pharmaceuticals

Review Targeting MMP-9 in Diabetic Foot Ulcers

Jeffrey I. Jones, Trung T. Nguyen, Zhihong Peng and Mayland Chang *

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA; [email protected] (J.I.J.); [email protected] (T.T.N.); [email protected] (Z.P.) * Correspondence: [email protected]; Tel.: +1-574-631-2965



Received: 1 May 2019; Accepted: 18 May 2019; Published: 22 May 2019


Abstract: Diabetic foot ulcers (DFUs) are significant complications of diabetes and an unmet medical need. Matrix metalloproteinases (MMPs) play important roles in the pathology of wounds and in the wound healing process. However, because of the challenge in distinguishing active MMPs from the two catalytically inactive forms of MMPs and the clinical failure of broad-spectrum MMP inhibitors in cancer, MMPs have not been a target for treatment of DFUs until recently. This review covers the discovery of active MMP-9 as the biochemical culprit in the recalcitrance of diabetic wounds to healing and targeting this proteinase as a novel approach for the treatment of DFUs. Active MMP-8 and MMP-9 were observed in mouse and human diabetic wounds using a batimastat affinity resin and proteomics. MMP-9 was shown to play a detrimental role in diabetic wound healing, whereas MMP-8 was beneficial. A new class of selective MMP-9 inhibitors shows clinical promise for the treatment of DFUs.

Keywords: matrix metalloproteinase-9; diabetic foot ulcers; wound healing; MMP-9 inhibitors

1. Introduction The skin, one of the largest human organs, is responsible for three critical roles: protection from the environment, maintaining homeostasis, and sensing the surroundings. When the skin is damaged, it undergoes a process termed wound healing. This is a progression of orderly and overlapping processes that culminates in repairing damage to the skin. The four stages of wound healing are inflammation, angiogenesis, re-epithelialization, and remodeling [1]. In an acute wound, there is a smooth and orderly progression through these stages, resulting in healing in a timely manner. Chronic wounds, in contrast, stall in the inflammation stage and do not heal [2]. Stress can adversely affect wound healing [3]. Cortisol, a steroid hormone, is elevated during stress, as well as in chronic inflammation [4], and delays wound healing [5]. CYP11B1, the enzyme that catalyzes biosynthesis of cortisol [6], has been found to be strongly linked to failure of diabetic foot ulcer (DFU) to heal [7]. Elevated MMP-9 levels have been associated with increased cortisol in patients with coronary artery disease [8]. Prostaglandin E2 is known to stimulate cortisol secretion and induce MMP-9 [9]. One of the culprits in the recalcitrance is the elevated activity of matrix metalloproteinases (MMPs) [10]. MMPs are a family of zinc-dependent endopeptidases first discovered in tadpoles in 1960 [11]. There are 24 different MMPs in humans, with a wide range of substrates and functions [12]. One of the ways MMPs are classified is based on their preferred substrates, such as the gelatinases (MMP-2 and MMP-9), collagenases (MMP-1, MMP-8, and MMP-13), and stromelysins (MMP-3 and MMP-10). Due to the high structural similarities among MMPs, these enzymes share significant overlap in their substrate preferences. All MMPs are produced in inactive zymogen form that requires cleavage of the prodomain to expose the active site [13,14] (Figure1). Cleavage to the active form by proteases, other MMPs, and post-translational modifications, and allosterically induced self-cleavage, is a secondary method of regulation alongside transcription for MMPs [15–17]. MMP activity is further regulated by

Pharmaceuticals 2019, 12, 79; doi:10.3390/ph12020079 www.mdpi.com/journal/pharmaceuticals PharmaceuticalsPharmaceuticals2019 2019, 12, 12, 79, x FOR PEER REVIEW 2 2 of of 21 20

regulated by inhibition of the active forms by tissue inhibitors of metalloproteinases (TIMPs), inhibitionresulting of in the three active forms forms of byMMPs tissue in inhibitors vivo (Figure of metalloproteinases 1), of which only (TIMPs),the active, resulting uncomplexed in three MMP forms is ofcatalytically MMPs in vivo competent(Figure1 ),[18]. of which only the active, uncomplexed MMP is catalytically competent [ 18].

