Mechanisms of Macromolecular Protease Inhibitors
Christopher J. Farady[a, b]and Charles S. Craik*[a, b]
Proteolytic enzymes are ubiquitous in all organisms and consti-
tute 2–4% of encoded gene products. They are critical for
diverse biological processes such as digestion, blood clotting,
host defense, pathogenic infection, viral replication, wound
healing, and disease progression, to name but a few. Because
proteases trigger an irreversible event—the cleavage of a pro-
tein—their activity must be tightly controlled. Dysregulated
proteolytic activity causes a disruption in the homeostatic bal-
ance of a biological system and can result in any number of
poor biological outcomes. As a result, nature has developed a
number of strategies to control proteolysis, including spatial
and temporal regulation, zymogen activation and protease
degradation, and through the inhibition of proteases by mac-
romolecular inhibitors. Somewhat surprisingly, relatively few
design principles underlie the mechanisms of inhibition of a
myriad range of macromolecular protease inhibitors. Signifi-
cant engineering efforts have gone into modifying and im-
proving inhibitor potency and specificity, and to a large extent,
the same design principles that work well for naturally occur-
ring protease inhibitors have proved valuable for inhibitors
developed in the laboratory.
This review aims to survey the mechanisms by which macro-
molecular protease inhibitors function. To do this, inhibitors
have been divided into categories based on their mechanism
in order to illustrate that a relatively small number of design
principles can be combined to develop new and effective pro-
tease inhibitors. These divisions are not strict, and many inhibi-
tors could be grouped in a number of classes. The list of mech-
anisms presented here is not exhaustive in its treatment of all
inhibitors, but aims to be illustrative of the many ways proteas-
es can be inhibited. For more information on genome-wide
protease mining,protease mechanism,preclinical inhibi-
tion,and drug-discovery efforts,the reader is directed to ex-
cellent reviews that have been written in recent years. Figure 1
provides an overview of basic substrate and protease nomen-
clature that will be used in this review.
The vast majority of protease inhibitors are competitive. De-
spite divergent targets and different mechanisms of inhibition,
most protease inhibitors bind a critical portion of the inhibitor
in the active site in a substrate-like manner (Figure 2). This is
an effective paradigm for potent inhibition, but because relat-
ed proteases often show a high degree of homology in the
active site, substrate-like binding often leads to inhibitors that
can potently inhibit more than one target protease. This inhibi-
tor promiscuity is evidenced by the fact that there are 115 an-
notated human protease inhibitors responsible for regulating
the activity of the 612 known human proteases. Though these
numbers will change as more refinement of the protease and
inhibitor families is achieved, the ratio of approximately one
Figure 1. A) Diagram of a protease active site. A protease, which has a num-
ber of specificity subsites that determine its specificity, cleaves a peptide at
the scissile bond. Substrates bind to a protease with their nonprime residues
on the N-terminal side of the scissile bond and their prime-side residues C-
terminal to the scissile bond. The catalytic residues determine the class of
protease. Serine, cysteine, and threonine proteases hydrolyze a peptide
bond via a covalent acyl-enzyme intermediate, and aspartic, glutamic and
metalloproteases activate a water molecule to hydrolyze the peptide bond
in a noncovalent manner. B) A serine protease (matriptase/MT-SP1, PDB ID:
1EAX) with the catalytic triad in yellow and the surface loops that surround
the active site in blue. While the catalytic architecture of proteases is remark-
ably conserved, the surface loops are areas of high sequential and structural
[a] C. J. Farady, Prof. C. S. Craik
Graduate Group in Biophysics, University of California–San Francisco
600 16th Street Genentech Hall, San Francisco, CA 94143-2240 (USA)
[b] C. J. Farady, Prof. C. S. Craik
Department of Pharmaceutical Chemistry
University of California–San Francisco
600 16th Street Genentech Hall, San Francisco, CA 94143-2280 (USA)
ChemBioChem 2010, 11, 2341–2346 ? 2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
protease inhibitor to five proteases is likely to remain con-
The most thoroughly studied mechanism of protein pro-
tease inhibitors is that of the standard (or canonical, or Las-
