Triple-helical transition state analogues: A new class of selective matrix metalloproteinase inhibitors

Article abstract of DOI:10.1021/ja0715849

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen

Full text of DOI:10.1021/ja0715849

Published on Web 08/02/2007
Triple-Helical Transition State Analogues: A New Class of
Selective Matrix Metalloproteinase Inhibitors
Janelle Lauer-Fields,†,‡ Keith Brew,‡ John K. Whitehead,§ Shunzi Li,§
Robert P. Hammer,§ and Gregg B. Fields*,†
Contribution from the Department of Chemistry and Biochemistry and College of Biomedical
Sciences, Florida Atlantic UniVersity, Boca Raton, Florida 33431 and Department of Chemistry,
Louisiana State UniVersity, Baton Rouge, Louisiana 70803
Received March 6, 2007; E-mail: fieldsg@fau.edu
Abstract: Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have
been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of
extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen
triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic
activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition
state analogue has been incorporated within a triple-helical peptide template. The template sequence was
based on the R1(V)436-450 collagen region, which is hydrolyzed at the Gly439-Val440 bond selectively by
MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the
water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide
bond between Gly-Val in the P1-P1′ subsites of the triple-helical peptide. Inhibition studies revealed Ki
values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8
and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-
helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state
analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new
class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime
active site subsites as well as with secondary binding sites (exosites).
Introduction
The first generation of MMP inhibitors were peptidic, broad
spectrum compounds, whereas the second generation were non-
peptidic compounds designed based on MMP active site
structural features.3,7 However, in general, neither generation
of MMP inhibitors were successful in clinic trials. Compounds
either showed no significant therapeutic advantage or had
considerable side effects, such as musculoskeletal syndrome
(MSS).7 One problem was the design of the clinic trials
themselves. MMP inhibitors had been successful in animal
models of early stage disease but were only tested in late-stage
disease in clinic trials.3 There were also concerns over whether
adequate doses of inhibitors were given.2 The lack of selectivity
of the first generation of MMP inhibitors may well have
contributed to MSS.3 In addition, some MMPs have host-
beneficial functions, making them antitargets.2 To date, the vast
majority of MMP inhibitors contain a hydroxamic or carboxylic
Proteolysis has often been cited as an important contributor
to cancer initiation and progression.1 The 565 proteases identi-
fied in humans constitute 1.7% of coding regions in the human
genome.2 The identification and validation of specific proteases
as anticancer targets and the development of appropriate
inhibitors are thus daunting tasks. Alterations in activities of
one family of proteases, the matrix metalloproteinases (MMPs),
have been implicated in primary and metastatic tumor growth,
angiogenesis, and pathological degradation of extracellular
matrix (ECM) components, such as collagen and laminin.3 In
fact, the destruction of collagen by tumor cell extracts was
observed 30 years ago.4 MMP inhibitor programs began in
earnest in the 1980s, using the destruction of ECM components
as a model for inhibitor design.5 Most of these programs
evaluated MMP inhibitors for treatment cancer or other inflam-
matory diseases such as arthritis.6,7
acid group which chelates the active site Zn2+ 5,7-10
However,
.
the hydroxamic or carboxylic acid usually represents a terminal
point in the chain, and thus residues that interact with only one
side of the enzyme active site can be incorporated into the
inhibitor. Hydroxamates may also chelate Zn2+ too strongly,
† Department of Chemistry and Biochemistry, Florida Atlantic University.
‡ College of Biomedical Sciences, Florida Atlantic University.
§ Louisiana State University.
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J. AM. CHEM. SOC. 2007, 129, 10408-10417
10.1021/ja0715849 CCC: $37.00 © 2007 American Chemical Society

Triple-Helical Transition State Analogues
A R T I C L E S
Figure 1. Nomenclature used for enzyme and substrate subsites.87 The arrow marks the site of protease hydrolysis.
Figure 2. Tetrahedral intermediate (boxed) and statine and phosphorus-based transition state analogue inhibitors.
overwhelming contributions (and hence specificity elements)
from the rest of the compound.11,12 This may be why some small
molecule MMP inhibitors bind to other, unrelated metallopro-
teases, such as neprilysin, leucine aminopeptidase, and dipep-
tidylpeptidase.13 An additional concern is that hydroxamates are
known to have unfavorable pharmacokinetics and poor solubili-
ties and may be metabolically activated.7,11,14 Attempts to
produce selective MMP inhibitors have been somewhat thwarted
by flexibility in MMP active site subsites, particularly S1′.7,11
One way to circumvent the selectivity problem is to add
sequence diversity, using an inhibitory molecule that, rather than
terminate a chain, can be incorporated within a chain. This
allows for inhibitor interaction with both the primed and
nonprimed sides of the active site (Figure 1).3,7,11 Additionally,
use of a zinc binding group (ZBG) with lesser affinity than a
hydroxamate may be advantageous.
Two classes of proteases, the aspartyl proteases and the
metallo(zinc)-proteases, use the nucleophilic attack of a water
molecule as one of the steps of amide bond hydrolysis.14 The
tetrahedral intermediate that results from water addition to the
amide carbonyl has been the focus of many protease inhibitor
designs and has given rise to two robust classes of inhibitors,
namely the statines and phosphorus-based amide bond replace-
ments (Figure 2). An effective enzyme substrate almost invari-
ably produces an inhibitor of the enzyme by incorporation of a
statine (mainly for aspartyl proteases) or phosphorus (aspartyl
and Zn2+ proteases) tetrahedral intermediate mimic.
as transition state analogues. Unfortunately, phosphonamidates
tend to be the least stable of the three phosphorus-based
inhibitors. A phosphonate peptide inhibitor has been described
for MMP-8,20,21 but the selectivity was not investigated.
Phosphinic peptides have also been shown to behave as
transition state analogue inhibitors of MMPs.22 Phosphinic
peptide combinatorial libraries have been used to design
selective MMP-12 inhibitors with Ki values below or in the low
nanomolar range.23-25 Phosphinic peptide inhibitors have also
been described for MMP-1, MMP-2, MMP-3, MMP-7, MMP-
8, MMP-9, MMP-11, MMP-13, and MT1-MMP.5,16,22,26-32 The
most effective of these inhibitors had Ki values in the low
nanomolar range but offered varying degrees of selectivity
between the nine aforementioned MMPs.
Phosphinates exhibit weaker binding to Zn2+ than hydrox-
amates and, thus, may limit off target inhibitor binding.12
Density functional theory calculations have indicated that
hypophosphite will bind to the Zn2+ in MMPs in a half-bidentate
(18) Bartlett, P. A.; Marlowe, C. K.; Giannousis, P. P.; Hanson, J. E. Cold Spring
Harbor Symp. Quant. Biol. 1987, 52, 83-90.
(19) Mookhtiar, K. A.; Marlowe, C. K.; Bartlett, P. A.; Van Wart, H. E.
Biochemistry 1987, 26, 1962-1965.
(20) D’Alessio, S.; Gallina, C.; Gavuzzo, E.; Giordano, C.; Gorini, B.; Mazza,
F.; Paglialunga Paradisi, M.; Panini, G.; Pochetti, G.; Sella, A. Bioorg.
Med. Chem. Lett. 1999, 7, 389-394.
(21) Gavuzzo, E.; Pochetti, G.; Mazza, F.; Gallina, C.; Gorini, B.; D’Alessio,
S.; Pieper, M.; Tschesche, H.; Tucker, P. A. J. Med. Chem. 2000, 43, 3377-
3385.
(22) Gall, A. L.; Ruff, M.; Kannan, R.; Cuniasse, P.; Yiotakis, A.; Dive, V.;
Rio, M. C.; Basset, P.; Moras, D. J. Mol. Biol. 2001, 307, 577-586.
(23) Buchardt, J.; Schiodt, C. B.; Krog-Jensen, C.; Delaisse´, J.-M.; Foged, N.
T.; Meldal, M. J. Comb. Chem. 2000, 2, 624-638.
