Structure-based design of residue 1 analogs of the direct thrombin inhibitor pentapeptide FM 19

Article abstract of DOI:10.1111/j.1747-0285.2009.00915.x

Myocardial ischemia and other acute coronary syndromes are leading causes of death worldwide, and often result from a thrombus that blocks an atherosclerotic coronary artery. A key enzyme in thrombus formation is the serine protease thrombin, which is responsible for both the conversion of soluble fibrinogen into insoluble fibrin, as well as the activation of the GPCRs, PAR1 and PAR4, which stimulate platelet aggregation. Thus, thrombin is an attractive target for anticoagulant and antithrombotic therapy. Previous studies in our laboratory led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH₂), which shows modest potency as a thrombin inhibitor. The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed potential sites for modification to improve potency. This study reports replacements to the first residue (d-Arg ¹) of FM 19, which seek to improve potency by removing the N-terminal amine to eliminate an adverse electrostatic interaction, and alterations to the length of the side chain to eliminate an unfavorable eclipsed conformation observed in the X-ray structure. This study produced two compounds, 1 and 9, with improved α-thrombin inhibition (IC₅₀ values of 0.66 ± 0.20 μm and 0.57 ± 0.12 μm, respectively).

Full text of DOI:10.1111/j.1747-0285.2009.00915.x

ª 2009 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00915.x
Chem Biol Drug Des 2010; 75: 35–39
Research Article
Structure-Based Design of Residue 1 Analogs of
the Direct Thrombin Inhibitor Pentapeptide FM 19
that blocks a coronary artery (1,3,4). A key enzyme in thrombus
formation is the serine protease thrombin, which is responsible for
two major clotting events. First, thrombin cleaves an extracellular
portion of the protease-activated receptors 1 and 4 (PAR1 and
PAR4), which are G protein-coupled receptors (GPCRs) found on
platelets. Once this fragment is cleaved, the newly uncovered N-ter-
minus binds intramolecularly to stimulate platelet activation (3).
Secondly, thrombin converts soluble fibrinogen into insoluble fibrin,
the protein that cross-links with platelets to form a thrombus (1,5).
Thus, thrombin is an attractive target for both antiplatelet and anti-
thrombotic therapy.
Elizabeth A. Girnys, Katarzyna
Sobczyk-Kojiro and Henry I. Mosberg*
Department of Medicinal Chemistry, College of Pharmacy, University
of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA
*Corresponding author: Henry I. Mosberg, him@umich.edu
Myocardial ischemia and other acute coronary
syndromes are leading causes of death worldwide,
and often result from a thrombus that blocks an
atherosclerotic coronary artery. A key enzyme in
thrombus formation is the serine protease throm-
bin, which is responsible for both the conversion
of soluble fibrinogen into insoluble fibrin, as well
as the activation of the GPCRs, PAR1 and PAR4,
which stimulate platelet aggregation. Thus, throm-
bin is an attractive target for anticoagulant and
antithrombotic therapy. Previous studies in our
laboratory led to the development of lead com-
pound FM 19 (D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH2),
which shows modest potency as a thrombin inhibi-
tor. The recently determined X-ray structure of FM
19 in the active site of thrombin has revealed
potential sites for modification to improve pot-
ency. This study reports replacements to the first
residue (D-Arg1) of FM 19, which seek to improve
potency by removing the N-terminal amine to elim-
inate an adverse electrostatic interaction, and
alterations to the length of the side chain to elimi-
nate an unfavorable eclipsed conformation obser-
ved in the X-ray structure. This study produced
two compounds, 1 and 9, with improved a-throm-
bin inhibition (IC50 values of 0.66 0.20 lM and
0.57 0.12 lM, respectively).