kowski) mechanism inhibitors of serine proteases.These in-
hibitors include the Kazal, Kunitz, and Bowman-Birk family of
inhibitors and bind in a lock-and-key fashion. Standard-mecha-
nism inhibitors insert into the active site of the protease a
reactive loop that is complementary to the substrate specificity
of the target protease and binds in an extended b-sheet with
the enzyme in a substrate-like manner (Figure 2A, B). While
bound to the protease, the “scissile bond” of standard-mecha-
nism inhibitors is hydrolyzed very slowly, but products are not
released, and the amide bond can be religated.[7,8]The stan-
dard mechanism is an efficient way to inhibit serine proteases,
and is thus used by many structurally disparate protein scaf-
folds to create potent inhibitors. However, the majority of stan-
dard-mechanism protease inhibitors tend to have relatively
broad specificity within subclasses of serine proteases. For
example, bovine pancreatic trypsin inhibitor (BPTI) efficiently
inhibits almost all trypsin-fold serine proteases with P1-Arg
specificity with sub-nanomolar potency, and can also potently
inhibit chymotrypsin (Phe P1 specificity) with a KIof 10 nm.
The majority of protease inhibitors bind in and block access
to the active site of their target protease, but do not bind in a
strictly substrate-like manner. Instead they interact with the
protease subsites and catalytic residues in a noncatalytically
competent manner. This differentiates them from standard-
mechanism inhibitors; however, like standard-mechanism in-
hibitors, they get most of their potency from interactions with-
in the protease active site, and tend to potently inhibit many
The cystatins, a superfamily of proteins that inhibit papain-
like cysteine proteases, are a classic example of these inhibitors
(Figure 2A, C). The cystatins insert into the V-shaped active site
of a cysteine protease a wedge-like face of the inhibitor that
consists of the protein N terminus and two hairpin loops. The
N-terminal residues bind in the S3–S1 pockets in a substrate-
like manner, but the peptide then turns away from the catalyt-
ic residues and out of the active site. The two hairpin loops
bind to the prime side of the active site, which provides the
majority of the binding energy for the interaction. Thus, both
the prime and nonprime sides of the active site are occupied,
but no interactions are actually made with the catalytic machi-
nery of the enzyme.
The four human tissue inhibitors of metalloproteases (TIMPs)
are responsible for the inhibition of dozens of extracellular
metalloproteases (Figure 2A). They bind to their target en-
zymes in a two-step mechanism similar to that of the cystatins.
While the N-terminal residues of cystatins bind to the non-
prime side of cysteine proteases, TIMPs N termini bind in the
P1-P3’ pockets of the protease, coordinate the catalytic Zn2+
ion, and exclude a catalytic water molecule from the active
site. Meanwhile a second loop of the TIMP binds both in the
P3 and P2 pockets, and binds to the N terminus of the matrix
metalloprotease (MMP). Despite the similarities in mechanistic
architecture between TIMPs and cystatins (hairpin loops and
N-terminal residues in substrate binding pockets), TIMPs inter-
fere with the catalytic machinery of MMPs by chelating the cat-
Competitive Inhibition with Exosite Binding
A number of protease inhibitors are competitive, and bind in
the protease active site, but also have secondary binding sites
outside the active site that are critical to inhibition. Exosite
binding provides two major benefits; it increases the surface
area of the protein–protein interaction, leading to a greater
affinity, and it can have a significant effect on the specificity of
Many blood-meal parasites have evolved protease inhibitors
that take advantage of exosites to prevent host blood clotting.
These inhibitors often use similar competitive inhibitory mech-
anisms to those described above, but have domains that bind
to protease exosites and provide a high degree of target spe-
cificity. Rhodniin, a thrombin inhibitor from the assassin bug
Rhodnius prolixus has two Kazal-type inhibitory domains, a
common standard-mechanism serine protease inhibitor do-
main (Figure 3A). While the N-terminal domain binds and in-
hibits through the standard mechanism, the second Kazal-type
domain has evolved to bind to exosite I on thrombin. The
binding affinities of the individual domains are roughly addi-
tive, and the resultant inhibitor has a KIof 0.2 pm and exquisite
specificity for thrombin.Therefore, the inhibitor gains both
potency and specificity from exosite binding.