(24) Schiodt, C. B.; Buchardt, J.; Terp, G. E.; Christensen, U.; Brink, M.; Larsen,
Y. B.; Meldal, M.; Foged, N. T. Curr. Med. Chem. 2001, 8, 967-976.
(25) Devel, L.; Rogakos, V.; David, A.; Makaritis, A.; Beau, F.; Cuniasse, P.;
Yiotakis, A.; Dive, V. J. Biol. Chem. 2006, 281, 11152-11160.
(26) Reiter, L. A., et al. Bioorg. Med. Chem. Lett. 1999, 9, 127-132.
(27) Buchardt, J.; Ferreras, M.; Krog-Jensen, C.; Delaisse, J.-M.; Foged, N. T.;
Meldal, M. Chem.sEur. J. 1999, 5, 2877-2884.
There are three general types of phosphorus-based inhibi-
tors: phosphonamidates [Ψ{PO2H-NH}; Y ) NH in Figure
2]; phosphonate esters [Ψ{PO2H-O}; Y ) O in Figure 2]; and
phosphinic peptides/phosphinates [Ψ{PO2H-CH2}; Y ) CH2
in Figure 2]. Phosphonamidates have been shown previously
to inhibit MMP-1,5,15,16 MMP-3,17 and MMP-818,19 by behaving
(28) Vassiliou, S.; Mucha, A.; Cuniasse, P.; Georgiadis, D.; Lucet-Levannier,
K.; Beau, F.; Kannan, R.; Murphy, G.; Knauper, V.; Rio, M. C.; Basset,
P.; Yiotakis, A.; Dive, V. J. Med. Chem. 1999, 42, 2610-2620.
(29) Matziari, M.; Georgiadis, D.; Dive, V.; Yiotakis, A. Org. Lett. 2001, 3,
659-662.
(11) Cuniasse, P.; Devel, L.; Makaritis, A.; Beau, F.; Georgiadis, D.; Matziari,
M.; Yiotakis, A.; Dive, V. Biochimie 2005, 87, 393-402.
(12) Overall, C. M.; Kleifeld, O. Br. J. Cancer 2006, 94, 941-946.
(13) Saghatelian, A.; Jessani, N.; Joseph, A.; Humphrey, M.; Cravatt, B. F. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 10000-10005.
(30) Cursio, R.; Mari, B.; Louis, K.; Rostagno, P.; Saint-Paul, M.-C.; Giudicelli,
J.; Bottero, V.; Anglard, P.; Yiotakis, A.; Dive, V.; Gugenheim, J.;
Auberger, P. FASEB J. 2002, 16, 93-95.
(14) Babine, R. E.; Bender, S. L. Chem. ReV. 1997, 97, 1359-1472.
(15) Kortylewicz, Z. P.; Galardy, R. E. J. Med. Chem. 1990, 33, 263-273.
(16) Galardy, R. E.; Grobelny, D.; Kortylewicz, Z. P.; Poncz, L. Matrix Suppl.
1992, 1, 259-262.
(31) Makaritis, A.; Georgiadis, D.; Dive, V.; Yiotakis, A. Chem.sEur. J. 2003,
9, 2079-2094.
(32) Bianchini, G.; Aschi, M.; Cavicchio, G.; Crucianelli, M.; Preziuso, S.;
Gallina, C.; Nastari, A.; Gavuzzo, E.; Mazza, F. Bioorg. Med. Chem. 2005,
13, 4740-4749.
(17) Izquierdo-Martin, M.; Stein, R. L. Bioorg. Med. Chem. 1993, 1, 19-26.
9
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A R T I C L E S
Lauer-Fields et al.
Figure 3. Synthesis of (R,S)-2-isopropyl-3-((1-(N-(9-fluorenylmethoxycarbonyl)amino)methyl)adamantyloxyphosphinyl) propanoic acid.
fashion; i.e., one oxygen coordinates directly with the Zn2+ while
the other does so partially.33 This prediction is borne out by
X-ray crystallographic analyses of phosphinic peptides bound
to MMPs. For phosphinic acid peptide inhibitors of MMP-1,
MMP-7, and MMP-11, the solvent accessible phosphinate
oxygen has been found to bind to the active site Zn2+ while the
buried phosphinate oxygen is hydrogen bonded to the active
site Glu.14,22,26 The buried phosphinate oxygen also interacts
triazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP),
1-hydroxybenzotriazole (HOBt), Fmoc-amino acid derivatives, and
MMP inhibitor III (a hydroxamic acid-Leu-homoPhe dipeptide) were
obtained from Novabiochem (San Diego, CA). Amino acids are of
L-configuration (except for Gly). Mca-Lys-Pro-Leu-Gly-Leu-Lys(Dnp)-
Ala-Arg-NH2 was synthesized according to methods described previ-
ously.37,38
For the synthesis of the protected Fmoc-GlyΨ{PO2H-CH2}Val
1
building block, intermediates and products were characterized by H,
with the MMP-11 active site Zn2+ 22
Overall, phosphinic acid
.
13C, and 31P NMR spectroscopy, mass spectrometry, and elemental
analysis. Assignment of the NMR signals was accomplished with the
aid of DEPT and HSQC experiments. 1H NMR spectra were recorded
at 250 or 300 MHz. 13C NMR spectra were recorded at 63 or 75 MHz.
31P NMR spectra were recorded at 101 or 202 MHz. 13C and 31P NMR
spectra were fully proton decoupled. Chemical shifts were expressed
peptides have been shown to be good analogues of the H2O-
bound tetrahedral transition state upon binding to MMPs.22
The present study has utilized the phosphinate tetrahedral
intermediate mimic to create a potentially selective MMP
inhibitor. The phosphinate moiety has been incorporated within
a triple-helical peptide template. We had previously described
a triple-helical substrate that was selective for MMP-2 and
MMP-9.34 The substrate sequence was based on residues 436-
450 from R1(V) collagen, where the Gly439-Val440 bond is
cleaved. To create an MMP-2/MMP-9 inhibitor, a 9-fluorenyl-
methoxycarbonyl (Fmoc)-GlyΨ{PO2H-CH2}Val building block
was synthesized (Figure 3) and used in the assembly of a triple-
helical collagen mimic sequence. Selectivity of the inhibitor may
originate from both the sequence and triple-helical structure,
as these elements were found to play a role in the selectivity of
the substrate.34 To evaluate the role of triple-helicity in MMP
inhibition, activity has been examined as a function of triple-
helical content. This has required the use of “peptide-am-
phiphiles,” in which the thermal stability of the triple-helix is
enhanced by pseudo-lipids attached to the N-terminus of the
peptide.35,36
1
in parts per million (δ) relative to tetramethylsilane for H NMR and
d6-DMSO or CDCl3 for 13C NMR. 31P-chemical shifts were reported
downfield from 85% H3PO4. Electrospray mass spectrometry (ES-MS)
was performed on a Hitachi MS-8000 3DQ LC-ion trap mass
spectrometer by the LSU Department of Chemistry Mass Spectrometry
Facility. Elemental analyses were obtained from M-H-W Laboratories
(Phoenix, AZ).
Synthesis of (R,S)-2-Isopropyl-3-((1-(N-(Fmoc)amino)methyl)-
adamantyloxyphosphinyl) Propanoic Acid (1). The synthesis of the
protected Fmoc-GlyΨ{PO2H-CH2}Val building block (1) is outlined
in Figure 3. The procedure follows the strategy devised by Yiotakis
and co-workers,39 which requires a Michael addition of the Arbuzov-
like nucleophile, 1-(N-(9-Fmoc)amino)methyl phosphinic acid (5), to
the acceptor, 2-isopropylacrylic acid allyl ester (6).
1-(N-(9-Fmoc)amino)methyl phosphinic acid (5) was prepared as
recently described.40 Briefly, the phosphinate analogue of Gly, 1-ami-
nomethylphosphinic acid (4), was prepared from the slow addition of
N-tritylmethanimine (3) to the in situ formed bistrimethylsilylphosphinic
acid, followed by hydrolysis to cleave the trityl group.41 Next, following
a modification of the Bolin procedure for amino acid protection,40 amino
acid (4) was silylated with trimethylsilyl chloride (TMS-Cl) and N,N-
Materials and Methods
All standard chemicals were peptide synthesis or molecular biology
grade and purchased from Fisher Scientific. O-(Benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), (7-azabenzo-
(37) Nagase, H.; Fields, C. G.; Fields, G. B. J. Biol. Chem. 1994, 269, 20952-
(33) Cheng, F.; Zhang, R.; Luo, X.; Shen, J.; Li, X.; Gu, J.; Zhu, W.; Shen, J.;
Sagi, I.; Ji, R.; Chen, K.; Jiang, H. J. Phys. Chem. B 2002, 106, 4552-
4559.
