cells

Review
The Rebirth of Matrix Metalloproteinase Inhibitors:
Moving Beyond the Dogma
Gregg B. Fields 1,2
1 Institute for Human Health & Disease Intervention, Department of Chemistry & Biochemistry, and the
Center for Molecular Biology & Biotechnology, Florida Atlantic University, Jupiter, FL 33458, USA;
[email protected]; Tel.: +1-561-799-8577
2 Department of Chemistry, The Scripps Research Institute/Scripps Florida, Jupiter, FL 33458, USA




Received: 2 August 2019; Accepted: 26 August 2019; Published: 27 August 2019


Abstract: The pursuit of matrix metalloproteinase (MMP) inhibitors began in earnest over three decades
ago. Initial clinical trials were disappointing, resulting in a negative view of MMPs as therapeutic
targets. As a better understanding of MMP biology and inhibitor pharmacokinetic properties emerged,
it became clear that initial MMP inhibitor clinical trials were held prematurely. Further complicating
matters were problematic conclusions drawn from animal model studies. The most recent generation
of MMP inhibitors have desirable selectivities and improved pharmacokinetics, resulting in improved
toxicity profiles. Application of selective MMP inhibitors led to the conclusion that MMP-2, MMP-9,
MMP-13, and MT1-MMP are not involved in musculoskeletal syndrome, a common side effect observed
with broad spectrum MMP inhibitors. Specific activities within a single MMP can now be inhibited.
Better definition of the roles of MMPs in immunological responses and inflammation will help inform
clinic trials, and multiple studies indicate that modulating MMP activity can improve immunotherapy.
There is a U.S. Food and Drug Administration (FDA)-approved MMP inhibitor for periodontal disease,
and several MMP inhibitors are in clinic trials, targeting a variety of maladies including gastric cancer,
diabetic foot ulcers, and multiple sclerosis. It is clearly time to move on from the dogma of viewing
MMP inhibition as intractable.

Keywords: matrix metalloproteinase; protease inhibitor; cancer; arthritis; wound healing

1. Introduction
Activities of the matrix metalloproteinase (MMP) family (Figure 1) have long been correlated
to disease initiation and progression [1–6]. For example, collagenolytic activity had been observed
in tumor tissues, cells, and extracts since the late 1960s [7–12]. Initially, MMPs were considered
as potential targets in cancer and osteoarthritis [13–16]. Subsequently, roles for MMPs emerged in
neurodegenerative, infectious, and cardiovascular diseases [17]. MMPs can be present in three forms,
latent (zymogen), activated, and activated but in complex with natural inhibitors such as tissue inhibitor
of metalloproteinases (TIMPs). During conditions where active MMPs are overproduced, the regulation
by TIMPs may be overwhelmed [18]. Developing inhibitors of the transcription, activation, or activity
of MMPs thus represents an attractive therapeutic prospect [19].
Members of the MMP family are also essential for the maintenance of healthy processes, such as
wound healing (e.g., MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, and MT1-MMP),
inflammatory response (e.g., MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP,
MMP-19, and MMP-28), and angiogenesis (e.g., MMP-2, MMP-9, and MT1-MMP) [5,20–23]. Thus,
inhibiting all MMP family members at the same time would be detrimental, as indicated by early
clinical trials conducted with broad spectrum MMP inhibitors [24,25]. In addition to problems of
selectivity, the low bioavailability and poor metabolic profile of broad spectrum MMP inhibitors led

Cells 2019, 8, 984; doi:10.3390/cells8090984 www.mdpi.com/journal/cells

Cells 2019, 8, 984 2 of 24

to limited beneficial effect and thus did not justify the further pursuit of clinical trials [18]. Overall,
the previous poor performance of MMP inhibitors in clinical trials has been attributed to (a) inhibition
of other metalloenzymes, (b) lack of specificity within the MMP family, (c) poor pharmacokinetics,
(d) dose-limiting side effects/toxicity, (e) in vivo instability, and (f) low oral availability/inability to assess
inhibition efficacy [26,27]. New classes of MMP inhibitors need to address these factors. In addition,
as the role of MMPs may change from detrimental to beneficial, or vice versa, during the course of
disease, the mechanism and/or substrate profile of MMPs needs to be clearly identified when designing
clinical trial protocols. Finally, there needs to be a precise understanding as to which MMPs are relevant
in specific
Cells 2019, 8,diseases,
x FOR PEERand the forms (active versus inactive) that these MMPs may be present in. 2 of 24
REVIEW

Figure 1. Schematic depiction of the domains of a representative MMP family member, membrane
Figure 1. Schematic depiction of the domains of a representative MMP family member, membrane
type 1 matrix metalloproteinase (MT1-MMP).
type 1 matrix metalloproteinase (MT1-MMP).
2. Problems Associated with MMP Inhibition, Real and Imagined
Members of the MMP family are also essential for the maintenance of healthy processes, such as
2.1. MSShealing
wound Induced(e.g.,
by Broad Spectrum
MMP-1, MMP-2,Inhibitors
MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, and MT1-MMP),
inflammatory response (e.g., MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MT1-
Initial strategies for designing MMP inhibitors focused on the necessity of Zn2+ in the catalytic
MMP, MMP-19, and MMP-28), and angiogenesis (e.g., MMP-2, MMP-9, and MT1-MMP) [5,20–23].
mechanism [14,28]. Small molecules such as hydroxylamine, which has the property of chelating
Thus, inhibiting all MMP family members at the same time would be detrimental, as indicated by
Zn2+ , were utilized as templates to create inhibitors with strong Zn2+ chelating properties [13,14].
early clinical trials conducted with broad spectrum MMP inhibitors [24,25]. In addition to problems
Unfortunately, clinical trials using broad spectrum inhibitors often led to severe side effects and had
of selectivity, the low bioavailability and poor metabolic profile of broad spectrum MMP inhibitors
to be discontinued [18,29,30]. More specifically, the poor selectivity of broad spectrum inhibitors
led to limited beneficial effect and thus did not justify the further pursuit of clinical trials [18]. Overall,
resulted in musculoskeletal triad (arthralgia, myalgia, tendinitis)/musculoskeletal syndrome (MSS)
the previous poor performance of MMP inhibitors in clinical trials has been attributed to (a) inhibition
and gastrointestinal disorders [31]. Development of roughly thirty MMP inhibitor-based anti-arthritic
of other metalloenzymes, (b) lack of specificity within the MMP family, (c) poor pharmacokinetics,
drugs were discontinued in clinical trials due to the occurrence of MSS [32–34].
(d) dose-limiting side effects/toxicity, (e) in vivo instability, and (f) low oral availability/inability to
MSS does not appear to be caused by the off-target inhibition of a single enzyme, but rather inhibition
assess inhibition efficacy [26,27]. New classes of MMP inhibitors need to address these factors. In
of a combination of several MMPs and/or possibly other related enzymes [35]. Among enzymes initially
addition, as the role of MMPs may change from detrimental to beneficial, or vice versa, during the
proposed to play role in MSS development were MMP-1 [35], MMP-2 [36], MMP-9 [37], MT1-MMP [38], and
course of disease, the mechanism and/or substrate profile of MMPs needs to be clearly identified
ADAM family members [35]. MSS was also attributed to combined inhibition of MMP-1 and ADAM17 [39]
when designing clinical trial protocols. Finally, there needs to be a precise understanding as to which
or ADAM10 and ADAM17 [30]. A pyrimidine-2,4,6-trione derivative that inhibited MT1-MMP, MMP-2,
MMPs are relevant in specific diseases, and the forms (active versus inactive) that these MMPs may
and MMP-9 was not associated with MSS [40], and no MSS was observed in animals treated with the
be present in.
MMP-13 selective inhibitor ALS 1-0635, even at an ALS 1-0635 concentration 200-fold greater than that of
marimastat known to induce this condition [41]. Thus, MMP-2, MMP-9, MMP-13, and MT1-MMP are
2. Problems Associated with MMP Inhibition, Real and Imagined
no longer considered to be involved in MSS. MSS has been evaluated in rat models, which may have
certain
2.1. MSScaveats (asbyrodents
Induced have MMP-1a
Broad Spectrum as a homolog to human MMP-1 [42]), but broad spectrum
Inhibitors
Initial strategies for designing MMP inhibitors focused on the necessity of Zn2+ in the catalytic
mechanism [14,28]. Small molecules such as hydroxylamine, which has the property of chelating Zn2+,
were utilized as templates to create inhibitors with strong Zn2+ chelating properties [13,14].
Unfortunately, clinical trials using broad spectrum inhibitors often led to severe side effects and had
Cells 2019, 8, 984 3 of 24