In contrast, the E. coli serine protease inhibitor ecotin uses
its exosites to provide binding energy and to actually broaden
the inhibitor promiscuity to protect the bacteria from diverse
host proteases (Figure 3A, B). Ecotin is a dimeric protein that
inhibits trypsin-fold serine proteases, regardless of primary spe-
cificity. It inhibits using a standard mechanism at its primary
Figure 2. Competitive, active-site inhibitors of proteases. A) Inhibitors bind
in the active site, but not in a substrate-like manner. Peptide extensions
bind in specificity subsites, and can interact with the catalytic residues (rec-
tangle). Crystal structures of B) a serine protease (matriptase/MT-SP1, PDB
ID: 1EAW) in complex with the standard-mechanism inhibitor aprotinin, and
C) the cystatin stefin A in complex with a cysteine protease (cathepsin H,
PDB ID: 1NB5). The portion of stefin A that interacts with the protease is col-
ored in green. Both inhibitors bind in the active-site groove of their targets.
? 2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2010, 11, 2341–2346
C. S. Craik et al.
binding site, but also has a secondary binding site that can
contribute up to 5 kcalmol?1of binding energy to the very
tight enzyme–inhibitor complex. Surprisingly, the individual
binding energies of the two binding sites are not additive; the
effect of the secondary binding site on affinity was found to
be inversely proportional to the strength of binding at the pri-
mary site. The secondary binding site seems to provide com-
pensatory effects that can overcome suboptimal binding at
the primary binding site; if binding at the primary site is not
optimal, the secondary binding interaction tends to be stron-
ger. In this way, the exosite actually makes the inhibitor less
specific, or more capable of inhibiting a broad range of pro-
teases, and allows one bacterial inhibitor to protect against a
number of host proteases.
The recent structures of the calcium-dependent protease
calpain 2 in complex with the inhibitory domain CAST4 of cal-
pastatin (Figure 3A, C) reveal another unique use of binding
sites outside the active site.[14,15]Free calpastatin is intrinsically
unstructured,but upon binding to calpain, the polypeptide
forms three helices, strung across the surface of the enzyme,
and binds in the protease active site to act as a competitive
inhibitor. Incredibly, CAST4 buries 2800 ?2of surface area on
calpain, this is approximately three times the surface area that
standard-mechanism protease inhibitors,
TIMPsbury when complexed with their respective sub-
strates. The majority of competitive protease inhibitors do not
show much induced fit upon binding, and thus do not require
a lot of buried surface area. In contrast, CAST4 uses this large
amount of buried surface area outside the protease active site
to compensate for the entropic penalty of ordering the inhibi-
tor upon binding, and still allows for a KIin the low-nanomolar
range. As discussed later, the use of exosites to increase the
surface area of a protease inhibitor, and allow for structural
arrangements to take place upon binding, is a theme that has
been extremely useful in the design of novel protein protease
Sometimes called suicide substrates, a handful of protease in-
hibitors use proteolytic activation by an enzyme to covalently
modify and thereby inhibit it. This sort of activity-dependent
inhibition is powerful and fundamentally different from the
competitive mechanisms outlined above; the inhibitor acts as
a substrate, then utilizes the enzymes’ catalytic machinery to
trap and inhibit the enzyme.
The inhibitor a-2-macroglobin (a2M) and its relatives are re-
sponsible for clearing excess proteases from plasma. Less an
inhibitor than a “protease sponge”, a2M is a large protein, a
tetramer of about 600 kD that has four bait loops on its sur-
face. When a protease cleaves one of these reactive loops, it
triggers a conformational change, and the protease becomes
crosslinked to the inhibitor through surface lysines and argi-
nines. The enzyme is still active; small-molecule substrates can
be hydrolyzed by proteases complexed with a2M, but protein
substrates are occluded from the active site, and the complex
is quickly cleared from the blood.