20957.
(38) Neumann, U.; Kubota, H.; Frei, K.; Ganu, V.; Leppert, D. Anal. Biochem.
2004, 328, 166-173.
(34) Lauer-Fields, J. L.; Sritharan, T.; Stack, M. S.; Nagase, H.; Fields, G. B.
J. Biol. Chem. 2003, 278, 18140-18145.
(39) Georgiadis, D.; Matziari, M.; Yiotakis, A. Tetrahedron 2001, 57, 3471-
3478.
(35) Yu, Y.-C.; Tirrell, M.; Fields, G. B. J. Am. Chem. Soc. 1998, 120, 9979-
(40) Li, S.; Whitehead, J. K.; Hammer, R. P. J. Org. Chem. 2007, 72, 3116-
9987.
3118.
(36) Minond, D.; Lauer-Fields, J. L.; Nagase, H.; Fields, G. B. Biochemistry
2004, 43, 11474-11481.
(41) Jiao, X. Y.; Verbruggen, C.; Borloo, M.; Bollaert, W.; Groot, A. D.;
Dommisse, R.; Haemers, A. Synthesis 1994, 23-24.
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Triple-Helical Transition State Analogues
A R T I C L E S
diisopropylethylamine (DIEA) in CH2Cl2 and then treated with Fmoc-
solvents were removed in Vacuo, and the residue was treated with
diethylether. The silver bromide and the excess of silver oxide were
removed by filtration through Celite. The filtrate was concentrated to
dryness, and the residue was purified by column chromatography (98:2
chloroform/2-propanol) to give (R,S)-2-isopropyl-3-((1-(N-(Fmoc)-
amino)methyl)adamantyloxyphosphinyl) propanoic acid, allyl ester (8)
(451 mg) in 97% yield. 1H (250 MHz, CDCl3) δ 7.8 (m, 3H), 7.6 (m,
2H), 7.4 (m, 6H), 5.9 (m, 1H), 5.3 (m, 2H), 4.6 (m, 2H), 4.4 (m, 2H),
4.2 (m, 1H), 3.6 (m, 2H), 2.1 (m, 15H), 1.7 (m, 17H), 1.2 (m, 1H), 0.9
(m, 6H); 13C (63 MHz, CDCl3) δ 173.9, 173.7, 156.2, 147.1, 146.8,
141.2, 140.6, 140.5, 132.0, 127.7, 127.4, 127.0, 125.3, 125.1, 124.8,
119.9, 119.8, 119.7, 118.6, 83.5, 83.3, 68.1, 67.3, 67.1, 65.4, 64.3, 47.1,
Cl to yield 1-(N-(9-Fmoc)amino)methyl phosphinic acid (5).28
2-Isopropylacrylic acid allyl ester (6) was prepared as follows.42 To
a suspension of t-BuOK (8.5 g, 72.0 mmol) in THF (200 mL) was
added allyl acetoacetate (9.9 mL, 70.4 mmol) at 0 °C. The resulting
clear solution was stirred for 30 min, and then iodopropane (7.9 mL,
77.7 mmol) was added to the solution. The solution was stirred at 70
°C for 12 h. The reaction was quenched with water, and then a saturated
aqueous sodium bicarbonate solution was added. The aqueous layer
was extracted with diethyl ether (3 × 100 mL). The combined organic
extracts were dried over MgSO4, filtered, and concentrated to give a
yellow oil. The crude product was purified by silica gel column
chromatography (1:5 EtOAc/hexanes) to give 2-isopropyl-3-oxobutyric
45.3, 44.3, 41.5, 40.9, 36.0, 35.6, 31.1, 30.7, 25.3, 19.7, 19.4, 19.3; 31
P
1
(101 MHz, CDCl3) δ 47.9, 47.4, 46.5, 46.3 (Supporting Information,
Figures S11-S13); [M + H]+ 606.0 Da (calculated 606.3 Da), [M +
Na]+ 628.3 (calculated 628.3).
acid allyl ester (2) (9.4 g) in 72% yield. H (250 MHz, CDCl3) δ 5.9
(m, 1H), 5.3 (m, 2H), 4.6 (d, J ) 5.7 Hz, 2H), 3.2 (d, J ) 9.5 Hz, 1H),
2.4 (m, 1H), 2.2 (s, 3H), 1.0 (dd, J ) 6.7 Hz, 11.0 Hz, 3H each); 13C
(63 MHz, CDCl3) δ 202.4, 168.4, 131.3, 118.4, 67.0, 65.2, 28.8, 28.3,
20.1, 20.0 (Supporting Information, Figures S4 and S5); [M + H]+
184.1 Da.
Removal of the allyl ester proceeded as follows:43 [CpRu(CH3CN)3]-
PF6 (8.7 mg, 0.02 mmol), quinaldic acid (3.5 mg, 0.02 mmol), and
ethanol (3 mL) were placed in a 10 mL two-neck round-bottom flask
under an argon stream. After standing for 30 min at 25 °C, the reddish
brown solution was transferred into a 25 mL two-neck round-bottom
flask containing (R,S)-2-isopropyl-3-((1-(N-(Fmoc)amino)methyl)ada-
mantyloxyphosphinyl) propanoic acid, allyl ester (8) (1.0 g, 1.65 mmol),
and ethanol (15 mL). The brown solution was stirred for 12 h at 70
°C. The reaction mixture was concentrated under reduced pressure to
give a crude product. This was purified by column chromatography
(95:5 chloroform/2-propanol) to give (R,S)-2-isopropyl-3-((1-(N-(Fmoc)-
amino)methyl)adamantyloxyphosphinyl) propanoic acid (1) (332 mg)
in 35% yield. 1H (250 MHz, CD3SOCD3) δ 7.9 (m, 2H), 7.7 (m, 2H),
7.4 (m, 4H), 4.3 (m, 3H), 2.0 (m, 16H), 1.5 (m, 11H), 1.2 (m, 1H), 0.8
(m, 7H); 13C (75 MHz, CD3SOCD3) δ 174.8, 174.7, 156.2, 143.7, 140.7,
133.3, 131.9, 131.5, 131.4, 128.7, 128.6, 127.6, 126.9, 125.1, 120.1,
115.3, 111.5, 80.9, 80.8, 79.1, 65.9, 46.6, 45.2, 43.4, 35.8, 35.3, 30.4,
29.9, 19.3; 31P (101 MHz, CD3SOCD3): δ 47.7, 47.4 (Supporting
Information, Figures S1-S3); [M + H]+ 567.0 Da (calculated 566.3
Da), [M + Na]+ 588.2 (calculated 588.3). Elemental analysis for C32H40-
NO6P: C, 66.75% (calculated 67.95%); H, 7.05% (calculated 7.13%);
N, 2.34% (calculated 2.48%).
Peptide Synthesis. Peptide-resin assembly of the triple-helical
peptide (THP) [(Gly-Pro-Hyp)4-Gly-Pro-Pro-GlyΨ{PO2H-CH2}Val-
Val-Gly-Glu-Gln-Gly-Glu-Gln-Gly-Pro-Pro-(Gly-Pro-Hyp)4-NH2] was
performed by Fmoc solid-phase methodology44 in continuous-flow
mode on an Applied Biosystems Pioneer Peptide Synthesizer. The
peptide was synthesized as a C-terminal amide using Fmoc-PAL-PEG-
PS resin (0.16 mmol/g initial loading) to prevent diketopiperazine
formation.45 Standard Fmoc-amino acid coupling utilized 4 equiv each
of amino acid, TBTU, and HOBt (0.25 M final concentration of each)
dissolved in 0.5 M DIEA in DMF for 1 h. For difficult couplings (Pro14,
Pro26, and Hyp39), 4 equiv of each amino acid and PyAOP (0.25 M
final concentration of each) were dissolved in 0.5 M DIEA in DMF
and double-coupled for 1 h each time. For addition of (R,S)-2-isopropyl-
3-((1-(N-(Fmoc)amino)methyl)adamantyloxyphosphinyl) propanoic acid
(1), 2 equiv of 1 and PyAOP (0.25 M final concentration of each) were
dissolved in 0.5 M DIEA in DMF and coupled in a shaker for 12 h.