MMP inhibition results in fibroproliferation affecting the patellar tendon and other intra- and periarticular
connective tissues in rats, mimicking MSS.

2.2. Off-Target Interactions of Hydroxamic Acid-Based Inhibitors

Hydroxamic acid-based inhibitors designed for MMPs, such as BB-94, were found to inhibit
ADAM family members, such as ADAM17, more potently then MMPs [43]. Hydroxamic acids
have poor selectivity for Zn2+ over other divalent first row transition metals [44]. Activity-based
protein profiling probes [45] were designed based on GM6001, a hydroxamic acid that chelates Zn2+
and was thought to selectively inhibit MMPs. When analyzing the metalloproteinases increased in
invasive melanoma, the enzymes that were affinity-purified were neprilysin (an integral membrane
metalloproteinase), leucine aminopeptidase, and dipeptidylpeptidase III [45]. These results suggest
that nonspecific inhibition of metalloproteinases (not limited to the MMP family) may explain the
toxicity observed with MMP inhibitors in clinical trials [33,46,47].
A strong chelator does not necessarily result in nonspecific metalloenzyme inhibition [48]. A variety
of chelators with greater selectivity for Zn2+ (compared with hydroxamic acids) have been developed
as MMP inhibitors [48,49].

2.3. Metabolic Instability of Hydroxamic Acid-Based Inhibitors

In addition to a lack of selectivity, hydroxamic acids are problematic compounds due to their
susceptibility to a variety of metabolic events. Hydroxamic acids can be hydrolyzed to carboxylic
acids [50,51], which are typically less active than the parent compound. In addition, hydrolysis results
in the production of hydroxylamine, a toxic byproduct [52]. Hydroxamic acids may also be reduced to
amides [53], or O-glucuronylated or O-sulfated [51,54,55]. Hydroxamic acid stability in plasma is
dependent upon esterase activity [56], specifically arylesterases and carboxylesterases [57]. The stability
of hydroxamic acid-containing compounds is dependent upon (a) the accessibility of the esterase to
the hydroxamic acid, (b) favorable or unfavorable interactions of the esterase with other constituents
present in the compound, (c) compound hydrophilicity (logP), and (d) number of hydrogen bond
donors in the compound [57]. Significant differences have also been noted in hydroxamic acid stability
in rat versus human plasma [57].

2.4. Animal Models

Genetically modified (knockout) mice have been utilized to delineate the roles MMPs in normal
physiological function and pathological states. Unfortunately, the identification of passenger mutations
that accompanied MMP knockouts raised serious concerns as to the interpretation of results from
disease models in which MMPs were implicated [58]. For example, the protection of Mmp7, Mmp8,
or Mmp13 null mice from lipopolysaccharide lethality [59–61] may well be due to a Casp11-inactivating
passenger mutation [58,62].
The passenger mutation effect may lead to questions about knockout animal results when, in fact,
the results have been interpreted correctly. For example, MMP-8 knockout mice were used to determine the
role of MMP-8 in wound healing [63]. MMP-8 was found to contribute to the resolution of inflammation
and promote re-epithelization, aiding wound repair [63]. Transplantation of bone marrow from wild-type
animals restored normal wound healing in MMP-8 knockout mice [63]. As indicated above, interpretation
of activity alterations in Mmp8 null mice may be complicated due to Casp11 inactivation. However,
the positive contribution of MMP-8 to diabetic wound healing was confirmed by chemical inhibition
approaches [20,64].
High levels of MMP-8 have been correlated with fatal outcomes in sepsis [65,66]. Subsequently, MMP-8
knockout mice demonstrated increased survival compared to wild-type animals when subjected to the
cecal ligation and puncture (CLP) model of sepsis [66]. Survival was also improved upon the application of
an MMP inhibitor ((3R)-(+)-[2-(4-methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]
or GlyΨ{PO2 H-CH2 }Ile-His-Lys-Gln THPI) to wild-type mice subjected to CLP [66,67], and transplantation
Cells 2019, 8, 984 4 of 24