The serpins are a family of inhibitors that covalently and ir-
reversibly inhibit primarily serine proteases(the serpin Crm1
inhibits cysteine proteases). Serpins have a large reactive
center loop (RCL) that is presented to a protease for proteolyt-
ic processing. Upon productive cleavage of the RCL, the N-ter-
minal half of the RCL, still attached to the protease as an acyl-
enzyme intermediate, is inserted into a b-sheet in the body of
the inhibitor. The resulting free-energy change is enough to
translocate the protease (still covalently attached to the RCL)
to the distal side of the inhibitor, and the resulting steric colli-
sions completely deform the protease active site, thus leaving
the protease tethered to the serpin and completely inactive
(Figure 4). The serpin inhibitory mechanism is completely irre-
versible. Because of the drastic nature and irreversibility of this
mechanism, serpins function as protease scavengers, protect-
ing cells and tissues from unwanted proteolytic activity.
These types of inhibitors, which take advantage of the cata-
lytic activity of a protease to trap and inhibit the enzyme, are
effective and powerful inhibitors that are responsible for pro-
tecting the organism from aberrant proteolytic activity from a
Figure 3. Inhibitors that take advantage of exosite binding. A) Most exosite
inhibitors are competitive inhibitors that prevent substrate binding at the
active site. In the case of ecotin (bound to trypsin, PDB ID: 1EZU), the exo-
sites provide binding energy and allow for broad specificity (B), while calpa-
statin (C) gains binding energy and specificity by forming critical interactions
across the calpain protease surface (PDB ID: 3BOW).
Figure 4. Serpins inhibit serine proteases by binding a reactive center loop
in the active site, forming a covalent complex with the enzyme, undergoing
a large conformational change, and irreversibly distorting the active site of
the protease (PDB IDs: 2GD4 and 1EZX).
ChemBioChem 2010, 11, 2341–2346 ? 2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
Macromolecular Protease Inhibitors
wide range of proteases. Thus, they tend to be relatively non-
specific. As reviewed by Powers et al,many small-molecule
covalent and/or irreversible inhibitors have been developed
that rely on the same fundamental mechanism of using
enzyme activity to trap and inactivate a protease. These mole-
cules use a reactive warhead that binds to the catalytic machi-
nery of the enzyme, and specificity elements are often linked
to the warheads to gain selectivity for one target. However,
due to the reactive nature of the warhead and the mechanistic
and structural similarities displayed by protease families,
absolute specificity is difficult to achieve.
Inhibitors Developed by Protein Engineering
Protein engineering has allowed for the development of new
protease inhibitors with increased potency, altered specificity,
and diverse mechanisms of action. Because proteases tend to
have relatively shallow active sites, a high degree of homology,
and can have broad specificity, macromolecules are attractive
inhibitors in that they can bury more surface area upon bind-
ing, and hopefully gain more potency and specificity than a
small-molecule inhibitor. Moreover, from a functional point of
view, the extracellular localization of many proteases make
them amenable to regulation by proteins and other macromo-
lecules in vivo. Structure-based modifications of naturally
occurring protease inhibitorsor other scaffoldshave been
successful, but the evolution of phage display (and similar
technologies) has revolutionized our ability to specifically in-
hibit individual proteases.
The combinatorial nature of phage display allows for the op-
timization of a number of individual positions on an inhibitor
at once, and early protein engineering efforts focused on im-
proving the specificity of naturally occurring protease inhibi-
tors. In an early example of this, Dennis et al. used phage-dis-
played libraries to randomize residues that interact with the
protease subsites to modify and improve the specificity of the
Kunitz-type serine protease inhibitor APPI. Using a series of
competitive selection strategies, the authors were able to engi-
neer potent inhibitors that were selective for the clotting
enzyme factor VIIa (FVIIa), and lost inhibitory potency against
homologous enzymes.[24,25]While modifying the residues that
interact with the protease active site has a drastic effect on in-
hibitor affinity, in naturally occurring inhibitors specificity tends
to be gained through the evolution of secondary interactions.