Capping was performed following the coupling of 1 by addition of
acetic anhydride (Ac2O)-DIEA-DMF (0.5:0.6:8.9). Washings between
reactions were carried out with DMF. Fmoc group removal was
achieved with piperidine-1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-
DMF (1:1:48) for 5 min. A portion of the peptide-resin was lipidated
with 20 equiv of hexanoic acid [CH3(CH2)4CO2H, designated C6] and
To a stirred solution of 2-isopropyl-3-oxobutyric acid allyl ester (2)
(4.61 g, 25.0 mmol) in THF (170 mL) was added lithium hexameth-
yldisilazide (LiHMDS) (27.5 mL, 27.5 mmol, and 1.0 M solution in
THF) at -78 °C. The solution was stirred for 30 min, and then
paraformaldehyde (3.5 g, excess) was added as a solid in one portion.
The resulting suspension was stirred at room temperature for 12 h and
then filtered through Celite to remove the excess paraformaldehyde.
The filtrate was concentrated, and the residue was purified by column
chromatography (1:9 EtOAc/hexanes) to give 2-isopropylacrylic acid
1
allyl ester (6) (2.7 g) in 69% yield. H (300 MHz, CDCl3) δ 6.2 (s,
1H), 6.0 (m, 1H), 5.5 (s, 1H), 5.3 (m, 2H), 4.7 (m, 2H), 2.8 (m, 1H),
1.1 (d, J ) 6.8 Hz, 6H); 13C (75 MHz, CDCl3) δ 166.7, 146.9, 132.2,
121.7, 117.7, 65.0, 29.2, 21.6 (Supporting Information, Figures S6 and
S7); [M + H]+ 154.1 Da.
Condensation of 1-(N-(9-Fmoc)amino)methyl phosphinic acid (5)
and 2-isopropylacrylic acid allyl ester (6) proceeded as follows.39 To
an ice cold suspension of 5 (532 mg, 1.68 mmol) in CH2Cl2 (8 mL),
DIEA (0.94 mL, 5.38 mmol) and TMS-Cl (0.68 mL, 5.38 mmol) were
added under an argon atmosphere. This solution was stirred for 3 h at
room temperature. Then, the mixture was cooled to 0 °C, and compound
6 (310 mg, 2.02 mmol) was added dropwise for 30 min. When the
addition was over, the solution was stirred for 36 h at 40 °C. Then,
absolute ethanol (2 mL) was added dropwise, and the mixture was
stirred for 15 min. The solvent was evaporated. To the residue H2O
was added, and the resulting suspension was acidified with 1 M HCl
to pH 1 and extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were dried over Na2SO4 and concentrated to dryness.
The oily residue was purified by column chromatography (7:0.4:0.4
chloroform/methanol/acetic acid) to give (R,S)-2-isopropyl-3-((1-(N-
(Fmoc)amino)methyl)phosphinic acid) propanoic acid allyl ester (7)
1
(423 mg) in 54% yield. H (300 MHz, CD3SOCD3) δ 7.9 (m, 11H),
7.7 (m, 6H), 7.4 (m, 15H), 5.9 (m, 1H), 5.3 (m, 2H), 4.5 (d, J ) 5.6
Hz, 2H), 4.3 (m, 8H), 2.0 (m, 6H), 0.8 (d, J ) 5.1 Hz, 6H); 13C (75
MHz, CD3SOCD3) δ 173.1, 170.2, 156.3, 147.4, 143.7, 140.6, 139.9,
132.7, 127.5, 127.0, 125.4, 125.2, 120.0, 119.7, 117.7, 65.8, 64.4, 59.7,
46.6, 44.7, 41.5, 32.3, 31.2, 27.4, 26.2, 20.7, 19.6, 19.1, 14.0; 31P (101
MHz, CD3SOCD3) δ 44.0, 43.8 (Supporting Information, Figures S8-
S10); [M + H]+ 472.2 Da (calculated 472.2 Da), [M + Na]+ 494.5
(calculated 494.2).
Phosphinate dipeptide (7) (360 mg, 0.77 mmol) and 1-adamantyl
bromide (199 mg, 0.92 mmol) were dissolved in chloroform (10 mL),
and the reaction mixture was refluxed. To this refluxing mixture silver
oxide (214 mg, 0.92 mmol) was added in five equal portions over 50
min. The reaction mixture was refluxed for an additional 2 h. Then the
(43) Tanaka, S.; Saburi, H.; Ishibashi, Y.; Kitamura, M. Org. Lett. 2004, 6,
1873-1875.
(44) Lauer-Fields, J. L.; Broder, T.; Sritharan, T.; Nagase, H.; Fields, G. B.
Biochemistry 2001, 40, 5795-5803.
(45) Fields, G. B.; Lauer-Fields, J. L.; Liu, R.-q.; Barany, G. In Synthetic
Peptides: A User’s Guide, 2nd Edition; Grant, G. A., Ed.; W.H. Freeman
& Co.: New York, 2001; pp 93-219.
(42) Lee, H.-S.; Park, J.-S.; Kim, B. M.; Gellman, S. H. J. Org. Chem. 2003,
68, 1575-1578.
9
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A R T I C L E S
Lauer-Fields et al.
PyAOP (0.25 M final concentration each) dissolved in 0.5 M DIEA in
DMF for 12 h to create a peptide-amphiphile.35,46 Cleavage and side-
chain deprotection of the peptide-resin and peptide-amphiphile-resin
proceeded for 2 h using 10 mL of triisopropylsilane-phenol-water-
trifluoroacetic acid (TFA) (2:5:5:88),47 followed by filtration of the resin.
The resin was rinsed with 2 mL of TFA, and the combined filtrates
were reduced under pressure at 35 °C to half the original volume. The
remaining filtrate was precipitated by dropwise addition into a 10-fold
excess of diethylether. The peptide and peptide-amphiphile were
allowed to precipitate at -27 °C for 24 h. The peptide/ether solution
was centrifuged, and the resulting pellet was washed 3 times with
diethylether and dried for 4 h under vacuum.
Peptide Analyses. Analytical RP-HPLC was performed on a Waters
600E multisolvent delivery system with a model 486 tunable dectector
controlled by Empower Software, equipped with a Vydac C18 RP
column (15 mm particle size, 300 Å pore size, 100 mm × 8 mm).
Eluants were 0.1% TFA in water (A) and 0.1% TFA in acetonitrile
(B). The elution gradient was 15-35% B in 60 min with a flow rate
of 1.0 mL/min. Detection was at λ ) 220 nm. Fractions from analytical
RP-HPLC were analyzed by matrix-assisted laser desorption-ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) on a Bruker
Proflex III instrument with XMASS software. Mass values were as
follows: f1, [M + H]+ 3579.2 Da (theoretical 3577.9 Da); f2, [M +
H]+ 3579.2 Da (theoretical 3577.9 Da); f3, [M + H]+ 3673.4 Da
(theoretical 3676.1 Da); and f4, [M + H]+ 3673.4 Da (theoretical 3676.1
Da). For size-exclusion chromatography (SEC), peptides were analyzed
in TSB (50 mM Tris‚HCl, pH 7.5 containing 100 mM NaCl, 10 mM
CaCl2, 0.05% Brij-35, pH 7.5) on an AKTA Purifier (GE Biosciences)
equipped with a Superdex 75 (GE Biosciences) column at a flow rate
of 1 mL/min. Both solvent and column were maintained at the
appropriate temperature, either 10 or 37 °C, for at least 2 h prior to
injection. Retention times of standard proteins did not vary with changes
in temperature.