of bone marrow from wild-type animals into MMP-8 knockout mice subjected to CLP compared with
transplantation of bone marrow from MMP-8 knockout mice into wild-type animals subjected to CLP [68].
In the case of inhibition, the inhibitory compounds may not have been selective for just MMP-8. The effect
in sepsis may be due to a combination of, for example, MMP-8 and MMP-13 inhibition. Along these lines,
a bispecific nanobody that inhibited MMP-8 and tumor necrosis factor receptor 1 offered complete protection
in mice subjected to endotoxemia and CLP [69].
Comparison of MMP-9 knockout and wild-type mice found 34 plasma glycoproteins significantly
different between the two, including Ecm1, periostin, and fibronectin [70]. The differing proteome
background between the MMP-9 knockout and wild-type mice suggested that disease models utilizing
pharmacological inhibition versus knockout of target enzymes may have different downstream
results [70]. Indeed, it was found that CD36 (a phagocytic marker in macrophages) was reduced
post-myocardial infarction in animals treated with an MMP-9 inhibitor but increased post-myocardial
infarction in MMP-9 knockout mice [70]. It is important to note that in the knockout mice, MMP-9 was
completely removed, while the MMP inhibitor reduced MMP activity by 30% [70]. Such differences
in relative MMP-9 inhibition, as well as the aforementioned differences in proteome background,
can produce different outcomes in the two animal model systems.
An additional concern with the application of MMP knockout mice for disease models are
compensatory effects on other MMPs. For example, in the aforementioned study of the role of MMP-8
in wound healing, MMP-8 knockout mice were found to have increased levels of MMP-9 in the wound
area compared with wild-type mice [63]. In contrast, the expression pattern of MMP-13 had a more
restricted distribution in the MMP-8 knockout mice compared with the wild-type mice [63]. MMP-13
knockout mice exhibited enhanced expression of MMP-8 in wound areas compared with wild-type
mice [71]. These compensatory effects complicate interpretation of results in which inhibitor-treated
wild-type animals are compared with knockout animals.
Beyond knockout studies, there are examples of where initial interpretation of animal models
has provided incorrect results for evaluating the roles of MMPs in disease. Based on animal studies,
MMP-9 was viewed as an appropriate target for modulating colitis [72]. Clinical trials of a monoclonal
antibody that inhibits MMP-9 (GS-5745/andecaliximab) for ulcerative colitis (UC) were subsequently
undertaken [73,74]. A later study utilizing three different mouse models of colitis indicated that MMP-9
upregulation was a consequence, rather than a cause, of intestinal inflammation [75]. Clinical remission
or response was not observed in the UC trial [74]. In addition to the initial colitis animal model results
being misleading, the lack of success of GS-5745/andecaliximab in the UC clinical trial may also have
been due to the antibody having higher affinity for proMMP-9 (KD = 0.008–0.043 nM) compared with
active MMP-9 (KD = 2.0–6.6 nM) [76], and thus much of the applied inhibitor may not have been bound
to the active form of the enzyme.

2.5. The Complexity of the Protease Web

MMPs operate in linear pathways (direct substrates), amplification cascades, and regulatory circuits,
resulting in a complex “protease web” (Figure 2) [33]. Therapeutic intervention should restore a normal
MMP protease web, but is daunting in terms of possible side effects due to MMP inhibition [77].
It has been recommended that a systems biology approach be utilized to predict consequences of
MMP inhibition [77]. Although many substrates have been identified and validated for MMPs [17,78],
the kinetics for substrate turnover are often not reported. In addition, in vitro activity towards a substrate
does not automatically indicate the processing of the substrate occurs in vivo. As has been noted, although
MMPs can process chemokines, lack of proteolysis of a specific chemokine has not been observed in
MMP knockout mice [79]. Since chemokine substrates of MMPs are often cleaved by multiple proteases,
proteolytic redundancy may explain why chemokines are still processed in MMP knockout mice.
MT1-MMP has a significant role in angiogenesis, whereby it can exhibit both pro-angiogenic and
anti-angiogenic behaviors [80–88]. These contrasting behaviors point to the importance of spatial
and temporal expression of MT1-MMP. Similar concerns have arisen for MMP inhibition following
Cells 2019, 8, 984 5 of 24

Cells 2019, 8,
ischemic x FOR PEER
stroke, whereREVIEW 5 of 24
MMPs can disrupt the neurovascular system and blood–brain barrier, leading
to cell death and cerebral edema, but can also contribute to neurorepair via angio-vasculogenesis,
vivo. As has been noted, although MMPs can process chemokines, lack of proteolysis of a specific
gliogenesis, and neurogenesis [89]. The same is true in neurological infectious diseases and multiple
chemokine has not been observed in MMP knockout mice [79]. Since chemokine substrates of MMPs
sclerosis, where inhibition during acute inflammation can be beneficial while inhibition during repair
are often cleaved by multiple proteases, proteolytic redundancy may explain why chemokines are
processes can be detrimental [6].
still processed in MMP knockout mice.