As such, randomization of the secondary binding sites of
protease inhibitors can greatly increase the specificity of an
inhibitor. The standard-mechanism serine protease inhibitors
ecotinand eglin chave both been refined at both their
primary and secondary interaction sites, which has drastically
improved their specificities for plasma kallikrein and kexin 2,
respectively. Functioning with inhibitory mechanisms similar to
that of rhodniin, the engineering of secondary sites on stan-
dard-mechanism protein scaffolds results in potent and selec-
tive inhibitors that allow for the modulation of a single pro-
tease in complex biological processes.
Another strategy has been to develop polypeptide-based
proteases inhibitors. Typically consisting of 10–20 amino acids,
and often containing disulfide bonds or chemical scaffolding
componentsto rigidify the inhibitors and decrease the en-
tropic cost of binding, constrained peptides have been devel-
oped to inhibit aspartic, cysteine, serine, and threonine pro-
teases. While peptides are not thought to be ideal drug mole-
cules, due to their susceptibility to proteolysis, the relatively
small size of constrained peptides allows for the creation of
extremely diverse libraries. Furthermore, they are amenable to
the incorporation of non-natural or d-amino acids, thus greatly
increasing potential diversity. The mechanisms of action of
these inhibitors have sometimes mimicked known biological
mechanisms, and other times have been completely novel
(Figure 5A). Constrained-peptide phage-display libraries have
yielded standard-mechanism inhibitors of the serine proteases
and urokinase-type plasminogen activator
(uPA)with moderate potency and specificity. Cyclic peptides
have also been shown to competitively inhibit the aspartic
protease renin, and are also thought to bind to the enzyme in
a substrate-like manner.
Constrained peptides that mimic natural inhibitors are es-
sentially a reduction of naturally occurring inhibitors to just
their reactive elements. However, a number of allosteric pep-
tide inhibitors have been developed that have novel mecha-
nisms of inhibition; this reveals information about enzyme
function and suggest new ways of regulating proteolysis. Con-
strained peptide libraries have yielded two potent exosite in-
hibitors of the clotting enzyme factor VIIa (FVIIa).[33,34]The two
inhibitors bind to two different sites outside of the active site
of the enzyme, and have unique mechanisms of inhibition.
One inhibitor, A-183, functions by forcing a loop near the
active site into an inactive conformation and occluding sub-
strate binding to the enzyme. The other inhibitor, E-76 is a
noncompetitive inhibitor of FVIIa’s natural substrate, factor X,
and it seems to work by locking the enzyme in a zymogen-like
conformation. In another example of allosteric inhibition, an a-
Figure 5. A) Inhibitory cyclic peptides bound to the surface of a serine pro-
tease. The cyclic peptide SFTI (purple, PDB ID: 1SFI) inhibits trypsin in a can-
onical manner, upain (orange, PDB ID: 2NWN) binds to the prime side of
uPA, and the FVIIa inhibitors E-76 (green, PDB ID: 1DVA) and A-183 (blue,
PDB ID: 1JBU) bind to protease exosites. B) Antibodies provide an alternative
scaffold on which to build protease inhibitors with a high degree of specific-
ity. The antibody E2 (purple) binds in the protease active site (MT-SP1, PDB
ID: 3BN9) in a noncanonical manner, while the Fab40 (orange, PDB ID:
3K2U) antibody inhibits HGFA while binding outside the protease active site
through an allosteric mechanism.