previously.52,53 ProMMP-1 was activated by reacting with 1 mM
4-aminophenyl mercuric acetate (APMA) and 0.1 equiv of MMP-
3(∆248-460) at 37 °C for 6 h. After activation, MMP-3(∆248-460) was
completely removed from MMP-1 by affinity chromatography using
an anti-MMP-3 IgG Affi-Gel 10 column. ProMMP-3 was activated by
reacting with 5 µg/mL chymotrypsin at 37 °C for 2 h. Chymotrypsin
was inactivated with 2 mM diisopropylfluorophosphate. ProMMP-2
was purified from the culture medium of human uterine cervical
fibroblasts54 and activated by incubating with 1 mM APMA for 2 h at
37 °C. ProMMP-8 was expressed in CHO-K1 cells as described
previously.55 ProMMP-8 was activated by incubating with 1 mM APMA
for 2 h at 37 °C. Recombinant proMMP-9 was purchased from
Chemicon International (Temecula, CA) and activated with 1 mM
APMA for 1 h at 37 °C. Recombinant proMMP-13 was purchased from
R&D Systems (Minneapolis, MN) and activated by incubating with 1
mM APMA for 2 h at 37 °C. The concentrations of active MMP-1,
MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13 were determined by
titration with recombinant TIMP-1 or N-TIMP-1 over a concentration
range of 0.1-3 µg/mL.56 Recombinant MT1-MMP with the linker and
C-terminal hemopexin-like domains deleted [residues 279-523; des-
ignated MT1-MMP(∆279-523)] was purchased from Chemicon. MT1-
MMP(∆279-523) was expressed and activated, resulting in Tyr112 at
the N-terminus. MT1-MMP(∆279-523), which, in contrast to MT1-MMP,
does not undergo rapid autoproteolysis, was used in the present studies
due to the relatively small differences in MT1-MMP(∆279-523) and MT1-
MMP triple-helical peptidase activities noted previously.57 The con-
centration of active MT1-MMP(∆279-523) was determined by titration
with recombinant N-TIMP-3.57 ProMMP-3(∆248-460) was expressed in
E. coli using the expression vector pET3a (Novagen), folded from
inclusion bodies and purified as described previously.58 The zymogen
was activated as described above for the full-length proMMP-3. Active
site titrations utilized either Mca-Lys-Pro-Leu-Gly-Leu-Lys(Dnp)-Ala-
Arg-NH2 or NFF-3 as substrate.37,38,59
Peptide Purification. Semipreparative RP-HPLC was performed on
a Waters Deltaprep System using a Vydac C18 RP column (15 µm
particle size, 300 Å pore size, 200 mm × 25 mm) at a flow rate of
10.0 mL/min. Eluants were 0.1% TFA in water (A) and 0.1% TFA in
acetonitrile (B). The elution gradient was 10-30% B in 80 min, with
detection at λ ) 220 nm. Isolated fractions were analyzed by PDA
RP-HPLC. PDA RP-HPLC was performed on a Waters 625 pump with
a model 996 diode array detector controlled by Millennium software,
using a Vydac C18 RP column (5 mm particle size, 120 Å pore size,
250 mm × 4.6 mm) at a flow rate of 1.0 mL/min. Eluants were 0.1%
TFA in water (A) and 0.1% TFA in acetonitrile (B). The elution gradient
was 10-30% B in 20 min, with detection at λ ) 200-400 nm.
Circular Dichroism (CD) Spectroscopy. CD spectra were recorded
over the range λ ) 190-250 nm on a JASCO J-810 spectropolarimeter
using a 0.1 cm path length quartz cell. Thermal transition curves were
obtained by recording the molar ellipticity ([Θ]) at λ ) 222 nm while
the temperature was continuously increased in the range 5-95 °C at a
rate of 0.2 °C/min. Temperature was controlled using a JASCO PFD-
425S temperature control unit. For samples exhibiting sigmoidal melting
curves, the inflection point in the transition region (first derivative) is
defined as the melting temperature (Tm). To compare transitions for
different triple-helical species, the highest [Θ]222 nm value was designated
as 100% folded and the lowest [Θ]222 nm value was designated as 0%
folded.48-51
Inhibition Kinetic Studies. Peptide substrates and inhibitors were
dissolved in TSB buffer. 1-2 nM enzyme was incubated with varying
concentrations of inhibitors for 2 h at room temperature. A 2 h
incubation was utilized based on the generally observed behavior of
slow on and off rates for tight-binding inhibitors60 and studies
demonstrating that high affinity phosphinate inhibitors of Zn2+ met-
alloproteinases are slow binding.61 Residual enzyme activity was
monitored by adding 0.1 volume of Mca-Lys-Pro-Leu-Gly-Leu-Lys-
(Dnp)-Ala-Arg-NH2 to produce a final concentration of <0.1 KM. Initial
velocity rates were determined from the first 20 min of hydrolysis when
product release is linear with time. Fluorescence was measured on a
Molecular Devices SPECTRAmax Gemini EM Dual-Scanning Micro-
plate Spectrofluorimeter using λexcitation ) 324 nm and λemission ) 393
nm. Apparent Ki values were calculated from the following formulas:
Vi/Vo ) {Et - It - Ki(app) + ((Et - It - Ki(app))2 + 4Et K(iapp) 0.5
Ki(app) ) Ki({At + KM}/KM)
)
}/2Et
(1)
(2)
where It is the total inhibitor concentration, Et is the total enzyme
concentration, At is the total substrate concentration, Vo is the activity
(51) Persikov, A. V.; Xu, Y.; Brodsky, B. Protein Sci. 2004, 13, 893-902.
(52) Chung, L.; Shimokawa, K.; Dinakarpandian, D.; Grams, F.; Fields, G. B.;
Nagase, H. J. Biol. Chem. 2000, 275, 29610-29617.
Matrix Metalloproteinases. ProMMP-1 and proMMP-3 were
expressed in E. coli and folded from the inclusion bodies as described
(53) Lauer-Fields, J. L.; Nagase, H.; Fields, G. B. J. Biomolecular Techniques
2004, 15, 305-316.
(46) Yu, Y.-C.; Berndt, P.; Tirrell, M.; Fields, G. B. J. Am. Chem. Soc. 1996,
118, 12515-12520.
(54) Itoh, Y.; Binner, S.; Nagase, H. Biochem. J. 1995, 308, 645-651.
(55) Pelman, G. R.; Morrison, C. J.; Overall, C. M. J. Biol. Chem. 2005, 280,
2370-2377.
(47) Sole´, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403.
(48) Beck, K.; Chan, V. C.; Shenoy, N.; Kirkpatrick, A.; Ramshaw, J. A. M.;
Brodsky, B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4273-4278.
(49) Persikov, A. V.; Ramshaw, J. A. M.; Brodsky, B. Biopolymers (Peptide
Sci.) 2000, 55, 436-450.
(56) Huang, W.; Suzuki, K.; Nagase, H.; Arumugam, S.; Van Doren, S.; Brew,
K. FEBS Lett. 1996, 384, 155-161.
(57) Hurst, D. R.; Schwartz, M. A.; Ghaffari, M. A.; Jin, Y.; Tschesche, H.;
Fields, G. B.; Sang, Q.-X. A. Biochem. J. 2004, 377, 775-779.
(58) Suzuki, K.; Kan, C.-C.; Huang, W.; Gehring, M. R.; Brew, K.; Nagase, H.
Biol. Chem. 1998, 379, 185-191.
(50) Persikov, A. V.; Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B.
Biochemistry 2000, 39, 14960-14967.