Figure 2. The “protease web”, in which proteases act in linear pathways, amplification cascades (with
aFigure 2. The
one-way flow“protease web”, inorwhich
of information), proteases
in circuits (with act in linear pathways,
information amplification
feedback) [33]. cascades (with
Figure reproduced with
a one-way of
permission flow
theof information),
Nature or in
Publishing circuits (with information feedback) [33]. Figure reproduced
Group.
with permission of the Nature Publishing Group.
The complexity of the protease web is not necessarily a deterrent for inhibitor development.
Angiotensin-converting enzyme (ACE)
MT1-MMP has a significant roleis
inwell recognizedwhereby
angiogenesis, for converting
it canangiotensin
exhibit bothI to angiotensin II and
pro-angiogenic and
degrading bradykinin. ACE has an extensive substrate profile [90], including degrading
anti-angiogenic behaviors [80–88]. These contrasting behaviors point to the importance of spatial and acetyl-Pro-Gly-Pro.
Acetyl-Pro-Gly-Pro
temporal expression serves to reduce neutrophilic
of MT1-MMP. Similar concerns inflammation
have arisen and for
matrix
MMP degradation
inhibition infollowing
chronic
obstructive pulmonary
ischemic stroke, wheredisease
MMPs can (COPD) [91].the
disrupt ACE also converts
neurovascular β-amyloid
system protein 42 (Aβ42,
and blood–brain barrier,which
leadingis
neurotoxic) to Aβ40 (which is neuroprotective) [92]. Despite a complex protease web
to cell death and cerebral edema, but can also contribute to neurorepair via angio-vasculogenesis, for ACE, clinical
applications
gliogenesis, of
and ACE inhibitors have
neurogenesis existed
[89]. The same forisdecades. A recent concern
true in neurological has diseases
infectious arisen based on ACE
and multiple
inhibition resulting in increased brain amyloid deposition and apoptotic neurons in mice
sclerosis, where inhibition during acute inflammation can be beneficial while inhibition during repair [93]. Whether
this translates
processes cantobehuman disease[6].
detrimental remains to be seen.
The complexity of the protease web is not necessarily a deterrent for inhibitor development.
3. Development of Improved MMP Inhibitors
Angiotensin-converting enzyme (ACE) is well recognized for converting angiotensin I to angiotensin
II and
3.1. degrading
Creating bradykinin.
Selective ACE has an extensive substrate profile [90], including degrading acetyl-
MMP Inhibitors
Pro-Gly-Pro. Acetyl-Pro-Gly-Pro serves to reduce neutrophilic inflammation and matrix degradation
The high
in chronic homologypulmonary
obstructive within the disease
MMP family
(COPD) has[91].
made the development
ACE also converts of selective protein
β-amyloid inhibitors42
difficult. Ideally, an MMP inhibitor should exhibit a ~3 orders of magnitude difference
(Aβ42, which is neurotoxic) to Aβ40 (which is neuroprotective) [92]. Despite a complex protease in K i between
web
the targetclinical
for ACE, MMP and other MMPs
applications of ACE[32,48]. Numerous
inhibitors strategies
have existed have been
for decades. suggested
A recent concern forhas
creating
arisen
selective MMP inhibitors, including (a) application of endogenous-like inhibitors
based on ACE inhibition resulting in increased brain amyloid deposition and apoptotic neurons in (TIMP analogs),
(b)
miceexosite targeting this
[93]. Whether (i.e.,translates
HPX domain or MMP-2/MMP-9
to human fibronectin
disease remains type II inserts), (c) combination
to be seen.
of exosite binding and metal chelation, (d) function blocking antibodies, and (e) disrupting MMP
interactions
3. Development with ofcellImproved
surface binding partners (Figure 3) [94].
MMP Inhibitors
Modification of TIMPs has been explored in an attempt to design selective MMP inhibitors [95].
It3.1.
was Creating
initiallySelective
observed MMP thatInhibitors
mutation of Thr2 to Ala in TIMP-1 resulted in an inhibitor that was much
less effective towards MMP-1 compared with MMP-2 and MMP-3 [96] Additional mutations of Thr2
The high homology within the MMP family has made the development of selective inhibitors
furthered the concept that TIMPs could be selectively targeted [97]. Mutation of residues 2, 4, and/or
difficult. Ideally, an MMP inhibitor should exhibit a ~3 orders of magnitude difference in Ki between
68 produced a TIMP-1 variant (Thr2Arg/Val4Ile) that effectively inhibited MMP-2 and MMP-3, but did
the target MMP and other MMPs [32,48]. Numerous strategies have been suggested for creating
not inhibit MMP-1 [98]. In turn, mutation of Thr98 to Leu converted TIMP-1 to an effective inhibitor of
selective MMP inhibitors, including (a) application of endogenous-like inhibitors (TIMP analogs), (b)
MT1-MMP [99], although multiple substitutions were required to fully explain why wild-type TIMP-1
exosite targeting (i.e., HPX domain or MMP-2/MMP-9 fibronectin type II inserts), (c) combination of
exosite binding and metal chelation, (d) function blocking antibodies, and (e) disrupting MMP
interactions with cell surface binding partners (Figure 3) [94].
Cells 2019, 8, 984 6 of 24

was a poor inhibitor of MT1-MMP [99,100]. The TIMP-1 triple mutant Val4Ala/Pro6Val/Thr98Leu, a very
effective inhibitor of MT1-MMP (Ki = 1.66 nM) [99], was fused to a glycosyl-phosphatidyl inositol anchor
for targeting to the cell membrane [101]. Mutation of Thr2 to Gly resulted in a TIMP-1 variant with
virtually complete selectivity between MMP-9 (Ki = 2.1 nM) and MMP-2 (Ki > 40 µM), while mutation
of Thr2 to Arg and replacement of the TIMP-1 AB loop with the TIMP-2 AB loop produced a TIMP-1
variant with selectivity for MMP-2 and MMP-9 [100]. The TIMP-2 mutant Ser2Asp/Ser4Ala was
effective against MMP-1 (Ki = 34 nM), while poorly inhibiting MMP-3, MMP-7, and MT1-MMP [102].
The TIMP-2 mutant Ser4Asp/Ile35Leu/Asn38Ser/Ser68Asp/Val71Ser/His97Ser/Thr99Phe had greatly
enhanced specificity for MT1-MMP compared with MMP-9, while the TIMP-2 mutant
Ser4Pro/Ile35Pro/Asn38Trp/Ser68Asn/His97Lys/Thr99Lys offered the opposite specificity [103].
Computational and mutational analysis of TIMP-2 interactions with multiple MMPs indicated that
TIMP-2 was not optimized to bind to any specific MMP, and thus many affinity-enhancing mutations
were possible [104]. Five mutations in TIMP-2, designed to optimize interactions with MT1-MMP
(Ile35Met/Asn38Asp/Ser68Asn/Val71Gly/His97Arg), resulted in an inhibitor with Ki = 0.9 pM and
selectivity over MMP-2 and MMP-10 [105]. While much work has focused on mutations in the
N-terminal domain of TIMPs, the C-terminal domain also impacts MMP selectivity. Replacement of the
TIMP-1 C-terminal domain with the TIMP-2 C-terminal produced a chimera (T1:T2) that was a much
more effective inhibitor of MT1-MMP and MMP-19 than the wild-type TIMP-1 [106]. Subsequent
studies led to the identification of residues in both the N- and C-termini of TIMP-1 that impacted
binding to MMP-3 [107]. The importance of cooperativity between TIMP-1 N- and C-terminal domains
was clearly demonstrated, and set the stage for additional considerations in the design of mutant
TIMPs as selective metalloproteinase inhibitors. TIMP-2 mutagenesis has been used to engineer
a bi-functional protein that binds to the αv β3 integrin with KD = 130 nM and inhibited MT1-MMP
with Ki = 77 pM [108]. Concerns exist about the short half-life of TIMPs in vivo, but conjugation of
20 kDa mPEG chains to TIMP-1 Lys residues dramatically extended the half-life in mouse plasma
(from 1.1 to 28 h), yet did not significantly impact inhibitory activities either in vitro or in vivo [109].
An additional concern raised about TIMP-based inhibitors is in their ability to penetrate cartilage [110],
as needed
Cells 2019, 8, xfor
FORosteoarthritis
PEER REVIEWapplications. 6 of 24

Figure 3. Strategies for creating selective MMP inhibitors. (A) endogenous-like inhibitors, (B) exosite
Figure 3. Strategies
targeting inhibitors,for
(C)creating selective
combination MMP inhibitors.
of exosite (A) metal
binding and endogenous-like inhibitors,
chelating inhibitor, (B)function
(D) exosite
targeting inhibitors, (C) combination of exosite binding and metal chelating inhibitor, (D) function
blocking antibodies, and (E) inhibitor disrupting MMP interactions with cell surface binding partners.
blocking antibodies, and (E) inhibitor disrupting MMP interactions with cell surface binding partners.