Bradykinin, a nonapeptide cleavage product of high-molecular-
weight kininogen with the sequence RPPGFSPFR (Arg-Pro-Pro-Gly-
Phe-Ser-Pro-Phe-Arg), increases vascular permeability and has car-
dioprotective effects (6–8). Bradykinin can be further cleaved by
angiotensin-converting enzyme to produce a pentapeptide with the
sequence RPPGF (Arg-Pro-Pro-Gly-Phe) (4). RPPGF has been shown
to inhibit the actions of thrombin in two ways (4). First, RPPGF
binds to the active site of thrombin in a retro-binding fashion. That
is, RPPGF binds to the thrombin active site in an opposite sense (in
terms of linear sequence) compared to thrombin substrates (4). Sec-
ondly, RPPGF has been shown to bind to the thrombin cleavage site
on PAR1, preventing cleavage by thrombin, and therefore inhibiting
platelet activation (4). Thus, peptide RPPGF serves as a starting
point for the development of a direct thrombin inhibitor, as well as
an inhibitor of platelet activation.
SAR studies were carried out to follow-up on RPPGF antithrombot-
ic activity and culminated in the development of lead compound,
FM 19, with the sequence D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH2,
where Oic is ((2S,3aS,7aS)-octahydroindole-2-carboxylic acid)
(Figure 1) (9). Although FM 19 is more potent than RPPGF
(Ki = 4.4 lM versus 1.75 mM), FM 19 is still only a modest inhibi-
tor of thrombin activity (10). The crystal structure of FM 19 in
thrombin's active site has recently been determined, showing that,
like RPPGF, FM 19 is a retrobinder (Figure 2) (10). This crystal
structure provides insight into potential modifications that can be
made to the FM 19 structure to increase binding interactions and
potency. The crystal structure shows that the side chain of the
D-Arg1 residue adopts a high-energy eclipsed conformation, which
places the positively charged guanidino group of the side chain in
close proximity to the N-terminal amine, creating an unfavorable
electrostatic interaction. We report here initial replacements to the
Key words: antithrombotic therapy, peptide synthesis, protease-
activated receptor, structure-based design, thrombin
Received 15 October 2009, revised and accepted for publication 27
October 2009
Acute coronary syndrome (ACS) is a broad term that can refer to
many different disease states, such as unstable angina and myocar-
dial infarction (MI), which is a leading cause of death worldwide
(1,2). MIs occur when blood supply to the heart is interrupted,
which is most commonly caused by the formation of a thrombus
Editor’s invited manuscript to celebrate the 4th Anniversary of Chemical Biology & Drug Design.
35

Girnys et al.
Amino acids and peptides
All Fmoc-protected amino acids (Fmoc-Phe(4-Me)-OH, Fmoc-Oic-OH,
Fmoc-Pro-OH, Fmoc-D-Ala-OHÆH2O and Fmoc-Gly-OH) were pur-
chased from Advanced Chem Tech (Louisville, KY, USA). Boc-7-
aminoheptanoic acid (N-tert-butoxycarbonyl-7-aminoheptanoic acid)
was purchased from Anaspec (San Jose, CA, USA). 4-Aminobutyric
acid and 6-aminohexanoic acid were purchased from Eastman
Kodak Co. (Rochester, NY, USA). 5-Aminovaleric acid and b-alanine
were purchased from Sigma-Aldrich. The p-aminobenzoic acid was
a generous gift from the laboratory of Dr. Scott Larsen, University
of Michigan, College of Pharmacy.
Figure 1: Structure of FM 19 (D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-
NH2
G218
Amine protection
The free amino groups on the side chains of 5-aminovaleric
acid, b-alanine, 4-aminobutyric acid, 6-aminohexanoic acid and
p-aminobenzoic acid were protected before peptide synthesis with
a Boc (N-tert-butoxycarbonyl) group as previously reported (11).
D189
Synthesis of N, N’-di-Boc-4-guanidinobenzoic
acid
N, N '-di-Boc-4-guanidinobenzoic acid was synthesized using a slight
modification of a previously described procedure (12). One equiva-
lent of N, N '-di-Boc- N '-triflylguanidine (Fluka, Buchs, Switzerland)
was dissolved in dioxane in the presence of a threefold excess of
triethylamine, and a 10% excess of p-aminobenzoic acid was
added. After 3 h, an additional twofold excess of triethylamine was
added, and the reaction was stirred for 8–19 days at room temper-
ature. The solution was then concentrated in vacuo and dissolved
in ethyl acetate and H2O. The aqueous layer was removed, acidified
to pH 1.5, and extracted 5 times with ethyl acetate. Saturated NaH-
CO3 was added to the combined organic layers, prompting precipita-
tion of N, N '-di-Boc-4-guanidinobenzoic acid as a white solid. This
product was then used to make compound 9.
