04-07-2013, 03:36 PM
Combinatorial Library of Serine and Cysteine Protease Inhibitors That Interact with Both the S and S¢ Binding Sites
[attachment=56432]
ABSTRACT
A combinatorial library of 400 inhibitors has been synthesized and screened against several
serine and cysteine proteases including plasmin, cathepsin B, and papain. The inhibitors are
based upon a cyclohexanone nucleus and are designed to probe binding interactions in the S2
and S2¢ binding sites. This methodology has led to the discovery of inhibitor 15A, which
incorporates Trp at both the P2 and P2¢ positions and has an inhibition constant against plasmin
of 5 íM. Data from screening of the library shows that plasmin has a strong specificity for Trp
at the S2 subsite and prefers to bind hydrophobic and aromatic amino acids such as Ile, Phe,
Trp, and Tyr at the S2¢ subsite. In contrast, the S2¢ subsites of cathepsin B and papain do not
show a strong preference for any particular amino acid.
Introduction
Combinatorial chemistry has emerged as a powerful
method for generating lead compounds for drug discovery
and for optimizing the biological activity of those
leads.1 This technique has been used to develop new
ligands for a variety of biological targets including
proteases, kinases, various receptors, and antibodies,
among others. Proteases are particularly interesting
targets because they are involved with a wide variety
of important diseases that include AIDS, cancer, and
malaria. Many of the libraries that have been generated
for screening against proteases incorporate a chemical
functionality that mimics the tetrahedral intermediate
that occurs during enzyme-catalyzed peptide hydrolysis.
For example, phosphonic acids have been screened
against the metalloprotease thermolysin,2 and statine,3
(hydroxyethyl)amine,4 and diamino diol5 isosteres have
been used to synthesize libraries against the aspartic
protease HIV protease. In addition, a peptide aldehyde
library has been targeted against the cysteine protease
interleukin-1â converting enzyme.
Results and Discussion
Design and Synthesis of the Library. Before we
began constructing the library, we needed to devise an
efficient synthesis of a building block such as compound
4 (Scheme 1). This molecule incorporates the cyclohexanone
nucleus, is amenable to solid-phase peptide
synthesis, and carries the ketone functionality in a
suitably protected form. We have reported previously
that compound 2 can be converted to 4 by oxidative
cleavage of the double bond and replacement of the Boc
protecting group with Fmoc.10 However, we have found
that on larger scale, reaction of the amino acid with
Fmoc chloride or Fmoc N-hydroxysuccinimide ester
under a variety of conditions gave relatively low and
inconsistent yields of 4. This problem can be circumvented
by switching the protecting groups first and then
oxidizing the alkene to the acid as shown in Scheme 1.
Compound 4 is a mixture of two diastereomers, each of
which has the substituents on the cyclohexanone ring
in the 2,6-cis configuration.
Conclusions
In this paper we have described the construction and
screening of a combinatorial library of protease inhibitors
that extend into both the S and S¢ specificity sites.
Using this method we have found an inhibitor that has
significant affinity for the serine protease plasmin. In
addition, this work has shown that combinatorial chemistry
is an efficient method for probing the specificity
of the S2¢ subsite. Currently there is little information
available concerning the binding specificity of this
position for many proteases.
Our data have shown that for plasmin, the S2¢ subsite
prefers to bind hydrophobic and especially aromatic
amino acids. Furthermore, binding of such hydrophobic
residues in this site significantly increases the affinity
of the inhibitor for the enzyme. On the other hand, the
S2¢ subsites of cathepsin B and papain do not appear
to have strong preferences for any particular amino acid,
and binding in this position leads to only incremental
increases in affinity. Concerning the S2 subsites, the
data that we have presented are consistent with the
known specificity of cathepsin B and papain, which
prefer hydrophobic amino acids at this position.18 For
plasmin, it has been believed that Phe binds well in the
S2 subsite.
Experimental Section
General Methods. NMR spectra were recorded on Bruker
Avance-300 or Avance-400 instruments. Spectra were calibrated
using TMS (ä ) 0.00 ppm) for 1H NMR. Mass spectra
were recorded on a Kratos MS 80 under fast-atom bombardment
(FAB) conditions or were performed using electrospray
ionization at the Harvard University Chemistry Department
Mass Spectrometry Facility. HPLC analyses were performed
on a Rainin HPLC system with Rainin Microsorb silica or C18
columns and UV detection. Semipreparative HPLC was performed
on the same system using semipreparative columns
(21.4 250 mm). UV spectra were recorded on a Perkin-Elmer
8452A diode array UV-vis spectrophotometer.