FigureFigure 1. 1.Three Three forms forms of of matrix matrix metalloproteinases metalloproteinases (MMPs), (MMPs), as as demonstrated demonstrated for for MMP-2: MMP-2: inactive inactive zymogenzymogen (pro-MMP), (pro-MMP), active active MMP,and MMP, inactive and inactive tissue inhibitor tissue ofinhibitor metalloproteinases of metalloproteinases (TIMP)-complexed (TIMP)- MMP.complexed Cleavage MMP. of the Cleavage prodomain of the (red) prodomain allows access (red) to allows the catalytic access site,to the while catalytic TIMPs site, block while access TIMPs to inactivateblock access the MMP.to inactivate Reproduced the MMP. from Re Nguyenproduced et al. from [13 ].Nguyen et al. [13].

DueDue to to the the limitations limitations of of these these methods methods in in studying studying MMPs, MMPs, the the identification identification of of active active MMPs MMPs involvedinvolved in in the the pathology pathology of of cancers cancers was was not not possible possible decades decades ago. ago. MMP MMP inhibitors inhibitors (MMPIs) (MMPIs) were were rushedrushed to to clinical clinical trials trials in in cancer, cancer, based based on on overexpression overexpression of of MMPs MMPs in in cancer cancer cell cell lines lines [19 [19]] and and the the protectiveprotective e ffeffectsects observed observed with with overexpression overexpression of of TIMPs TIMPs [20 [20].]. The The first-generation first-generation MMPIs MMPIs were were broad-spectrumbroad-spectrum zinc zinc chelators chelators with with poor poor bioavailability, bioavailability, which which led led to to a a second second generation generation of of orally orally bioactivebioactive broad-spectrum broad-spectrum inhibitors inhibitors[ [21].21]. However,However, indiscriminateindiscriminate inhibition inhibition of of all all MMPs MMPs resulted resulted in inundesirable undesirable side side effects, effects, such such as musculoskeletal painpain andand inflammationinflammation observedobserved during during phase phase I I clinicalclinical studies studies [22 [22].]. While While these these proved proved to to be be reversible reversible with with brief brief drug drug holidays, holidays, it it led led to to limitations limitations onon dose dose size size in in subsequent subsequent trialstrials [[21].21]. Combination phase phase II/III II/III studies studies had had disappointing disappointing results results as aswell, well, ranging ranging from from no no effect effect to decreased to decreased survival survival [23]. [23 Overall,]. Overall, more more than than50 MMPIs 50 MMPIs failed failedin clinical in clinicaltrials for trials cancer for cancer [24]. It [24 is]. now It is believed now believed that nonspecific that nonspecific chelation chelation and other and other off-target off-target effects eff ectswere wereresponsible responsible for these for these failures failures [22,25], [22, 25as], well as well as the as thelack lack of understanding of understanding that that some some MMPs MMPs can canplay playa beneficial a beneficial role role in cancer in cancer [26]. [26 As]. negative As negative results results continued continued to mount, to mount, clinical clinical trials trials of MMPIs of MMPIs came ® cameto a halt, to a halt,with withonly onlyone being one being licen licensedsed in the in United the United States, States, Periostat Periostat® (doxycycline),(doxycycline), a weak a weakbroad- broad-spectrumspectrum MMPI, MMPI, for treatment for treatment of periodontal of periodontal disease disease [27]. [However,27]. However, MMPs MMPs have have been been found found to be todysregulated be dysregulated in a wide in a widevariety variety of diseases of diseases beyond beyond cancer, cancer, such as such stroke as stroke[28–30] [ 28and–30 chronic] and chronic wounds wounds[31–33], [ 31which–33], present which present a new avenue a new avenuefor MMPIs for MMPIsto move to to move the clinic. to the This clinic. review This will review focus will on focusDFUs, onone DFUs, of the one most of thecommon most common forms of formschronic of wounds. chronic wounds. ChronicChronic wounds wounds are are defined defined as as those those that that fail fail to to go go through through the the healing healing process process in in a a timely timely manner,manner, and and are are a a significant significant burden burden on on the the healthcare healthcare system, system, costing costing an an estimated estimated 2–3% 2–3% of of the the healthcarehealthcare budget budget in in developed developed countries countries [34 [34].]. DFUs DFUs alone alone cost cost an estimatedan estimated $9–13 $9–13 billion billion annually annually in thein Unitedthe United States States [35 ].[35]. However, However, despite despite this this major major expenditure, expenditure, there there is is a lacka lack of of treatment treatment options, options, withwith debridement, debridement, offl offloading,oading, and and infection infection control cont beingrol thebeing standard-of-care. the standard-of-care. Assessment Assessment of wound of severitywound in severity DFUs is in made DFUs using is made the Wagner using grade the Wagner system, ranginggrade system, from 1, ranging indicating from a superficial 1, indicating ulcer, a tosuperficial 5, denoting ulcer, a complete to 5, denoting foot gangrene a complete [36 ].foot In ga casesngrene that [36]. progress In cases to grades that progress 4 and 5, to the grades recourse 4 and is5, sadly the recourse amputation, is sadly which amputation, has significant which e ffhasects significant on the quality effects of on life the and quality also is of associated life and also with is highassociated mortality, with 44% high after mortality, one year [44%34,37 after]. Chronic one year wounds [34,37]. typically Chronic stall wounds in the inflammation typically stall stage in ofthe woundinflammation healing, stage where of it wound is believed healing, that excessivewhere it is MMP believed activity that plays excessive a role MMP in preventing activity theplays wound a role fromin preventing healing [38 the,39 ].wound from healing [38,39].