? 2010 Wiley-VCH Verlag GmbH&Co. KGaA, WeinheimChemBioChem 2010, 11, 2341–2346
C. S. Craik et al.
helical peptide was designed to disrupt the dimerization of the
protease from Kaposi’s sarcoma-associated herpes virus (KSHV)
and thus prevent its activation.These allosteric peptide
inhibitors lock their target enzymes in an inactive, closed, or
zymogen-like state that prevents substrate binding and impairs
catalytic activity, and thus they are usually mixed-type inhibi-
tors. The allure of allosteric inhibition is founded in the idea
that there are multiple sites on an enzyme that regulate activi-
ty, and because allosteric sites are less likely to show sequence
conservation than the protease active site, allosteric inhibitors
will hopefully show a higher degree of specificity. Nature uti-
lizes a number of techniques to allosterically regulate proteas-
es. Zymogen activation, dimerization, and the binding of pro-
teins, cofactors, or metal ions are all ways to more finely regu-
late proteases, and localize proteolytic activity to a specific
spatial or temporal location. But as discussed above, with very
few exceptions (such as the BIR3 domain inhibition of caspase-
9), naturally occurring protease inhibitors do not take ad-
vantage of the allosteric movements in proteins, and instead
are potent competitive or irreversible inhibitors that complete-
ly ablate proteolytic activity. Taking advantage of the chemical
diversity of phage-display libraries and screening libraries of
peptides or antibodies against targets has allowed the isola-
tion of these allosteric inhibitors and the elucidation of mecha-
nisms of allosteric regulation that have not been seen in
nature and might not have been discovered by using tradition-
al high-throughput-screening techniques.
A third approach has been to evolve specific protease inhibi-
tors on other natural protein scaffolds. An enzyme–substrate
interaction is really a protein–protein interaction, so any pro-
tein that can disrupt the protein–protein interaction can be an
effective inhibitor. The antibody scaffold has been the most
popular to date, but ankyrin repeats,cysteine knots,and
aptamershave also been developed into protease inhibitors.
By using either hybridoma or phage-display technologies, anti-
body inhibitors have been raised against all four classes of pro-
teases, and biochemical and biophysical characterizations
reveal familiar mechanisms of inhibition. The majority of anti-
body-based protease inhibitors work by binding to surface
loops (see Figure 1)—thus gaining specificity and occluding
macromolecular substrates from the protease active site.[40–43]
The antibody inhibitors that have been shown to bind in the
protease active site do so by binding antibody hypervariable
loops in a non-substrate-like manner in the substrate binding
pockets (Figure 5B).[44,45]In a manner analogous to the cysta-
tins or calpastatin, they adopt conformations that avoid direct
interaction with the catalytic nucleophile, thus avoiding hydrol-
ysis. Antibodies directed to the dimer interface of HIV pro-
teaseor caspase 1have been used to sequester mono-
meric forms of the protease, and thus, are able to inhibit their
target protease with a mixed-type mechanism (both competi-
tive and noncompetitive inhibition). Antibodies are attractive
inhibitors because they are exquisitely specific—antibodies
having evolved specifically to bind to their antigen—and are
useful biological tools for imaging and in vivo experiments.
That they have been able to incorporate many of the design
aspects that make naturally occurring protease inhibitors effec-
tive suggests that specific inhibitors on alternate protein scaf-
folds can be developed for many proteases.
Conclusions and Outlook
Relatively few design principles underlie the mechanisms of
inhibition of a diverse range of protease inhibitors. Protease
inhibitors tend to compete with substrate binding, either
through direct competition or deformation of the protease
active site. While protein inhibitors can gain potency through
the burial of a large surface area and specificity through con-
tacts with specific exosites, small-molecule inhibitors primarily
gain potency through interactions with the catalytic machinery
of the enzyme, and specificity through interactions with the
substrate binding sites. While there are several examples of
successful small-molecule protease inhibitors in the clinic,
selectivity and potency can be significant challenges when
targeting particular protease family members. The search for
novel modes of enzyme control, such as allosteric regulation, is
therefore particularly exciting, with the hope that these regula-
tory sites will be more amenable to the design of specific and
Our evolving understanding of macromolecular protease in-
hibitor mechanisms has allowed us to dissect complex biologi-
cal systems and to help determine the role of a single member
or an entire family of proteases in either homeostasis or in
disease states. Furthermore, protein protease inhibitors have
begun to make their way into the clinic. The serpins a1-anti-
trypsin and antithrombin III,purified from human plasma,
are used as replacement therapies for patients deficient in
those proteins. A recent illustration of how protein protease
inhibitors can translate into the clinic is the example of Kalbitor
(DX-88, Dyax Inc). The relatively unspecific standard-mecha-
nism Kunitz domain of tissue factor pathway inhibitor 1 was
matured by phage display to have a higher degree of selectivi-
ty for plasma kallikrein.In 2009, it was approved for the
treatment of hereditary angioedema, and is the first example
of an engineered protein therapeutic targeted against a pro-
tease. The extracellular location of many proteases, coupled
with the increase in knowledge and techniques that allows for
the development of more potent and specific inhibitors bodes
well for the future of macromolecular therapeutic protease
This work was funded by US National Institutes of Health Grants
AI067423 and CA128765.