A190
Figure 2: Crystal structure of FM 19 in thrombin's active site.
The D-Arg1 residue (yellow) interacts with G218, D189 and A190 of
thrombin (cyan). PDB: 3BV9, 1.80 ꢀ resolution.
D-Arg1 residue of the FM 19 sequence (Figure 3) aimed at elimi-
nating the unfavorable electrostatic interaction, and explore alter-
ing the side-chain length in an attempt to alleviate the eclipsed
conformation. Conversely, we also report an initial application of
conformational restriction to this side chain, to stabilize the
eclipsed conformation.
Materials and Methods
Peptide synthesis
Peptides were synthesized using standard solid phase Fmoc
(fluorenylmethyloxycarbonyl) chemistry on a CS Bio CS336X Peptide
Synthesizer (CS Bio Company, Menlo Park, CA, USA), using previously
described protocols (13). Rink resin (Advanced Chem Tech, Louisville,
KY, USA) was used to produce all peptides as C-terminal amides. A
20% solution of piperidine in NMP was used to remove the Fmoc-
protecting group from the Rink resin linker, and again to remove the
Fmoc-protecting group after each coupling cycle. Coupling was
performed using a fourfold excess of amino acid and a solution of
0.4 M hydroxybenzotriazole (HOBt; Advanced Chem Tech, Louisville,
Reagents
NMP (N-methyl-2-pyrrolidinone) and diisopropylethylamine were pur-
chased from Advanced Chem Tech (Louisville, KY, USA). Piperidine,
trifluoroacetic acid for cleavage and deprotection, dimethylforma-
mide, triethylamine and dioxane were purchased from Sigma-Aldrich
(St Louis, MO, USA). Diethyl ether, ethyl acetate and HPLC grade
solvents (water, acetonitrile and trifluoroacetic acid) were purchased
from Fisher Scientific (Pittsburg, PA, USA). Dichloromethane was
purchased from Acros Organics (Geel, Belgium).
Figure 3: Structures of replace-
ments to the D-Arg1 residue
36
Chem Biol Drug Des 2010; 75: 35–39

D-Arg Replacements to Thrombin Inhibitor FM 19
KY, USA) and O-benzotriazole- N, N, N ', N '-tetramethyl-uronium-
hexafluoro-phosphate (HBTU; Advanced Chem Tech, Louisville, KY,
USA) in dimethylformamide, in the presence of diisopropylethylamine.
After the synthesis was complete, the resin was washed with NMP
and then dichloromethane, and dried. The peptides were cleaved
from the resin and side-chain-protecting groups removed after
treatment for 3–4 h with a cleavage cocktail consisting of 9.5 mL
trifluoroacetic acid and 0.5 mL H2O. The solution was concentrated
in vacuo, and peptides were precipitated using cold diethylether. The
filtered crude material was then purified using a Waters semi-
preparative HPLC (Waters Corporation, Milford, MA, USA) with a
Vydac Protein and Peptide C18 column, using a linear gradient of 0%
or 10% Solvent B (0.1% trifluoroacetic acid in acetonitrile) in Solvent
A (0.1% trifluoroacetic acid in water) to 50% Solvent B in Solvent A,
at a rate of 1% per minute. The identity of each peptide was
determined by mass spectrometry using a Finnigan LCQ mass
spectrometer (ThermoFinnigan, San Jose, CA, USA) in positive mode.
The purity of all final peptides was determined using a Waters
Alliance 2690 Analytical HPLC (Waters Corporation, Milford, MA,
USA) and Vydac Protein and Peptide C18 reverse phase column, using
a linear gradient of 0–70% Solvent B in Solvent A in 70 min.