Modification of TIMPs has been explored in an attempt to design selective MMP inhibitors [95].
It was initially observed that mutation of Thr2 to Ala in TIMP-1 resulted in an inhibitor that was
much less effective towards MMP-1 compared with MMP-2 and MMP-3 [96] Additional mutations
 There are multiple examples of MMP-13 small molecule inhibitors that do not interact with the
active site Zn2+, but rather bind via so called secondary binding/exosites or allosteric sites [78,111].
Many of these inhibitors take advantage of the MMP-13 P1’ subsite. However, distinct drawbacks to
these inhibitors have been reported. For inhibitors presented as organic anions, binding to human
organic anion transporter 3 resulted in nephrotoxicity [112]. Inhibitors possessing carboxylic acids
Cells 2019, 8, 984 7 of 24
may generate reactive metabolites through protein conjugation of the resulting acyl glucuronide
[112,113]. Pyrimidine-2-carboxamide-4-one-based inhibitors have exhibited poor bioavailability, low
volume Thereof are
distribution, poor metabolic
multiple examples of MMP-13 stability, and/or P450
small molecule 3A4 inhibition
inhibitors that do not[114]. Obtaining
interact with the
appropriate
active site Zn 2+ , but
kinetic solubilities
rather bind forvia
MMP-13
so calledinhibitors
secondaryhas binding/exosites
proven challenging [115,116].sites [78,111].
or allosteric
Many Starting
of thesefrom the 2-(arylmethylthio)-cyclopentapyrimidin-4-one
inhibitors take advantage of the MMP-13 P1 ’ subsite. However, scaffold distinct
identified in a high
drawbacks to
throughput
these inhibitorsscreen [115,117,118],
have been reported.we synthesized
For inhibitorsmore than 130 as
presented compounds with 10d
organic anions, and (S)-17b
binding to human (as
numbered
organic anion when initially3 published)
transporter emerging as the
resulted in nephrotoxicity two
[112]. most promising
Inhibitors possessinginhibitors
carboxylic(Figure
acids may 4)
[119,120]. Both compounds inhibited MMP-13 with low nanomolar IC values
generate reactive metabolites through protein conjugation of the resulting acyl glucuronide [112,113].
50 and exhibited an
excellent selectivity profile within the MMP
Pyrimidine-2-carboxamide-4-one-based family have
inhibitors (>170-fold and >1000-fold
exhibited selectivity,low
poor bioavailability, respectively,
volume of
compared
distribution, poor metabolic stability, and/or P450 3A4 inhibition [114]. Obtaining appropriateplasma
to MMP-1, MMP-2, MMP-9, and MT1-MMP) [119]. Compound (S)-17b had a long kinetic
half-life (T1/2for
solubilities = 2.93
MMP-13h) and a low clearance
inhibitors has provenratechallenging
(Cl = 0.18 mL/min/kg)
[115,116]. after IV administration in rats
[120].Starting
Due to design
from the considerations based on activity profiles and prior scaffold
2-(arylmethylthio)-cyclopentapyrimidin-4-one data, 10didentified
and (S)-17bin aavoid
high
many of the aforementioned pitfalls of MMP-13 inhibitors, particularly poor
throughput screen [115,117,118], we synthesized more than 130 compounds with 10d and (S)-17b solubility and metabolic
stability,
(as numbered as well as the
when potential
initially for nephrotoxicity
published) emerging asand the generation of reactive
two most promising metabolites.
inhibitors (Figure 4) [119,
120].Compound
Both compounds inhibitedJNJ0966 MMP-13 [N-(2-((2-methoxyphenyl)amino)-4’-methyl-[4,5'-bithiazol]-2'-
with low nanomolar IC50 values and exhibited an excellent
yl)acetamide]
selectivity profile(Figure
within 4) the
inhibited the activation
MMP family (>170-fold ofand >1000-fold
proMMP-9 and the migration
selectivity, of HT1080
respectively, cells
compared
[121]. Intratumoral
to MMP-1, MMP-2,injections
MMP-9, and of compound
MT1-MMP) NSC405020 [3,4-dichloro-N-(1-methylbutyl)benzamide]
[119]. Compound (S)-17b had a long plasma half-life
(T1/2 = 2.93 h) and a low clearance rate (Cl = 0.18 mL/min/kg) after IV administration
(Figure 4), an MT1-MMP HPX domain binder, reduced MCF7-β3/MT tumor xenograft size
in rats [120].
significantly
Due to design[122]. Screening based
considerations of oligomer libraries
on activity comprised
profiles and priorofdata,
peptoid and (S)-17b
10d and peptideavoid
tertiary
many acidof
units led to the identification
the aforementioned pitfalls ofofMMP-13
chemically diverseparticularly
inhibitors, MT1-MMP inhibitors [123].and
poor solubility Onemetabolic
drawbackstability,
is that
these aforementioned
as well as the potentialcompounds typicallyand
for nephrotoxicity possess only modest
generation affinities.
of reactive metabolites.

Figure 4. Structures of small molecule MMP inhibitors (A) 10d, (B) (S)-17b, (C) JNJ0966 [N-(2-((2-
Figure 4. Structures of small molecule MMP inhibitors (A) 10d, (B) (S)-17b, (C) JNJ0966 [N-(2-((2-
methoxyphenyl)amino)-4’-methyl-[4,5’-bithiazol]-2’-yl)acetamide], (D) NSC405020 [3,4-dichloro-N-
methoxyphenyl)amino)-4’-methyl-[4,5'-bithiazol]-2'-yl)acetamide], (D) NSC405020 [3,4-dichloro-N-
(1-methylbutyl)benzamide], (E) N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-
(1-methylbutyl)benzamide], (E) N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-
ylthio)butanamide, (F) doxycycline, (G) minocycline, and (H) (R)-ND-336.
ylthio)butanamide, (F) doxycycline, (G) minocycline, and (H) (R)-ND-336.