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Many MMPs have been identified in chronic wounds: MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, and MMP-14 [31,40]. However, these MMPs were identified using methods that do not distinguish among the three forms of MMPs. Only active MMPs in the absence of regulation by TIMPs are catalytically competent to play roles in the pathology of chronic wounds. In addition, not all identified MMPs play a deleterious role in the process of wound healing, as shown by treatment with the broad-spectrum inhibitor, GM 6001, which slows healing of human wounds [41]. Given the failure of MMPIs for treatment of cancer, it is important to have a solid understanding of what roles are played by which active unregulated MMP(s).

2. Effect of Treatments on MMPs in DFUs Treatments for DFUs that do not specifically target MMPs can have important effects on MMP expression and activity. As MMP-9 has long been of interest in wound healing, most studies have focused on the effects on this proteinase in particular [42]. These effects are summarized in Table1.

Table 1. Summary of the reported effects on treatments on MMPs and how the MMPs were measured.

Method of MMP Treatment Study Material Effect on MMPs Reference Measuring Vacuum-assisted Human chronic Gelatin zymography Reduced MMP-9 and MMP-2 [43] closure (VAC) wound fluid Human chronic Reduced MMP-9/TIMP-1 ratio, no VAC ELISA [44] wound fluid change in MMP-9 Mesenchymal stem Mouse model of Gelatin zymography, Reduced MMP-9 activity and [45] cells (MSC) diabetic wounds quantitative PCR expression HaCat cells treated N-acetyl cysteine Western blot Reduced MMP-9 expression [46] with MGO Human DFU Manuka honey Increased MMP-9 expression (no patient wound ELISA [47] wound dressing improvement on ulcer healing) fluid Decreased gelatinase activity, no In-situ zymography and Mouse model of effect on collagenase activity; Becaplermin batimastat affinity resin [48] diabetic wounds decreased active MMP-9, no effect coupled proteomics on active MMP-8 Mouse model of Batimastat affinity resin Increased active MMP-9, no effect Aclerastide [49] diabetic wounds coupled proteomics on active MMP-8

2.1. Vacuum-Assisted Closure A treatment option that has drawn attention since 1993 is vacuum-assisted closure (VAC), also known as topical negative pressure [50]. VAC involves placing foam around the wound, covering with a soft dressing, and then vacuum is applied to the covered wound via drainage tubes. Multiple studies have shown that VAC promotes wound healing in chronic wounds [50–52]. A small-scale study showed a reduction in MMP-9 and MMP-2 by gelatin zymography in chronic wounds of human patients treated with VAC, though the small number of subjects precludes any meaningful statistics [43]. This sparked interest in a larger-scale follow-up study, where it was observed that the increased MMP-9/TIMP-1 ratio in conventionally treated patients is not seen in patients treated with VAC. While there was no significant change in the reported MMP-9 over the course of the study, the use of an ELISA-based assay in addition to sample handling concerns support the argument that this is an artifact of the study [44].