Thrombin inhibition
Assays to measure the ability of synthetic peptides to inhibit human
a-thrombin's (Haematologic Technologies, Essex Junction, VT, USA)
cleavage of the chromogenic substrate Sar-Pro-Arg-pNA, where
pNA is p-nitroanilide (Bachem, Torrance, CA, USA) were adapted
from a previously described procedure (14). Experiments were car-
ried out in triplicate in 96-well plates, in the presence or absence
of synthetic peptide inhibitors. Controls included no enzyme, no
inhibitor, and lead compound FM 19. Enzyme was preincubated with
varying concentrations of inhibitor in assay buffer (10 mM Tris,
150 mM NaCl, pH 7.6) for 5 min at 37 ꢀC, and the experiment was
started with the addition of substrate. After 10 min, the OD405 was
plotted as a function of the negative log of the inhibitor concentra-
a
tion to generate a dose–response curve using GRAPHPAD PRISM .
From these data, an IC50 value was determined. Each compound
was tested in at least three independent experiments, and the aver-
age and standard deviation are reported.
Results and Discussion
Nine compounds with replacements for the D-Arg1 residue in the
FM 19 sequence were synthesized, based on observations from the
crystal structure (Figure 2). First, the side chain of D-Arg1 adopts a
high-energy, eclipsed conformation. Further, this conformation places
the N-terminal amine in close proximity (3.8 ꢀ) to the positively
charged nitrogen of the guanidino group of the side chain, resulting
in an additional energetic penalty via an unfavorable electrostatic
interaction. Since the guanidino group makes polar interactions with
the side chain of D189 as well as backbone carbonyls of G218 and
A190 (10), a positively charged moiety is desirable on this side
chain. The N-terminal amine, however, does not make any contacts
with thrombin, and so removal of this group (compound 1) would
be expected to eliminate the unfavorable electrostatic clash without
eliminating any favorable interactions and thus result in improved
potency. The length of the side chain was also investigated (2–5)
to potentially eliminate the eclipsed conformation of the side chain.
Additionally, replacement of the guanidino group with an amino
group on the side chain was explored (6–8), while maintaining the
same linker length between the backbone carbonyl carbon of the
residue and the positively charged moiety on the end of the side
chain (guanidino or amino). Finally, one conformationally restricted
analog was synthesized (9), in an attempt to lock in the eclipsed
conformation observed in the X-ray structure. Analytical data for all
nine compounds along with lead compound FM 19 are displayed in
Table 1. Thrombin inhibition data for all nine compounds as well as
lead compound FM 19 are shown in Table 2. Structures for all
replacements are shown in Figure 3.
Guanylated compounds 1–5
Precursors of 1–5 were synthesized first as peptides containing an
amino group in the place of the desired guanidino group. Each
peptide was then cleaved from the resin and had the Boc-protect-
ing group removed, as described earlier. Guanylation of the primary
amine was then carried out similarly as previously described (12).
One equivalent of N, N '-di-Boc-N '-triflylguanidine was dissolved in
dioxane in the presence of a threefold excess of triethylamine, and
a 10% excess of the amine-containing precursor peptide was
added. After 3 h, an additional twofold excess of triethylamine was
added, and the reaction was stirred for another 7–21 h at room
temperature. The solution was then concentrated in vacuo,
dissolved in ethyl acetate and washed twice each with 2 M KHSO4
and saturated NaHCO3. After drying over anhydrous MgSO4, the
ethyl acetate was removed in vacuo, and the remaining residue
was treated at room temperature with a deprotection solution
consisting of 9.5 mL trifluoroacetic acid and 0.5 mL H2O for 3–4 h.
This solution was then concentrated in vacuo, and the peptide was
precipitated with diethylether and filtered. The crude material was
then purified, identified, and final purity was determined as
described earlier.