Compound JNJ0966 [N-(2-((2-methoxyphenyl)amino)-4’-methyl-[4,5’-bithiazol]-2’-yl)acetamide]

(Figure 4) inhibited the activation of proMMP-9 and the migration of HT1080 cells [121]. Intratumoral
injections of compound NSC405020 [3,4-dichloro-N-(1-methylbutyl)benzamide] (Figure 4), an MT1-MMP
HPX domain binder, reduced MCF7-β3/MT tumor xenograft size significantly [122]. Screening of
oligomer libraries comprised of peptoid and peptide tertiary acid units led to the identification of
chemically diverse MT1-MMP inhibitors [123]. One drawback is that these aforementioned compounds
typically possess only modest affinities.
As discussed earlier, hydroxylamine was utilized as a template to create MMP inhibitors possessing
hydroxamic acid moieties. Hydroxamic acids bind Zn2+ in a bidentate fashion [44]. Carboxylic acids
bind Zn2+ in a monodentate fashion, and thus are more weakly bound to the MMP active site Zn2+ than
hydroxamic acids [44]. The weaker binding to Zn2+ could reduce nonspecific inhibition. Phosphinic
acids bind Zn2+ in a monodentate fashion, as one phosphinic oxygen binds the MMP active site
Cells 2019, 8, 984 8 of 24

Zn2+ while the second phosphinic oxygen hydrogen bonds to the active site Glu [44]. In addition to
a possible advantage due to weaker Zn2+ binding, phosphinic acids may have improved metabolic
stability compared with hydroxamic acids [48].
A variety of phosphorus-based peptides have been developed as MMP inhibitors, including
phosphonates/phosphonic acids [124–131], phosphoramidates [132–134], phosphonamidates [133,135,136],
and phosphinates/phosphinic peptides [136–148]. Phosphinates/phosphinic peptides contain a hydrolytically
stable, tetrahedral phosphinic pseudo-dipeptide moiety which mimics the tetrahedral intermediate formed
during Zn2+ metalloproteinase-catalyzed amide bond hydrolysis. Phosphinic peptides offer the possibility of
interacting with both the primed and unprimed sides of the MMP active site cleft [43]. We have reported that
phosphinic triple-helical peptides (THPs) behave as effective transition state analog inhibitors of collagenolytic
MMPs [67,149–152]. These triple-helical peptide inhibitors (THPIs) have proven effective in mouse models of
multiple sclerosis, sepsis, and myocardial infarction [67,153]. THPs have been found to be reasonably stable to
general proteolysis, as observed in vitro in mouse, rat, and human serum and/or plasma and in vivo in mice
and rats [154–159]. THPs were found to exhibit high stability (72%) when administered orally to rats [160].
Function blocking metallobodies (SDS3 and SDS4) were generated by immunization directed at
the catalytic Zn2+ and enzyme surface epitopes in activated MMP-9 [161]. SDS3 was shown, in both
prophylactic and therapeutic applications, to protect mice from dextran sodium sulfate-induced
colitis [161].
Numerous MMP inhibitory antibodies have been developed [23,26,95,162]. Mouse mAb REGA-3G12,
a selective inhibitor of MMP-9 [163], prevented interleukin-8-induced mobilization of hematopoietic
progenitor cells in rhesus monkeys [164]. Two monoclonal anti-MMP-9 antibodies, AB0041 and AB0046,
were shown to inhibit tumor growth and metastasis in a model of colorectal carcinoma [72]. A humanized
version of AB0041, GS-5745/andecaliximab, was generated for use in clinical trials [72]. As mentioned
earlier, GS-5745 inhibited proMMP-9 activation and non-competitively inhibited MMP-9 activity [76].
DX-2400, a selective, fully human MT1-MMP antibody inhibited metastasis in a breast cancer
xenograft mouse model [165]. DX-2400 also enhanced tumor response to radiation therapy [166].
Two humanized scFv antibodies, CHA and CHL, generated against the MT1-MMP HPX domain
inhibited HT1080 invasion of type I collagen [167]. Human scFv-Fc antibody E3 bound to the MT1-MMP
CAT domain and inhibited type I collagen binding [168]. A second generation E3 clone (E2_C6) inhibited
tumor growth and metastasis [168]. Human antibody Fab libraries were synthesized, where the Peptide
G sequence (Phe-Ser-Ile-Ala-His-Glu) [169] was incorporated into complementarity-determining region
(CDR)-H3 [170]. The resulting Fab 1F8 inhibited MT1-MMP CAT domain activity [170]. Screening of
a phage displayed synthetic humanized Fab library led to the identification of Fab 3369, which inhibited
the activity of the MT1-MMP CAT domain [171]. IgG 3369 treatment of MDA-MB-231 mammary
orthotopic xenograft mice reduced lung metastases, collagen processing, and tumor density of CD31+
blood vessels [171].
mAb 9E8 inhibited MT1-MMP activation of proMMP-2, but not other MT1-MMP catalytic
activities [172]. Antibody LOOPAb also inhibited MT1-MMP activation of proMMP-2 but not MT1-MMP
collagenolysis [173]. These antibodies represent approaches in which a specific activity of an MMP is
inhibited, which would prove especially beneficial in cases where maintaining certain activities of the
MMP are desirable to support normal physiological functions.
The LEM-2/15 antibody was generated using a cyclic peptide mimicking the MT1-MMP CAT
domain V-B loop (residues 218-233) [174]. A minimized Fab fragment of LEM-2/15 [175] significantly
increased the ability of virally infected mice to fight off secondary Streptococcus pneumoniae bacterial
infection [176].
It has been noted that antibody antigen binding sites are not complementary to the concave shape of
catalytic clefts, as antigen binding sites are planar or concave [95]. To overcome this, the convex-shaped
paratope of camelid antibodies was incorporated into the human antibody scaffold [177]. The resulting
Fab 3A2 was a competitive MT1-MMP inhibitor which reduced metastasis in a melanoma mouse
model [178].
Cells 2019, 8, 984 9 of 24

There are several inhibitors that act by disrupting interactions of MMPs with cell surface binding
partners. The MMP-9 HPX domain binds to B chronic lymphocytic leukemia (B-CLL) cells via the
α4β1 integrin [179]. A variant of the MMP-9 HPX domain containing blades III and IV (B3B4)
interacted with the α4β1 integrin, and a peptide derived from this region (peptide P3, residues
654-674, PFPGVPLDTHDVFQYREKAYFC) inhibited cell adhesion. A truncated version of peptide
P3 (P3a, FPGVPLDTHDVFQYREK) inhibited B-CLL and MEC-1 cell adhesion to proMMP-9 and
inhibited B-CLL cell transendothelial migration and intracellular survival signals. The compound
N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-ylthio)butanamide (Figure 4) prevented
association of proMMP-9 with the α4β1 integrin and CD44, resulting in the dissociation of epidermal
growth factor receptor (EGFR) from the β1 integrin subunit and CD44 [180].
Interaction of CD44 with MT1-MMP was found to require the outermost strand of HPX domain
blade I [181]. A peptide derived from this blade, IS4 (acetyl-VMDGYPMP-NH2 ), inhibited MT1-MMP
mediated cell migration and metastasis in vivo [181].