2.2. Mesenchymal Stem Cells Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are a promising strategy for promoting wound healing [53–55]. MSCs can be isolated from a number of sources, most importantly bone marrow and adipose tissue, the latter of which allows for easy collection with minimal discomfort [56]. Once isolated, the MSCs are re-injected back into the wound area and have been reported to significantly improve healing in a mouse model of diabetic ulcers [57]. Another Pharmaceuticals 2019, 12, 79 4 of 21 method of treatment is to impregnate a hydrogel with MSCs, an approach that shows efficacy in a mouse model of diabetic wounds [58]. Recently, treatment with MSCs in a diabetic animal model was shown to significantly reduce MMP-9 expression by gelatin zymography, and upregulate microRNA miR-29b, a known regulator for MMP-9 [45].

2.3. N-Acetyl Cysteine Another possible treatment to help improve healing in chronic wounds is N-acetyl cysteine (NAC), which has a long-track history of safe usage in a variety of diseases, ranging from chronic bronchitis to premature birth [59]. NAC has a strong anti-oxidant effect, which helps fight inflammation and combat oxidative stress [60]. In particular, it is believed to prevent the formation of advanced glycosylation end products (AGE) in diabetics [46]. There is mounting evidence that suggests AGEs are significant contributors to diabetic complications [61]. As a result, NAC has been shown to improve healing in a mouse model of diabetic wounds [62]. Methylglyoxal (MGO) is a key intermediate in AGE production [63]. Treating HaCaT cells with MGO, mimicking the condition found in diabetic tissue, induces MMP-9 expression. However, there was a significant reduction in the expression of MMP-9 by Western blot in cells treated with NAC [46].

2.4. Wound Dressings Advanced wound dressings are another possible method to help improve wound healing by combating infection given the rise of multidrug-resistant bacteria. A study looking at the effectiveness of nano-silver and Manuka honey wound dressings compared to traditional dressings in human DFUs demonstrated a small but significant reduction in ulcer size for the nano-silver dressing. The Manuka honey group did not show a significant improvement in healing, which might be explained by the dramatic increase in MMP-9 observed by ELISA, even relative to the control group [47].

2.5. Growth Factors Growth factors have been an area of considerable focus for drug development to treat DFUs, with at least 28 clinical trials conducted as of 2015. Meta-analysis of these trials found no clear evidence that treatment with growth factors can reduce the number of lower-limb amputations in DFUs [64]. Although human growth factors improve outcomes in complete wound closure, no benefits could be shown in the rate of adverse effects, in addition to concerns about bias in study design and reporting [64]. The sole Federal Drug Administration (FDA)-approved drug for treatment of DFUs is becaplermin, but a “black-box warning” was added in 2008 due to an increased rate of cancer and mortality [65], which was removed in January of 2019 after the manufacturer cited multiple studies that showed no increased safety risk. Becaplermin contains human-platelet-derived growth factor (PDGF)-BB, whereas PDGF-D has been shown to upregulate the expression of TIMP-1, which decreases MMP-2 and MMP-9 activity [66]. Recent results show that treatment of diabetic mouse wounds with becaplermim does improve healing compared to vehicle [48]. In addition, it was observed by in-situ zymography that there is a decrease in the activity of gelatinases, but no effect on collagenases [48], although dye-quenched (DQ)-gelatin is digested by gelatinases and collagenases. DQ-collagen type I fluorescein conjugate (D-12060) can be digested by MMP-1, MMP-2, and MMP-8; mice do not express MMP-1, and MMP-2 is not present in mouse diabetic wounds [67]. However, use of a batimastat affinity resin that binds only to the active forms of MMPs, followed by mass spectrometry (MS)/MS analysis, demonstrated that becaplermin decreases active MMP-9 and does not affect active MMP-8 levels [48]. Aclerastide, also known as NorLeu3-A(1–7), is a peptide analog of angiotensin II, a human growth factor [68]. Angiotensin II is known to have an important role in wound healing [69,70], and aclerastide has been reported to improve healing in diabetic mice when administered topically immediately after wound infliction once a day for 5 days [71]. A phase II clinical trial involving 77 patients receiving placebo or aclerastide showed differences in healing and time to ulcer healing in the per-protocol Pharmaceuticals 2019, 12, 79 5 of 21 group (p < 0.05), but not in the intend-to-treat population (p > 0.05) [72]. However, aclerastide failed phase III clinical trials in 2015 due to a lack of efficacy after topical administration once a day for 28 days. Recent research using a more clinically relevant dosing regimen (topical administration once a day for 14 days starting one day after wound infliction) showed that aclerastide did not accelerate wound healing in diabetic mice (p > 0.05) and that the lack of efficacy in human clinical trials is likely due to upregulation of active MMP-9 [49], as determined by a batimastat affinity resin coupled with proteomics. Previously, angiotensin II had been shown to increase the mRNA expression of MMP-9 by reverse transcription polymerase chain reaction (RT-PCR), as well as the protein expression by Western blotting [73], a method that does not distinguish between the three forms of MMP-9.