9
10412 J. AM. CHEM. SOC. VOL. 129, NO. 34, 2007

Triple-Helical Transition State Analogues
A R T I C L E S
in the absence of inhibitor, and KM is the Michaelis constant. In our
assays the value of Et/K(app) does not exceed 100 so that the inhibitor
i
is distributed in both free and bound forms, and K(iapp) can be
calculated by fitting inhibition data to eq 1. Because the substrate
concentration is less than KM/10, K(app) values are insignificantly
i
different from true Ki values. In cases where weak inhibition occurred,
K(iapp) values were calculated using Vi ) Vo/(1 + It/K(app)).
i
Results
Synthesis and Characterization of Triple-Helical Transi-
tion State Analogues. The type V collagen-derived sequence
Gly-Pro-Pro-Gly439-Val440-Val-Gly-Glu-Gln, when incorporated
into a triple-helical structure, is hydrolyzed efficiently by
MMP-2 and MMP-9 but not by MMP-1, MMP-3, MMP-13, or
MT1-MMP.34 The sequence was thus used as a template for
the design of a potentially selective MMP inhibitor. In order to
create the desired phosphinate transition state analogue, a GlyΨ-
{PO2H-CH2}Val building block needed to be synthesized. A
conjugate addition of 1-(N-(9-Fmoc)amino)methyl phosphinic
acid (5) to 2-isopropylacrylic acid allyl ester (6) was required
to prepare the building block (Figure 3). N-Protected R-ami-
nophosphinic acids are necessary intermediates in the synthesis
of a variety of transition state analogues such as phosphinate,
phosphonate, and phosphonamide dipeptides.23 The Fmoc
protecting group is desirable for peptide synthesis, but until
recently, there was no facile process reported for its incorpora-
tion onto R-aminophosphinic acids. The previous methods using
aqueous/organic solvent mixtures and classic Schotten-Bau-
mann conditions are successful62 but are not generally high
yielding due to hydrolysis side reactions and solubility problems.
An in situ silylation procedure popularized for Fmoc derivati-
zation of L-amino acids63 was utilized for protection of R-ami-
nophosphinic acid with an approximate yield of 80%.40
Mild silylation of Fmoc-aminophosphinic acid with TMS-
Cl and DIEA gave the trivalent phosphinate, which reacted
readily in an Arbusov-like reaction with allyl acrylate 664 to
give the phosphinic acid 7. Finally, the phosphinate dipeptide
analogue was protected as the adamantyl ester by reaction with
silver oxide and 1-adamantyl bromide, and the terminal allyl
ester was removed by CpRu(CH3CN)3PF6 catalysis to give the
protected phosphinate dipeptide mimic 1 [(R,S)-2-isopropyl-3-
((1-(N-(Fmoc)amino)methyl)adamantyloxyphosphinyl) propano-
ic acid]. The use of allyl protection for 2-isopropylacrylic acid,
and removal of the allyl group in the presence of the Fmoc
group, allowed for a more convenient preparation of the Fmoc-
phosphinate dipeptide building block than reported previ-
ously.27,29,39,65 The adamantyl ester in compound 1 does not
prematurely hydrolyze, but it is readily removed under TFA
cleavage conditions used in Fmoc solid-phase strategies.66,67
Figure 4. Semipreparative RP-HPLC analysis of (a) phosphinate triple-
helical peptide and (b) phosphinate triple-helical peptide-amphiphile.
Semipreparative RP-HPLC was performed using a C18 RP column, an
elution gradient of 10-30% B in 80 min, and a flow rate of 10.0 mL/min.
Eluants were 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B).
Detection was at λ ) 220 nm. Other conditions are given in Materials and
Methods.
(R,S)-2-Isopropyl-3-((1-(N-(Fmoc)amino)methyl)adamantyl-
oxyphosphinyl) propanoic acid was incorporated by solid-phase
methods to create (Gly-Pro-Hyp)4-Gly-Pro-Pro-GlyΨ{PO2H-
CH2}(R,S)Val-Val-Gly-Glu-Gln-Gly-Glu-Gln-Gly-Pro-Pro-(Gly-
Pro-Hyp)4-NH2. A portion of the peptidyl-resin was lipidated
on the N-terminus with hexanoic acid to create a peptide-
amphiphile construct.
RP-HPLC analysis of the triple-helical peptide and peptide-
amphiphile revealed two major peaks in each chromatogram
(Figure 4). The material in the major peaks (f1-f4) was isolated
and analyzed by RP-HPLC (Figure 5) and MALDI-TOF-MS.
Peaks f1 and f2 had masses that corresponded to the desired
triple-helical peptide, while f3 and f4 had masses that cor-
responded to the desired triple-helical peptide-amphiphile. Thus,
it appeared that in each case that the diastereomers, where the
Val analogue in the P1′ subsite was either the S or R
configuration, could be separated. Based on these data and
relative inhibitory activities (see later discussion), we hypoth-
esize that f1 and f3 contain the S configuration in the P1′ position
(which is equivalent to an L-amino acid) and that f2 and f4
contain the R configuration in the P1′ position (which is
equivalent to a D-amino acid). Prior studies on phosphinate
peptide inhibitors of MMPs indicated that the earlier eluting
(59) Knight, C. G.; Willenbrock, F.; Murphy, G. FEBS Lett. 1992, 296, 263-
266.
(60) Copeland, R. A. In EValuation of Enzyme Inhibitors in Drug DiscoVery;
Copeland, R. A., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2005; pp
178-213.
(61) Yiallouros, I.; Vassiliou, S.; Yiotakis, A.; Zwilling, R.; Sto¨cker, W. Biochem.
J. 1998, 331, 375-379.
(62) Dumy, P.; Escale, R.; Girard, J. P.; Parello, J.; Vidal, J. P. Synthesis 1992,
1226-1228.
(63) Bolin, D. R. Int. J. Pept. Protein Res. 1989, 33, 353-359.
(64) Huntington, K. M.; Yi, T.; Wei, Y.; Pei, D. Biochemistry 2000, 39, 4543-
4551.
(65) Matziari, M.; Yiotakis, A. Org. Lett. 2005, 7, 4049-4052.
(66) Yiotakis, A.; Vassiliou, S.; Jiracek, J.; Dive, V. J. Org. Chem. 1996, 61,
6601-6605.
(67) Georgiadis, D.; Dive, V.; Yiotakis, A. J. Org. Chem. 2001, 66, 6604-
6610.
9
J. AM. CHEM. SOC. VOL. 129, NO. 34, 2007 10413

A R T I C L E S
Lauer-Fields et al.
Figure 5. PDA RP-HPLC analysis of isolated (a) f1, (b) f2, (c) f3, and (d) f4 products (see designations in Figure 4). PDA RP-HPLC was performed using
a C18 RP column, an elution gradient of 10-30% B in 20 min, and a flow rate of 1.0 mL/min. Eluants were 0.1% TFA in water (A) and 0.1% TFA in
acetonitrile (B). Other conditions are given in Materials and Methods.
Figure 6. CD spectra of purified f1 (blue), f2 (green), f3 (yellow), f4 (orange), and the R1(V)436-450 THP substrate (red) in 0.25% (v/v) TSB buffer at
a substrate concentration of 25 µM. Molar ellipticity ([θ]) values are in units of deg‚cm2‚dmol-1 × 10-3
.
component by RP-HPLC contains the S configuration in the
P1′ position and is the more potent inhibitor.22,25,27,31
CD spectra indicated weak triple-helices for f1 and f2 and a
more pronounced triple-helical structure for f3 and f4 (Figure
6). To examine the thermal stability of each construct, the molar
ellipticity ([Θ]) at λ ) 225 nm was monitored as a function of
increasing temperature. All structures exhibited cooperative
transitions, indicative of the melting of a triple-helix to a single-
stranded structure (Figure 7). Melting temperatures (Tm values)
were then determined for f1-f4. The f1 and f2 peptides had
9
10414 J. AM. CHEM. SOC. VOL. 129, NO. 34, 2007

Triple-Helical Transition State Analogues
A R T I C L E S
Figure 7. Thermal transition curve for purified f1 (blue diamonds), f2 (green diamonds), f3 (yellow diamonds), f4 (orange diamonds), and the R1(V)-
436-450 THP substrate (red diamonds) in 0.25% (v/v) TSB buffer at a peptide concentration of 25 µM. Molar ellipticities ([θ]) were recorded at λ ) 225
nm while the temperature was increased from 5 to 85 °C.