3.2. Timing of MMP Inhibition

Often overlooked is that the timing of MMP inhibitor application may be critical to achieve the
desired therapeutic effect. Application of marimastat in combination with gemcitabine significantly
improved survival in pancreatic cancer patients with disease confined to the pancreas [182].
Presurgical treatment with the oral MMP inhibitor SD-7300 (N-hydroxy-1-(2-methoxyethyl)-4-
[4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl]sulfonylpiperidine-4-carboxamide, with high inhibitory
activity towards MMP-2, MMP-9, and MMP-13) improved survival from 67 to 92% in a mouse breast
cancer model and significantly decreased the risk of recurrence [183]. The “window of opportunity” for
MMP inhibitor application in cancer is often in premetastatic disease [30,184–186]. In the future, MMP
inhibitors may need to be considered in metastasis prevention trials.
MMP-9 is a key contributor to the “angiogenic switch” during carcinogenesis of pancreatic
islets [187]. However, MMP-9 deficiency in pancreatic ductal adenocarcinoma (PDAC) mouse models
resulted in more invasive tumors and an increase in desmoplastic stroma [188]. The absence of MMP-9
led to increased interleukin 6 levels in the bone marrow, which activated tumor cell STAT3 signaling
and promoted PDAC invasion and metastasis [188]. Thus, MMP-9 represents an anti-target in the later
stage of pancreatic cancer.

3.3. Side Effects of Selective MMP Inhibitors

The monoclonal anti-MMP-9 antibody GS-5745/andecaliximab has been evaluated under several
clinical trials. The outcome of a randomized placebo controlled phase 1b single and multiple ascending
dose-ranging clinical trial on 72 patients diagnosed with moderately to severely active UC showed
that GS-5745/andecaliximab was safe and well tolerated [73]. A phase 2/3 UC study with 165 patients
treated over 8 weeks further indicated that GS-5745/andecaliximab was well tolerated [74]. A phase
1b trial investigating the safety, pharmacokinetics, and disease-related outcomes for 15 rheumatoid
arthritis patients demonstrated that GS-5745/andecaliximab was safe, with adverse events that were
only grade 1 or 2 in severity and no indication of MSS [189]. A phase 1 GS-5745/andecaliximab
dose-finding trial of 13 patients with advanced solid tumors found no dose-limiting toxicity at any
concentration (200, 600, or 1800 mg IV every 2 weeks), with adverse events that were only grade 1 or 2
in severity [190]. These clinical trials provide further evidence that selective MMP inhibitors can be
developed without inducing MSS.

4. The Outlook for Current MMP Inhibitors

The lack of success in early MMP clinical trials has created a strong negative bias toward MMP
inhibition as a clinical strategy. In actuality, MMP clinical trials were undertaken prematurely [191,192],
due to insufficient knowledge of MMP biology and the choice of hydroxamic acid as the active
component of the inhibitors. Unique modes of action have been used to develop the next generation
Cells 2019, 8, 984 10 of 24

of MMP inhibitors. Clinical trials utilizing antibodies have provided evidence that selective MMP
inhibitors do not induce MSS. The therapeutic potential of anti-MMP antibodies has yet to be realized,
with one concern being limited extravascular distribution [189,193]. Antibodies are also subject to
proteolysis, may be removed from circulation rapidly, and are costly. Nonetheless, antibodies have
provided truly selective, high affinity MMP inhibitors. In addition, selective, high affinity inhibitors
can be developed for MMPs based on triple-helical structure. Triple-helical peptide inhibitors (THPIs)
have excellent pharmacokinetic properties compared with other peptide-based therapeutics.

4.1. MMP Inhibition and the Immune System

It has been proposed that the major role of MMPs is in modulation of inflammatory and immune
processes [17,79]. MMP processing can either activate or inhibit a variety of chemokines and cytokines [79,194].
For example, MMP-9 cleaves interleukin-8 and increases the activity of the chemokine 10-fold for neutrophil
activation and chemotaxis [195]. In turn, neutrophils release a TIMP-free form of MMP-9 which promotes
angiogenesis [196]. Overall, MMPs can cleave virtually all human chemokines [197]. A better understanding
of MMPs in the immune system and in inflammatory diseases may be necessary before proceeding with
cancer-based clinical trials [191].
Insight into the roles of MMPs in the immune system has revealed potential applications of
MMP inhibitors to improve immunotherapy. Monoclonal MMP-9 antibody AB0046, which inhibited
tumor growth and metastasis in a surgical orthotopic xenograft model of colorectal carcinoma [72],
improved immune responses to tumors, as the inhibition of MMP-9 reversed MMP-9 inactivation of
T-cell chemoattractant CXCR3 ligands (CXCL9, CXCL10, and CXCL11) [198]. MT1-MMP shed tumor
cell MHC class I chain-related molecule A (MICA) [199]. Engagement of MICA to NKG2D stimulated
natural killer (NK) and T-cell antitumor activity [199]. Protection of MICA stimulated antitumor
immunity and reduced metastasis in a humanized melanoma mouse model [200].
Examination of tumor cryosections in an MT1-MMP antibody treated MDA-MB-231 triple-negative
breast cancer xenograft mouse model revealed an increased density of iNOS+ cells (a marker of anti-tumor
M1 tumor-associated macrophages) and Granzyme B+ cells [171]. Application of an MT1-MMP antibody
to a 4T1 triple-negative breast cancer mouse model shifted macrophages towards the antitumor M1-like
phenotype and reduced activated TGFβ (an immunosuppressive cytokine) [166].

4.2. MMP Inhibitors in the Clinic and in Clinical Trials

There is an established history of clinically successful MMP inhibitors. Periostat, which is doxycycline
(Figure 4) formulated at a sub-antimicrobial dose, is a U.S. Food and Drug Administration (FDA)-approved
MMP inhibitor for periodontal disease treatment [201–204]. Doxycycline is a broad-spectrum MMP
inhibitor with IC50 values in the range of 2–50 µM [18]. Tetracyclines have shown promise in other MMP
indications. Treatment of 16 relapsing–remitting multiple sclerosis patients with a doxycycline/interferon
combination for 4 months (ClinicalTrials.gov Identifier NCT00246324) reduced brain lesions and MMP-9
serum levels and improved Expanded Disability Status Scale values [205]. The treatment was considered
effective, safe, and well tolerated [205,206]. In other trials for relapsing–remitting multiple sclerosis,
minocycline (Figure 4) treatment resulted in no new active lesions after 1 month with detection up to
6 months for 10 patients [207] while conversion from a first demyelinating event (clinically isolated
syndrome) to multiple sclerosis was lowered by 18.5% over 6 months but was not lowered over 24 months
for 72 patients [208]. Combination treatment of minocycline with glatiramer acetate, compared with
glatiramer alone, in 44 patients showed the combination therapy reduced the number of lesions by
63–65% and lowered the risk of relapse after 8–9 months [209]. The treatment was considered safe and
well tolerated [209]. Additional clinical trials using minocycline for multiple sclerosis treatment are
reportedly upcoming [6]. In similar fashion to doxycycline, minocycline is a broad-spectrum MMP
inhibitor, with IC50 values in the 100–300 µM range [18].
Minocycline has also been examined for improving neurological outcomes following stroke [210,211].
MMP-9 levels are increased following cerebral ischemia, and are further enhanced by tissue plasminogen
Cells 2019, 8, 984 11 of 24