Keywords: inhibitors · mechanisms · proteases · protein
engineering · specificity
 X. S. Puente, L. M. S?nchez, C. M. Overall, C. L?pez-Ot?n, Nat. Rev. Genet.
2003, 4, 544–558.
 A. J. Barrett, G. Salvesen in Research Monographs in Cell and Tissue Physi-
ology, Vol. 12, Elsevier, Amsterdam, 1986, p. 661.
ChemBioChem 2010, 11, 2341–2346 ? 2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
Macromolecular Protease Inhibitors
 J. C. Powers, J. L. Asgian, O. D. Ekici, K. E. James, Chem. Rev. 2002, 102, Download full-text
 B. Turk, Nat. Rev. Drug Discovery 2006, 5, 785–799.
 N. D. Rawlings, A. J. Barrett, A. Bateman, Nucleic Acids Res. 2010, 38,
 M. Laskowski, Jr., I. Kato, Annu. Rev. Biochem. 1980, 49, 593–626.
 J. A. Luthy, M. Praissman, W. R. Finkenstadt, M. Laskowski, Jr., J. Biol.
Chem. 1973, 248, 1760–1771.
 E. Zakharova, M. P. Horvath, D. P. Goldenberg, Proc. Natl. Acad. Sci. USA
2009, 106, 11034–11039.
 M. J. Castro, S. Anderson, Biochemistry 1996, 35, 11435–11446.
 W. Bode, R. Huber, Biochim. Biophys. Acta Protein Struct. Mol. Enzymol.
2000, 1477, 241–252.
 K. Brew, D. Dinakarpandian, H. Nagase, Biochim. Biophys. Acta Protein
Struct. Mol. Enzymol. 2000, 1477, 267–283.
 A. Roussel, M. Mathieu, A. Dobbs, B. Luu, C. Cambillau, C. Kellenberger,
J. Biol. Chem. 2001, 276, 38893–38898.
 C. T. Eggers, S. X. Wang, R. J. Fletterick, C. S. Craik, J. Mol. Biol. 2001, 308,
 R. A. Hanna, R. L. Campbell, P. L. Davies, Nature 2008, 456, 409–412.
 T. Moldoveanu, K. Gehring, D. R. Green, Nature 2008, 456, 404–408.
 A. Wendt, V. F. Thompson, D. E. Goll, Biol. Chem. 2004, 385, 465–472.
 A. J. Scheidig, T. R. Hynes, L. A. Pelletier, J. A. Wells, A. A. Kossiakoff, Pro-
tein Sci. 1997, 6, 1806–1824.
 S. Jenko, I. Dolenc, G. Gunc ˇar, A. Dobers ˇek, M. Podobnik, D. Turk, J. Mol.
Biol. 2003, 326, 875–885.
 K. Maskos, R. Lang, H. Tschesche, W. Bode, J. Mol. Biol. 2007, 366, 1222–
 S. J. Kolodziej, T. Wagenknecht, D. K. Strickland, J. K. Stoops, J. Biol.
Chem. 2002, 277, 28031–28037.
 P. G. Gettins, Chem. Rev. 2002, 102, 4751–4804.
 G. Izaguirre, A. R. Rezaie, S. T. Olson, J. Biol. Chem. 2009, 284, 1550–
 E. Kohfeldt, W. Gohring, U. Mayer, M. Zweckstetter, T. A. Holak, M. L.
Chu, R. Timpl, Eur. J. Biochem. 1996, 238, 333–340.