Figure 9. Inhibition of MMP-2 by f4 at varying temperatures. MMP-2
Figure 8. Inhibition of MMP-2 by f3 at varying temperatures. MMP-2
was incubated with varying concentrations of f3 at 10 (blue diamonds), 25
was incubated with varying concentrations of f3 at 10 (blue diamonds), 25
(pink squares), 30 (green triangles), or 37 °C (orange circles). Residual
(pink squares), 30 (green triangles), or 37 °C (orange circles). Residual
activity was monitored as indicated in Materials and Methods. Standard
activity was monitored as indicated in Materials and Methods. Standard
deviations are indicated with error bars.
deviations are indicated with error bars.
Table 1. Inhibition of MMP-2 and MMP-9
similar melting temperatures (Tm ∼17.5 °C), as did the f3 and
temp
C)
K(iapp)
(nM)
f4 peptide-amphiphiles (Tm ∼25 °C). The Tm values for the
enzyme
inhibitor
(
°
phosphinate peptide-amphiphiles were considerably lower than
MMP-2
f3
“
f4
“
10
37
10
37
10
37
25
25
10
37
10
37
4.14 ( 0.47
19.23 ( 0.64
5.48 ( 0.00
138.32 ( 27.85
3.17 ( 0.23
0.83 ( 0.03
50.05 ( 9.56
451.18 ( 89.48
1.76 ( 0.05
1.29 ( 0.00
2.20 ( 0.34
2.34 ( 0.23
that for the analogueous peptide-amphiphile substrate (Tm
)
“
“
“
“
“
“
“
49.5 °C).34 SEC analysis of f4 showed that, at 37 °C, f4 appears
as a single species of near identical molecular weight as that of
f2, but upon cooling to 10 °C both f4 and f2 shift to a trimeric
species (data not shown). The trimeric species of f4 had virtually
the identical retention time as that of the analogueous, trimeric
peptide-amphiphile substrate R1(V)436-450 THP.34 There were
no aggregates of trimeric species observed.
MMP inhibitor III
“
f1
f2
f3
“
MMP-9
“
“
“
f4
“
Inhibition of MMPs. The phosphinate peptide-amphiphiles
(f3 and f4) were initially tested against MMP-2 (Figures 8 and
9; Table 1) and MMP-9 (Table 1). Due to the low melting
temperatures of the potential inhibitors, Ki values were first
determined at 10 °C. Both f3 and f4 were found to be very
effective inhibitors of MMP-2 and MMP-9, with Ki values in
the 4-6 and 2 nM ranges, respectively. When inhibition assays
were repeated at 37 °C, Ki values increased for MMP-2 but not
for MMP-9 (Table 1). Thus, triple-helical structure modulated
inhibition of MMP-2 but not MMP-9. Inhibition of MMP-2 was
then examined with peptides f1 and f2 at 25 °C, which
corresponds to ∼15-20% folded conformation (see Figure 7).
9
J. AM. CHEM. SOC. VOL. 129, NO. 34, 2007 10415

A R T I C L E S
Lauer-Fields et al.
The Ki values for f1 and f2 (Table 1) were worse than those for
the analogous peptide-amphiphiles f3 and f4 at 37 °C (which
corresponds to ∼15-20% of their folded conformation).
Although the low melting temperatures of f1 and f2 precluded
their study as inhibitors when primarily in triple-helical
conformation, partially unfolded conditions demonstrated that
the more stable triple-helices (f3 and f4) were better MMP-2
inhibitors than their less stable counterparts (f1 and f2).
At 10 and 37 °C, f3 was a better inhibitor than f4 for both
MMP-2 and MMP-9. Interestingly, the increase in Ki value as
a function of temperature was not the same for f3 and f4. For
f3, the increase in temperature resulted in a 5-fold increase in
Ki for MMP-2. For f4, the increase in temperature resulted in a
25-fold increase in Ki for MMP-2. Removing the R configuration
of the isopropyl side chain from a triple-helical context made
f4 a considerably worse inhibitor than when removing the S
configuration from a triple-helical context (f3).
To determine if an increase in Ki as a function of temperature
was a general trend for inhibition of MMP-2, inhibition of
MMP-2 by MMP inhibitor III was examined. At 10 °C, the Ki
value for MMP-2 inhibition was 3 nM (Table 1). Increasing
the temperature to 37 °C decreased the Ki to 0.8 nM (Table 1).
Thus, for a small molecule inhibitor, an increase in temperature
slightly increased the affinity toward MMP-2, most likely due
to an enhancement of hydrophobic interactions. This further
suggests that the decreased inhibition of MMP-2 by f3 and f4
as a function of increasing temperature is due to unfolding of
the inhibitor triple-helical structure.
modeled after (Figure 7). It is likely that the GlyΨ{PO2H-
CH2}Val transition state analogue destabilizes the triple-helix
via charge repulsion and/or steric clashes. Alternatively, the
H2O-bound tetrahedral intermediate may destabilize native
collagen, which would explain why the MMP active site Zn2+
is required for triple-helix unwinding to proceed.70 It is possible
that MMPs utilize H2O to destabilize the substrate, thus allowing
for unwinding without an external source of energy (such as
ATP). Future experiments will address the role of the tetrahedral
intermediate in substrate unwinding.
The present THP inhibitors exhibit low nM Ki values for
MMP-2 and MMP-9 while having little or no activity against
collagenolytic MMPs (MMP-1, MMP-8, MMP-13, and MT1-
MMP) or MMP-3. This selectivity is important, as MMP-3 and
MMP-8 have been identified as antitargets for cancer thera-
peutics.2 It was previously noted that an MMP inhibitor should
be selective over antitarget MMPs by ∼3 log orders of
difference in Ki values,12 which is satisfied in the present case.
Prior work created a series of phosphonic acid pseudotripeptide
inhibitors of MMP-2 and MMP-9 with Ki values in the low
nanomolar range but offered little selectivity toward MMP-8
and MT1-MMP.28 Similarly, peptidyl biphenylalkylphosphinates
inhibited both MMP-2 and MMP-8 with IC50 values in the low
micromolar range.32
Phosphinic acid inhibitors of MMPs have often utilized
GlyΨ{PO2H-CH2}Leu transition state analogues. Ala-Gly-Pro-
Leu-GlyΨ{PO2H-CH2}Leu-Tyr-Ala-Arg-Gly was found to
inhibit MMP-9 with Ki values of 0.6 and 3.4 nM for the S and
R diastereomers, respectively.27 â-Napthoyl-GlyΨ{PO2H-
CH2}Leu-Trp-NHBzl had a Ki value of 10 nM for MMP-1.16
Selectivity among MMPs for these inhibitors was not reported.
Reiter et al. developed a pseudotripeptide phosphinic acid
MMP-1 inhibitor with an IC50 value of 60 nM; replacement of
the isobutyl side chain in the P1′ subsite with phenethyl or
phenoxybutyl resulted in inhibitors more selective for MMP-
13 than MMP-1 and with IC50 values of 14 and 30 nM,
respectively, for MMP-13.26 These studies did not examine
inhibitor selectivity for a variety of MMPs; an 85-fold selectivity
was observed between MMP-13 and MMP-3 for one inhibitor.
A latter study from the same group identified a phosphinic acid
MMP-13 inhibitor that had an IC50 of 4.5 nM for MMP-13 and
offered 270-360-fold selectivity over MMP-1 and MMP-3.71
However, low nanomolar IC50 values were also observed for
MMP-2, MMP-8, and MMP-12, and thus the inhibitor was only
partially selective.71
A solid-phase combinatorial chemistry approach, which
incorporated the GlyΨ{PO2H-CH2}Leu mimic with amino acid
substitutions in the P4, P3, P2, P2′, P3′, P4′, and P5′ subsites,
facilitated the development of inhibitors selective for MMP-
12.23,24 The best MMP-12 inhibitors had Ki values in the 6-25
nM range and exhibited up to 2000-fold selectivity for MMP-
12 over MMP-9 and 250-300-fold selectivity for MMP-12 over
MMP-13 and MT1-MMP.23,24 A selective MMP-12 inhibitor
was also obtained by screening peptide libraries of the general
structure p-Br-Ph-(PO2-CH2)-Xaa′-Yaa′-Zaa′.25 p-Br-Ph-
(PO2-CH2)-(3-[3-chlorobiphenyl-4-yl]isoxazol-5-yl)methyl-
MMP-1, MMP-3, MMP-8, MMP-13, and MT1-MMP were
tested for inhibition by f3 and f4. No inhibition of MMP-1,
MMP-3, or MT1-MMP was observed up to a concentration of
25 µM for either f3 or f4 (data not shown). MMP-8 and MMP-
13 were inhibited weakly, with Ki values in the range of 50
and 10 µM, respectively (data not shown).