Compound 9
Compound 9 followed the general peptide synthesis protocol
described earlier for the first four coupling and Fmoc-deprotection
cycles. A twofold excess of N,N'-di-Boc-4-guanidinobenzoic acid
was dissolved in NMP, and to this solution was added a fourfold
excess of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU, Advanced Chem Tech, Louisville, KY)
and a 26-fold excess of diisopropylethylamine. The solution was
then added to the tetrapeptide resin and allowed to shake
overnight. The resin was then rinsed with dichloromethane and
dried, and the peptide cleaved, precipitated and purified as
described earlier.
Compound 1 (5-guanidinopentanoic acid) differs from lead com-
pound FM 19 only in the removal of the N-terminal amine. Thus, its
side chain guanidino group maintains the same distance from the
backbone carbonyl carbon as in the D-Arg1 of FM 19. As seen in
Table 2, compound 1 shows considerable improvement over lead
compound FM 19 (over sixfold increase in potency). Thus, removal
of the N-terminal amine, and consequent elimination of the adverse
electrostatic interaction seen in the X-ray structure of FM 19 bound
to thrombin, does, as predicted, lead to an increase in potency.
Chem Biol Drug Des 2010; 75: 35–39
37

Girnys et al.
Table 1: Analytical data: D-argi-
nine replacements in the FM 19
sequence (X-Oic-Pro-D-Ala-Phe
(p-Me)-NH2)
Analytical HPLC
retention time (min)
Compound
D-Arg replacement (X)
Expected [M + H]
Found [M + H]
FM 19
D-Arg
654.4
639.4
597.3
611.4
625.4
653.4
597.4
611.4
625.4
659.4
654.4
639.4
597.3
611.2
625.3
653.5
597.2
611.4
625.3
659.3
26.5
32.6
30.1
30.1
31.4
33.9
30.6
31.6
33.5
31.6
1
2
3
4
5
6
7
8
9
5-Guanidinopentanoic acid
Guanidinoglycine
3-Guanidinopropionic acid
4-Guanidinobutyric acid
6-Guanidinohexanoic acid
5-Aminopentanoic acid
6-Aminohexanoic acid
7-Aminoheptanoic acid
p-Guanidinobenzoic acid
Table 2: Thrombin inhibition potencies of D-arginine replace-
ments in the FM 19 sequence (X-Oic-Pro-D-Ala-Phe(p-Me)-NH2)
compound 6 (5-aminopentanoic acid). Similarly, compound 4 (4-gua-
nidinobutyric acid) is more than 15 times more potent than
compound 7 (6-aminohexanoic acid). Finally, compound 1 (5-guanidi-
nopentanoic acid) is over 750 times more potent than compound 8
(7-aminoheptanoic acid). The comparison of these pairs of com-
pounds suggests that the guanidino group, and not merely a cat-
ionic moiety, is important for potency. The difference in potency
seen in the last pair of compounds (1 versus 8) is much larger than
that found in the other pairs, and might be attributed to entropic
effects, since these compounds have the longest side chain length.
It is possible that the entropic penalty encountered with constricting
compound 8 into a proper binding orientation is too large so that
the enthalpy gain upon binding is not enough to produce a similarly
potent compound. It has been suggested that delocalization of the
positive charge over more than one atom in a guanidino group
allows for greater polar contacts with the active site D189 than
a point charge on an amino group (15). The results for compounds
6–8 support this suggestion.