activator (tPA) stroke treatment [212–214]. Administered within 6 h of stroke symptom onset in 60 patients,
minocyline was well tolerated up to 10 mg/kg intravenous administration and had a half-life of 24 h [210].
Minocycline decreased MMP-9 levels at 24 h and to baseline in 72 h in 36 patients treated with tPA [211].
Incyclinide (a.k.a. Metastat, COL-3), a chemically modified tetracycline with no antibiotic activity,
showed a significant regression of AIDS-related Kaposi’s sarcoma with a significant reduction of MMP-2
and MMP-9 plasma levels [215].
Clinical trials using anti-MMP antibodies have moved forward. GS5745/andecaliximab in combination
with mFOLFOX6 (oxaliplatin, leucovorin, and 5-fluorouracil) was utilized in a phase I clinical trial of
40 patients with HER2-negative gastric and gastroesophageal junction adenocarcinoma [190]. The overall
response rate for all patients was 47.5% (7.5% complete response and 40% partial response), with no
observed MSS [190]. As described earlier, one concern about GS5745/andecaliximab is the much higher
affinity for proMMP-9 compared with active MMP-9 [76].
Topical application of MMP inhibitors may also prove to be efficacious. In human diabetic foot ulcers,
levels of active MMP-8 and MMP-9 are increased [216]. MMP-9 has been identified as a limiting factor in
healing, while MMP-8 facilitates wound repair [20,64,216]. The compound (R)-ND-336 (Figure 4) selectively
inhibits MMP-2, MMP-9, and MT1-MMP with Ki < 100 nM, while weakly inhibiting MMP-8 [216]. Thus,
(R)-ND-336 has the desired inhibitory profile for application to diabetic foot ulcers. Additionally, (R)-ND-336
is a slow binding inhibitor of MMP-2, MMP-9, and MT1-MMP with a long residence time [216]. (R)-ND-336
is more efficacious than becaplermin in animal models of wound healing, and thus represents a promising
topical treatment for diabetic foot ulcers [64,216]. Clinical trials for (R)-ND-336 utility are anticipated
(https://ideacenter.nd.edu/news-events/news/new-hope-for-the-treatment-of-diabetic-foot-ulcers/).
A related, promising therapeutic approach is the use of MMPs to activate prodrugs and imaging
agents or facilitate drug delivery [27,217–221]. A concern with these approaches is the “selectivity”
of the peptide sequences used for activation [217–219,222]. ICT2588 is a vascular disrupting agent
designed to be activated by MT1-MMP, containing an Arg-Ser-Cit-Gly~Hof-Tyr-Leu-Tyr sequence
(where Cit is citrulline and Hof is homophenylalanine) with azademethylcolchicine at the C-terminus
and fluorescein (FITC) at the N-terminus [223,224]. Azademethylcolchicine functions as the vascular
disrupting agent. Following hydrolysis at the Gly~Hof bond by MT1-MMP, exopeptidase activity
removes the remaining Hof-Tyr-Leu-Tyr sequence to liberate azademethylcolchicine [223]. ICT2588
produced a 90% decrease in functional tumor vasculature in HT1080 tumor-bearing mice, and
co-administration of ICT2588 and doxorubicin was significantly more effective in reducing tumor
volume compared with either ICT2588 or doxorubicin alone [223]. Conjugation of a variant of ICT2588
(FITC-βAla-Cys-Arg-Ser-Cit-Gly~Hof-Tyr-Leu-Tyr-azademethylcolchicine) with a cross-linked iron
oxide (CLIO) nanocarrier resulted in a theranostic that allowed for magnetic resonance imaging
of drug delivery and accumulation in tumors [220,225]. Co-administration of the CLIO-ICT with
temozolomide resulted in decreased tumor size and increased survival in glioblastoma xenograft
mice [226]. ICT2588 was scheduled to begin Phase 1 clinical trials in 2018 (http://www.incanthera.com/
what-we-do/technology-pipeline/).

5. Conclusions
There is considerable promise in the recent generation of MMP inhibitors. Valuable lessons were
learned from prior clinical trial failures, including the need for selective and metabolically stable inhibitors.
Proteomics approaches have better defined the impact of MMP activity on biological systems. Short-term
indications may be needed when anti-target activities are of concern. While there is great reliance on
mouse models for examining MMP in vivo behaviors, it has been recognized that animal models may not
clearly reflect human conditions and mechanism of disease. Consideration of MMP secondary binding
sites (exosites) offers the best opportunity for development of selective inhibitors. The FDA approval of
an MMP inhibitor and the advancement of numerous MMP inhibitors to clinical trials clearly indicates
that it is time to depart from the dogma of viewing MMP inhibition as intractable.
Cells 2019, 8, 984 12 of 24

Author Contributions: Writing—original draft preparation, review, and editing, G.B.F.

Funding: MMP inhibitor studies in the author’s laboratory have been supported by the National Institutes of Health
(CA098799, CA239214, AR063795, MH078948, and NHLBI Contract 268201000036C-0-0-1), the James and Esther
King Biomedical Research Program (8JK01), the US–Israel Binational Science Foundation (2015180), the Multiple
Sclerosis National Research Institute, the Center for Molecular Biology & Biotechnology at Florida Atlantic
University, and the State of Florida, Executive Office of the Governor’s Department of Economic Opportunity.
Acknowledgments: The author is most appreciative of valuable discussions, via the internet and in person, with
Paolo Mignatti, Chris Overall, Barbara Fingleton, James Quigley, Ghislain Opdenakker, Steve Weiss, Irit Sagi, and
Sharon Stack, and for the construction of Figures 1 and 3 by Ania Knapinska.
Conflicts of Interest: The author is Vice President of MMP Biopharma, Inc.

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