Discussion
The present study has utilized a GlyΨ{PO2H-CH2}Val
transition state analogue to bind selectively at the S1-S1′ site
of MMP-2 and MMP-9. MMP-2 has been validated as an
anticancer drug target, while MMP-9 inhibition may be useful
in treating early stage cancers.2 An MMP-2 and MMP-9
selective substrate was used as a framework for a phosphinic
acid-based inhibitor. Two variants of the phosphinate triple-
helical peptide were produced, one in which the P1′ isopropyl
side chain is in the S configuration and one in which the side
chain is in the R configuration. The difference in triple-helical
thermal stability between the S and R forms of the inhibitor is
negligible. As shown previously, the use of stabilizing regions
[such as (Gly-Pro-Hyp)n] on both the N- and C-termini can fold
and order a central non-Gly-Xxx-Yyy region, minimizing
relative thermal destabilization.68,69 Also, the presumed S
diastereomer (natural “L-amino acid like” stereochemistry) is a
slightly better inhibitor than the R diastereomer. This is
consistent with prior studies on inhibition of MMPs by P1′
subsite S versus R phosphinic peptides.22,25,27,31
The triple-helical transition state analogue had a Tm value
25-27 °C lower than that of the triple-helical substrate it was
(70) Chung, L.; Dinakarpandian, D.; Yoshida, N.; Lauer-Fields, J. L.; Fields,
G. B.; Visse, R.; Nagase, H. EMBO J. 2004, 23, 3020-3030.
(71) Reiter, L. A.; Mitchell, P. G.; Martinelli, G. J.; Lopresti-Morrow, L. L.;
Yocum, S. A.; Eskra, J. D. Bioorg. Med. Chem. Lett. 2003, 13, 2331-
2336.
(68) Mohs, A.; Popiel, M.; Li, Y.; Baum, J.; Brodsky, B. J. Biol. Chem. 2006,
281, 17197-17202.
(69) Hyde, T. J.; Bryan, M.; Brodsky, B.; Baum, J. J. Biol. Chem. 2006, 281,
36937-36943.
9
10416 J. AM. CHEM. SOC. VOL. 129, NO. 34, 2007

Triple-Helical Transition State Analogues
A R T I C L E S
Glu-Glu was found to inhibit MMP-12 with a Ki value of 0.19
nM and offer a 200-100 000-fold selectivity over other
MMPs.25 Combinatorial approaches were also utilized to create
isoxazole-containing phosphinic peptides with low nanomolar
Ki values toward MMP-13 and MT1-MMP.31 Finally, a series
of phosphinic peptides of general structure Z-Ala/PheΨ{PO2H-
CH2}Xaa′-Yaa′-NH2 or R-PheΨ{PO2H-CH2}Xaa′-Trp-NH2
were utilized to develop high affinity MMP-11 inhibitors (low
nanomolar and sub-nanomolar Ki values).28
reduce proteolytic processing of peptides in ViVo,26,80 and thus
its inclusion in a triple-helical context may create an inhibitor
that is reasonably stable to metabolic processes. The phosphinate
triple-helical inhibitor is also faster binding than the natural
MMP inhibitors, TIMPs.
The present inhibitors are too thermally unstable for in ViVo
applications but can be modified by inclusion of additional Gly-
Pro-Hyp repeats and/or longer N-terminal alkyl chains or
replacement of Hyp residues by 4-fluoroproline to improve
triple-helical stability.35,73,81-83 For other collagenolytic enzymes,
diversity of the phosphinate pseudodipeptide may be efficiently
achieved by a recently described three-component condensation
reaction.65 It has been proposed that the negative charge of the
phosphoryl group may prevent peptide membrane permeability
and uptake by liver cells and promote peptide interaction with
nonspecific proteases in the plasma.12,80 However, a phosphinic
MMP inhibitor has been to shown to decrease necrosis and
apoptosis in ischemic rat livers.30 In addition, monopril (fosi-
nopril sodium tablets; L-proline, 4-cyclohexyl-1-[[[2-methyl-1-
(1-oxopropoxy)propoxyl](4-phenylbutyl)phosphinyl]acetyl]-, so-
dium salt, trans-) is an orally bioavailable phosphinate tripeptide
analogue inhibitor of the angiotensin converting enzyme.84,85
However, because of the solid-phase methodology employed
here, one can potentially utilize new, higher affinity ZBGs7,86
to replace the phosphinate if needed.
There are neither prior examples of phosphinic peptides
offering the MMP-2/MMP-9 selectivity observed here nor
documented triple-helical phosphinic peptides. Triple-helical
peptide inhibitors of MMPs have been constructed previously
using a hydroxamate as the ZBG. In one study, (Pro-Pro-Gly)6-
NHOH and (Pro-Pro-Gly)12-NHOH were found to inhibit
MMP-2 with IC50 values of 160 and 90 µM, respectively.72
These constructs showed similarly weak inhibition of MMP-1
and MMP-3.72 The weak inhibition may have been the result
of interaction with only the S subsites of the enzyme and/or
poor alignment of the ZBG into the active site. It should be
noted that the melting temperatures of these inhibitors were not
evaluated. Inhibition assays were performed at 37 °C; since (Pro-
Pro-Gly)10 has a Tm value of 25 °C,73 the inhibitors were most
likely not triple-helical unless the hydroxamate group conferred
considerable stability to the system. A second study utilized a
solid-phase C-terminal branching protocol74 to incorporate a
hydroxamate-containing peptidomimetic onto the N-terminus of
(Gly-Pro-Hyp)4-Gly-Pro-Pro-Gly-Ser-Ser.75 Inhibition of MMP-1
was achieved with an IC50 value of ∼9 nM. Constructs in which
fewer Gly-Pro-Hyp repeats were present, and thus presumably
had less or no triple-helicity, exhibited IC50 values of ∼100-
500 nM. Thus, MMP-1 inhibition was dependent upon triple-
helical structure. While interesting, this particular inhibitor
would offer little selectivity among collagenolytic MMPs.
Phosphinate triple-helical MMP inhibitors have several
advantages over other inhibitor constructs. These analogues
allow incorporation of specificity elements for both the S and
S′ subsites of the enzyme. Although binding to the nonprimed
region of the active site is generally weaker than that to the
primed site to prevent product inhibition,14 it does add sequence
diversity and potential selectivity. The triple-helical structure
allows for interaction with both the active site and secondary
substrate binding sites (exosites).2,3,12 Inhibitors that recognize
exosites can enhance selectivity.76-78 The triple-helical confor-
mation is also less susceptible to proteolysis than peptides and
other folded proteins.79 On its own, a phosphinate seems to
The present MMP-2/MMP-9 selective transition state ana-
logue inhibitor has several potential applications. First, it can
be utilized for metalloproteinase structural studies and to
evaluate exosite interactions. Second, it can allow for the
mechanistic study of collagenolysis. Third, it can be evaluated
for inhibition of experimental metastasis. Finally, it could serve
as a molecular probe and a lead compound for drug develop-
ment.
Acknowledgment. We gratefully acknowledge Dr. Hideaki
Nagase for supplying MMP-1, MMP-2, and MMP-3 and Dr.
Christopher Overall for supplying MMP-8. This work was
supported by the National Institutes of Health (CA 98799 to
GBF/RPH), a Glenn/American Federation for Aging Research
(AFAR) Scholarship (to J.L.-F.), an ASBMB Travel Award (to
J.L.-F.), and an NSF IGERT (CHE-9987603) Fellowship (to
J.K.W.).
Supporting Information Available: 1H and 13C NMR spectra
for compounds 1, 2, 6, 7, and 8; 31P NMR spectra for
compounds 1, 7, and 8; and the complete ref 26. This material
is available free of charge via the Internet at http://pubs.acs.org.
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