3. Affinity Enrichment Approaches to Identify MMPs As evidence for a deleterious role of MMP-9 in wound healing and its correlation with wound healing increased, MMP-9 has been suspected of having a causal role in the delayed healing of DFUs [2,42,74,75]. However, the role of MMP-9 in DFUs had not been conclusively determined because the methods used do not distinguish between the three forms of MMP-9, of which only active MMP-9 in the absence of regulation by complexation with TIMP is catalytically competent to play a role in the pathology of DFUs. Homology between the three forms of MMPs presents a significant analytical challenge in measuring only the active MMPs [76,77]. Activity-based techniques, such as gelatin zymography, are semi-quantitative at best and cannot distinguish between the TIMP-inhibited and the active form of the proteinase due to dissociation of TIMP during analysis [76]. Measurements of expression via mRNA, made using RT-PCR, do not give information about the activation from the zymogen form nor inactivation via TIMPs. Antibody-based assays, such as ELISA or Western blots, use antibodies that are not necessarily specific to the active form; thus, there is cross-reactivity between pro-MMPs, active MMPs, and TIMP-complexed MMPs [76]. An additional drawback of these methods is that they require screening for a specific MMP rather than simultaneously identifying the MMP(s) that plays critical roles in the pathology and repair of DFUs. MS, the gold-standard method for protein quantitation [78], cannot differentiate between the three forms in a standard bottom-up proteomics experiment. A conventional proteomics strategy separates proteins in a biological sample extract by 1D/2D high performance liquid chromatography (HPLC) and identifies the trypsin-digested peptides by mass spectrometry (MS)/MS. This strategy identifies thousands of proteins. In order to enrich the proteinases, an MMPI has been covalently attached to a resin, allowing for isolation of the active MMP forms alone for identification and quantitation. Initially, a bifunctional probe HxBP-Rh based on the structure of the broad-spectrum MMPI illomastat was synthesized, which contained a fluorescent rhodamine group and a photoreactive benzophenone for covalent binding to the target MMPs [79]. HxBP-Rh was demonstrated to bind active MMP-2, MMP-7, and MMP-9. To enable affinity purification, a trifunctional probe of HxBP-Rx was synthesized incorporating biotin. Application of this method coupled to MS/MS identified the metalloendopeptidase neprilysin in invasive melanoma [79]. Another approach is the broad-spectrum MMPI TAPI-2 covalently attached to Sepharose resin (Figure2A) [ 80,81], which can be packed on a cartridge and proteinases are eluted with EDTA. Recoveries of MMP-1, -7, -8, -9, -10, -12, and -13 were >96% when injected in buffer at a concentration of 0.5 µg/mL. Synovial fluid from a patient with rheumatoid arthritis was analyzed by this method coupled with gelatin zymography, and showed enrichment of MMP-9. Pharmaceuticals 2019, 12, 79 6 of 21 Pharmaceuticals 2019, 12, x FOR PEER REVIEW 6 of 20

