioorg. Med. Chem. 1998, 6, 661-671. (b) Kuang, R.;
Venkataraman, R.; Ruan, S.; Groutas, W. C. Bioorg. Med. Chem. Lett. 1998,
8, 539-544. (c) Groutas, W. C.; Kuang, R.; Venkataraman, R. Biochem.
Biophys. Res. Commun. 1994, 198, 341-349.
teases;6 consequently, inhibitor scaffold (I) is expected to be a
general template capable of affording specific inhibitors of a wide
range of serine proteases by appending the corresponding recogni-
tion elements (P2-P2′) spatially at the three positions (P1, R2, L)
of the cyclic template.
We wish to describe herein the results of in vitro biochemical
studies, which demonstrate the generality of the aforementioned
heterocyclic scaffold (I) for the design of specific inhibitors of a
(3) Groutas, W. C.; Kuang, R.; Venkataraman, R.; Epp, J. B.; Ruan, S.;
Prakash, O. Biochemistry 1997, 36, 4739-4750.
(4) Bode, W.; Wei, A.-Z.; Huber, R.; Meyer, E. F.; Travis, J.; Neumann,
S. EMBO J. 1986, 5, 2453-2458.
(5) S1, S2, S3, ...Sn and S1′, S2′, S3′, ...Sn′ correspond to the enzyme subsites
on either side of the scissile bond. Each subsite accommodates a corresponding
amino acid side chain designated P1, P2, P3, ...Pn and P1′, P2′, P3′, ...Pn′ of the
substrate or inhibitor. S1 is the primary specificity site. (Schecter, I.; Berger,
A. Biochem. Biophys. Res. Commun. 1967, 27, 157-162).
(6) Bode, W.; Huber, R. Eur. J. Biochem. 1992, 204, 433-451. (b) Wibley,
K. S.; Barlow, D. J. J. Enzyme Inhib. 1992, 5, 331-338.
10.1021/ja990160e CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/21/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 35, 1999 8129
Table 1. Selectivity of Inhibitors (I) Against a Panel of Serine Proteases
kinact/Ki M-1s-1
compoundsa
P1
neutral
Cat Gb
basic
acidic
no.
R2
PR-3b
HLEb
chymase
R-CT
trypsin
tryptase
plasmin
granz B
1
2
3
4
5
6
7
8
n-propyl
n-propyl
isobutyl
isobutyl
benzyl
(CH2)4NH2
(CH2)4NH2
CH2CO2H
methyl
benzyl
methyl
benzyl
benzyl
methyl
benzyl
benzyl
1830
4960
2250
5200
na
na
na
780
7260
9490
95200
na
na
na
nac
130
na
110
11200
na
na
60
na
na
12500
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
500d
na
na
na
na
13500
30700
na
6380
12900
na
850
1310
na
na
na
na
na
na
na
na
na
160e
a L ) phenylsulfonyl. b Reference 2. c No activity. A compound was classified as being inactive when it gave less than 50% inhibition following
d
e
incubation with an inhibitor-to-enzyme ratio of 250. kobs/[I] M-1 s-1 determined by the incubation method. kobs/[I] M-1 s-1 determined by the
progress curve method.
Scheme 1
Scheme 2
individual inhibitors and libraries were chosen on the basis of
the known primary substrate specificities of HLE (Val, Leu),7
Cat G (Phe),8 and human chymase (Phe, Tyr, Leu)9 (vide infra).
All steps proceeded in high yield, and each library, consisting of
three pairs of racemic components, had a >95% purity.
Each library was assayed against HLE, Cat G, and chymase
using the progress curve method.2-3,10 The apparent second-order
rate constants kinact/Ki (M-1 s-1) serve as an index of inhibitory
potency and are summarized in Figure 1. As suggested by the
data, all three enzymes prefer the same R2 group (p-MeOPh-
CH2-). However, there are substantial differences in their tolerance
of leaving group L (Figure 1c), which implies that the S1′ and
S2′ subsites are more valuable secondary subsites for optimizing
selectivity than the S2 subsite. Indeed, the presence of an Arg-
143 residue in the S2′ subsite of chymase, as opposed to a Leu-
143 residue in the S2′ subsite of R-chymotrypsin, led to the design
of compound 9 (I, P1 ) R2 ) benzyl, L ) (p-carboxyphenyl)-
sulfonyl), which was found to be a highly efficient inhibitor of
recombinant chymase11 (kinact/Ki )186 000 M-1 s-1) but was
devoid of any inhibitory activity toward R-chymotrypsin.
In conclusion, the results presented herein demonstrate that the
1,2,5-thiadiazolidin-3-one 1,1 dioxide platform embodies a general
motif that renders the platform capable of binding to the active
site of many serine proteases with a (chymo)trypsin-like fold in
a predictable fashion and is amenable to the facile construction
of libraries for lead identification and optimization.
panel of serine proteases with widely differing specificities
(neutral, basic, acidic), as well as the development of a combi-
natorial approach for the rapid establishment of the inhibitory
profile of this template.
According to the design rationale, the nature of the P1 residue,
accommodated at the primary specificity site S1 of a target serine
protease, determines which type of protease will be inhibited. This
is indeed the case as suggested by the data in Table 1. For
instance, compounds with a basic P1 group (compounds 6 and 7)
are fairly effective inhibitors of trypsin, tryptase, and plasmin.
Likewise, an acidic P1 residue leads to inhibition of granzyme B
only, while a medium size alkyl or aromatic P1 residue gives rise
to inhibitors of proteinase 3 (PR 3) and human leukocyte elastase
(HLE), and chymase and R-chymotrypsin, respectively, in ac-
cordance with the known substrate specificity of each enzyme.
Thus, absolute specificity between neutral, basic, and acidic serine
proteases can be achieved by choosing an appropriate P1 residue.
Most importantly, the heterocyclic platform docks to the active
site of these serine proteases in a predictable fashion, namely,
with the P1 residue accommodated at the primary specificity site
(S1) and the R2 and L groups oriented toward the S2 and Sn′
subsites, respectively.
After establishing the generality of the 1,2,5-thiadiazolidin-3-
one 1,1 dioxide scaffold, we then pursued a library approach to
rapidly obtain the inhibitory profile of this template, with emphasis
on the exploitation of secondary recognition elements (P2, P1′ and
P2′) to achieve highly selective inhibition of members of a subset
of serine proteases with similar substrate specificity (for example,
chymase vs R-chymotrypsin or cathepsin G (Cat G); PR 3 vs
HLE; trypsin vs tryptase, etc.). Thus, as a first step toward this
goal, the solution phase construction of a series of small biased
libraries was carried out using Scheme 2 with diversity elements
derived from three racemic amino acids (Ala, Leu, and Phe) as
P1, three aldehydes (pivalaldehyde, p-methoxybenzaldehyde,
m-phenoxybenzaldehyde) for R2, and a range of leaving groups
for L. The amino acid ester precursors used in the synthesis of
Acknowledgment. This work was supported by Grants from NIH
(HL 57788 and AR 42931 to W.C.G. and N.M.S., respectively) and
SPARTA Pharmaceuticals, Inc.
JA990160E
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