Compound
D-Arg replacement (X)
IC50 (lM)
FM 19
D-Arg
4.4 € 1.3
0.66 € 0.20
36.8 € 9.4
6.5 € 0.6
8.4 € 2.0
25.6 € 6.8
241 € 41
130 € 31
498 € 14
0.57 € 0.12
1
2
3
4
5
6
7
8
9
5-Guanidinopentanoic acid
Guanidinoglycine
3-Guanidinopropionic acid
4-Guanidinobutyric acid
6-Guanidinohexanoic acid
5-Aminopentanoic acid
6-Aminohexanoic acid
7-Aminoheptanoic acid
p-Guanidinobenzoic acid
Compounds 2–5 explore the effects of a different linker length,
while still containing a guanidino group on the side chain and elim-
inating the N-terminal amine. Compounds 2 (guanidinoglycine), 3
(3-guanidinopropionic acid) and 4 (4-guanidinobutyric acid) incorpo-
rate a shorter linker, while compound 5 (6-guanidinohexanoic acid)
incorporates a longer one, compared to FM 19. All four analogs
show reduced potency compared to compound 1, with the shortest
(2) and longest (5), side chains displaying lower potency than the
intermediate analogs 3 and 4. Although compound 4 is over 10-
fold less potent than compound 1, it might be expected to be bet-
ter, since its shorter side chain would allow interaction with throm-
bin without the need for an eclipsed conformation. Presumably, the
guanidino group is not positioned correctly in 4 to make optimal
interactions. Compound 3, with a shorter, less flexible side chain
than 4 would be expected to suffer less of an entropy loss upon
binding. However, 3 shows similar inhibitor potency to 4, presum-
ably because the side chain of 3 is too short to perfectly position
the guanidino as in 1, similar to 4. Thus, even though it adopts a
strained eclipsed conformation, it appears as if compound 1 has
the optimal side-chain length of six atoms between the carbonyl
carbon and positively charged guanidino group on the end of the
side chain to provide favorable interaction with the thrombin active
site.
Finally, compound 9 (p-guanidinobenzoic acid) was synthesized in
an attempt to lock in the eclipsed conformation that D-Arg1 of FM
19 adopts upon binding to thrombin's active site. Like compound 1,
compound 9 contains no N-terminal amine and has an equivalent
side chain length, but in addition is conformationally constrained to
adopt an eclipsed orientation. Accordingly, compound 9 might be
expected to be more potent than compound 1. Instead, this com-
pound is equipotent to compound 1. A possible explanation could
be that, while the D-Arg1 side chain in the FM 19 crystal structure
is not completely planar, the p-guanidinobenzoic acid side chain of
compound 9 is. This could cause the guanidino group of compound
9 to point into a slightly different, less optimal position, resulting
in a lower than expected potency. It is also possible that steric
effects because of the remainder of the phenyl ring may lower
binding affinity for thrombin.
Conclusions
Two compounds, 1 and 9, have been synthesized that show a
significant increase in human a-thrombin inhibition over the previ-
ously reported lead compound FM 19. Both compounds eliminate
the N-terminal amine, which improves potency as predicted
from the crystal structure. Both compounds also contain a highly
basic guanidino group that increases potency of this class of
Next, replacement of the guanidino group with an amino group was
investigated, while maintaining the same linker length between the
backbone carbonyl carbon and the positively charged moiety. As
seen in Table 2, compound 3 (3-guanidinopropionic acid) is more
than 30 times more potent than its amino-containing counterpart,
38
Chem Biol Drug Des 2010; 75: 35–39

D-Arg Replacements to Thrombin Inhibitor FM 19
compounds. Compound 9 also adds conformational restriction to
the modified side chain to lock in the eclipsed conformation. Fur-
ther studies are underway that will explore additional conforma-
tional restriction on this side chain, as well as additional
guanidino modifications that maintain basicity as well as delocal-
ization of positive charge.
7. Hasan A.A., Amenta S., Schmaier A.H. (1996) Bradykinin and its
metabolite, Arg-Pro-Pro-Gly-Phe, are selective inhibitors of
a-thrombin-induced platelet activation. Circulation;94:517–528.
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J.O. (2000) Inactivation of bradykinin by angiotensin-converting
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Acknowledgments
10. Nieman M.T., Burke F., Warnock M., Zhou Y., Sweigart J., Chen
A., Ricketts D., Lucchesi B.R., Chen Z., DiCera E., Hilfinger J.,
Kim J.S., Mosberg H.I., Schmaier A.H., (2008) Thrombostatin
FM compounds: direct thrombin inhibitors – mechanism of
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This work was supported by a University of Michigan Rackham
Merit Fellowship to EAG. We would also like to thank Dr Mary
Divin for many helpful discussions. This work is dedicated to the
memory of Edward C. Girnys.
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