[attachment=56432]
ABSTRACT
A combinatorial library of 400 inhibitors has been synthesized and screened against several
serine and cysteine proteases including plasmin, cathepsin B, and papain. The inhibitors are
based upon a cyclohexanone nucleus and are designed to probe binding interactions in the S2
and S2¢ binding sites. This methodology has led to the discovery of inhibitor 15A, which
incorporates Trp at both the P2 and P2¢ positions and has an inhibition constant against plasmin
of 5 íM. Data from screening of the library shows that plasmin has a strong specificity for Trp
at the S2 subsite and prefers to bind hydrophobic and aromatic amino acids such as Ile, Phe,
Trp, and Tyr at the S2¢ subsite. In contrast, the S2¢ subsites of cathepsin B and papain do not
show a strong preference for any particular amino acid.
Introduction
Combinatorial chemistry has emerged as a powerful
method for generating lead compounds for drug discovery
and for optimizing the biological activity of those
leads.1 This technique has been used to develop new
ligands for a variety of biological targets including
proteases, kinases, various receptors, and antibodies,
among others. Proteases are particularly interesting
targets because they are involved with a wide variety
of important diseases that include AIDS, cancer, and
malaria. Many of the libraries that have been generated
for screening against proteases incorporate a chemical
functionality that mimics the tetrahedral intermediate
that occurs during enzyme-catalyzed peptide hydrolysis.
For example, phosphonic acids have been screened
against the metalloprotease thermolysin,2 and statine,3
(hydroxyethyl)amine,4 and diamino diol5 isosteres have
been used to synthesize libraries against the aspartic
protease HIV protease. In addition, a peptide aldehyde
library has been targeted against the cysteine protease
interleukin-1â converting enzyme.
Results and Discussion
Design and Synthesis of the Library. Before we
began constructing the library, we needed to devise an
efficient synthesis of a building block such as compound
4 (Scheme 1). This molecule incorporates the cyclohexanone
nucleus, is amenable to solid-phase peptide
synthesis, and carries the ketone functionality in a
suitably protected form. We have reported previously
that compound 2 can be converted to 4 by oxidative
cleavage of the double bond and replacement of the Boc
protecting group with Fmoc.10 However, we have found
that on larger scale, reaction of the amino acid with
Fmoc chloride or Fmoc N-hydroxysuccinimide ester
under a variety of conditions gave relatively low and
inconsistent yields of 4. This problem can be circumvented
by switching the protecting groups first and then
oxidizing the alkene to the acid as shown in Scheme 1.
Compound 4 is a mixture of two diastereomers, each of
which has the substituents on the cyclohexanone ring
in the 2,6-cis configuration.
Conclusions
In this paper we have described the construction and
screening of a combinatorial library of protease inhibitors
that extend into both the S and S¢ specificity sites.
Using this method we have found an inhibitor that has
significant affinity for the serine protease plasmin. In
addition, this work has shown that combinatorial chemistry
is an efficient method for probing the specificity
of the S2¢ subsite. Currently there is little information
available concerning the binding specificity of this
position for many proteases.
Our data have shown that for plasmin, the S2¢ subsite
prefers to bind hydrophobic and especially aromatic
amino acids. Furthermore, binding of such hydrophobic
residues in this site significantly increases the affinity
of the inhibitor for the enzyme. On the other hand, the
S2¢ subsites of cathepsin B and papain do not appear
to have strong preferences for any particular amino acid,
and binding in this position leads to only incremental
increases in affinity. Concerning the S2 subsites, the
data that we have presented are consistent with the
known specificity of cathepsin B and papain, which
prefer hydrophobic amino acids at this position.18 For
plasmin, it has been believed that Phe binds well in the
S2 subsite.
Experimental Section
General Methods. NMR spectra were recorded on Bruker
Avance-300 or Avance-400 instruments. Spectra were calibrated
using TMS (ä ) 0.00 ppm) for 1H NMR. Mass spectra
were recorded on a Kratos MS 80 under fast-atom bombardment
(FAB) conditions or were performed using electrospray
ionization at the Harvard University Chemistry Department
Mass Spectrometry Facility. HPLC analyses were performed
on a Rainin HPLC system with Rainin Microsorb silica or C18
columns and UV detection. Semipreparative HPLC was performed
on the same system using semipreparative columns
(21.4 250 mm). UV spectra were recorded on a Perkin-Elmer
8452A diode array UV-vis spectrophotometer.