 M. S. Dennis, R. A. Lazarus, J. Biol. Chem. 1994, 269, 22137–22144.
 M. S. Dennis, R. A. Lazarus, J. Biol. Chem. 1994, 269, 22129–22136.
 A. A. Stoop, C. S. Craik, Nat. Biotechnol. 2003, 21, 1063–1068.
 T. Komiyama, B. VanderLugt, M. Fug?re, R. Day, R. J. Kaufman, R. S.
Fuller, Proc. Natl. Acad. Sci. USA 2003, 100, 8205–8210.
 J. N. Lilla, R. V. Joshi, C. S. Craik, Z. Werb, J. Biol. Chem. 2009, 284,
 C. Heinis, T. Rutherford, S. Freund, G. Winter, Nat. Chem. Biol. 2009, 5,
 E. Dumez, J. S. Snaith, R. F. Jackson, A. B. McElroy, J. Overington, M. J.
Wythes, J. M. Withka, T. J. McLellan, J. Org. Chem. 2002, 67, 4882–4892.
 M. Hansen, J. Biol. Chem. 2005, 280, 38424–38437.
 C. R. Nakaie, M. C. Oliveira, L. Juliano, A. C. Paiva, Biochem. J. 1982, 205,
 M. S. Dennis, C. Eigenbrot, N. J. Skelton, M. H. Ultsch, L. Santell, M. A.
Dwyer, M. P. O’Connell, R. A. Lazarus, Nature 2000, 404, 465–470.
 M. Roberge, L. Santell, M. S. Dennis, C. Eigenbrot, M. A. Dwyer, R. A.
Lazarus, Biochemistry 2001, 40, 9522–9531.
 N. Shimba, A. M. Nomura, A. B. Marnett, C. S. Craik, J. Virol. 2004, 78,
 E. N. Shiozaki, Mol. Cell 2003, 11, 519–527.
 A. Schweizer, Structure 2007, 15, 625–636.
 H. Kolmar, FEBS J. 2008, 275, 2684–2690.
 S. C. Gopinath, Thromb. Res. 2008, 122, 838–847.
 S. Mat?as-Rom?n, B. G. G?lvez, L. Genis, M. Y?Çez-M?, G. de la Rosa, P.
S?nchez-Mateos, F. S?nchez-Madrid, A. G. Arroyo, Blood 2005, 105,
 N. Obermajer, A. Premzl, T. Zavasnik Bergant, B. Turk, J. Kos, Exp. Cell
Res. 2006, 312, 2515–2527.
 H. H. Petersen, M. Hansen, S. L. Schousboe, P. A. Andreasen, Eur. J. Bio-
chem. 2001, 268, 4430–4439.
 R. Ganesan, C. Eigenbrot, D. Kirchhofer, Biochem. J. 2010, 430, 179–189.
 C. J. Farady, P. F. Egea, E. L. Schneider, M. R. Darragh, C. S. Craik, J. Mol.
Biol. 2008, 380, 351–360.
 Y. Wu, Proc. Natl. Acad. Sci. USA 2007, 104, 19784–19789.
 P. Rezacova, J. Lescar, J. Brynda, M. Fabry, M. Horejsi, J. Sedlacek, G. A.
Bentley, Structure 2001, 9, 887–895.
 J. Gao, S. S. Sidhu, J. A. Wells, Proc. Natl. Acad. Sci. USA 2009, 106,
 B. Leader, Q. J. Baca, D. E. Golan, Nat. Rev. Drug Discovery 2008, 7, 21–
 A. Lehmann, Curr. Opin. Investig. Drugs 2006, 7, 282–290.
Received: August 4, 2010
Published online on November 4, 2010
? 2010 Wiley-VCH Verlag GmbH&Co. KGaA, WeinheimChemBioChem 2010, 11, 2341–2346
C. S. Craik et al.