Figure 2. Structures of (A) the TAPI-2 affinity resin and (B) the batimastat affinity resin used to capture Figure 2. Structures of (A) the TAPI-2 affinity resin and (B) the batimastat affinity resin used to active MMPs and related ADAMs (a disintegrin and metalloproteinase). The portion of the structure capture active MMPs and related ADAMs (a disintegrin and metalloproteinase). The portion of the based on TAPI-2 is indicated in red and that based on batimastat is shown in blue. (C) Recovery of structure based on TAPI-2 is indicated in red and that based on batimastat is shown in blue. (C) representative active MMPs and ADAMs by the batimastat affinity resin. Mouse tissue homogenate Recovery of representative active MMPs and ADAMs by the batimastat affinity resin. Mouse tissue was spiked with 10 pmol of proteinase and incubated with the resin. Recovery was determined from homogenate was spiked with 10 pmol of proteinase and incubated with the resin. Recovery was quantitation of the proteinase recovered after the clean-up procedure relative to the spiked amount; determined from quantitation of the proteinase recovered after the clean-up procedure relative to the mean SD, n = 3. spiked± amount; mean ± SD, n = 3. A third approach is the covalent tethering of the broad-spectrum inhibitor batimastat to Sepharose 4. The Roles of MMPs in DFUs resin (Figure2B) [ 81]. The batimastat affinity resin binds exclusively to the active forms of MMPs. As seenThe in batimastat Figure1, pro-MMPs affinity resin have is thethe prodomainonly approach (depicted that has in red)been covering applied to the the active identification site, while of TIMP-complexedactive MMPs in DFUs. MMPs Excisional have TIMP wounds (depicted in diabet in purple)ic mice blocking treated with the activethe batimastat site; only affinity the active resin MMPscoupled (Figure with 1a center) bottom-up have proteomics the active site approach available showed for binding upregulated of the a levelsffinity of resin. active Recoveries MMP-8 and of representativeMMP-9 compared MMPs to non-diabetic and ADAMs mouse (a disintegrin wounds and [67]. metalloproteinase) The question was what are > 75%roles (Figure these two2C). MMPs The limitplay of in quantitation DFUs. Answers is 6 femtomol to these [67 questions]. Application require of thisavailability method coupledof selective with MMP-8 gelatin zymographyand MMP-9 showedinhibitors. the presenceHowever, of because active MMP-2 of the structural in breast carcinoma similarity samplesbetween [ 81MMPs,]. selective inhibitors for a particular MMP are difficult to design. 4. TheThe Roles first of selective MMPs ininhibitor DFUs for the gelatinases (MMP-2 and MMP-9), referred to as SB-3CT, was reportedThe batimastat by Brown aetffi al.nity [82]. resin SB-3CT is the (Table only 2) approach inhibits thatMMP-2 has and been MMP-9 applied with to theKi values identification of 28 ± 7 ofnM active and MMPs400 ± 15 in nM, DFUs. respectively, Excisional and wounds inhibits in other diabetic MMPs mice poorly treated [82,83]. with The the selectivity batimastat of a SB-3CTffinity resintowards coupled the gelatinases with a bottom-up is attributed proteomics to mechanism- approachbased showed inhibition, upregulated in which levels Glu-404 of active at the MMP-8 active andsite MMP-9of the gelatinases compared abstracts to non-diabetic a proton mouse α to the wounds sulfone, [67 opening]. Thequestion the thiirane was ring what to a roles thiolate, these which two MMPsis a tight-binding play in DFUs. inhibitor Answers that to thesecoordinates questions with require zinc ion availability [84]. This of results selective in MMP-8residence and times MMP-9 (the inhibitors.time the inhibitor However, is bound because to of the the gelatinases) structural similaritythat are very between long, MMPs,as the revers selectiveal of inhibitors this reaction for ais particularslow. SB-3CT MMP inhibits are diffi MMP-8cult to design.as a non-competitive inhibitor with a Ki value of 2100 ± 400 nM [83], for aThe selectivity first selective towards inhibitor MMP-9 for relative the gelatinases to MMP-8 (MMP-2 of 3.5 (Table andMMP-9), 2). A drawback referred of to SB-3CT as SB-3CT, is its waspoor reportedwater solubility by Brown of et 2.3 al. μ [82g/mL]. SB-3CT [85]. (Table2) inhibits MMP-2 and MMP-9 with K values of 28 7 nM i ± Table 2. Inhibition constants for the small-molecule inhibitors, showing the progression to selectivity.

Pharmaceuticals 2019, 12, 79 7 of 21 and 400 15 nM, respectively, and inhibits other MMPs poorly [82,83]. The selectivity of SB-3CT ± towards the gelatinases is attributed to mechanism-based inhibition, in which Glu-404 at the active site of the gelatinases abstracts a proton α to the sulfone, opening the thiirane ring to a thiolate, which is a tight-binding inhibitor that coordinates with zinc ion [84]. This results in residence times (the time the inhibitor is bound to the gelatinases) that are very long, as the reversal of this reaction is slow. SB-3CT inhibits MMP-8 as a non-competitive inhibitor with a Ki value of 2100 400 nM [83], for a selectivity ± towards MMP-9 relative to MMP-8 of 3.5 (Table2). A drawback of SB-3CT is its poor water solubility µ of 2.3 g/mLPharmaceuticalsPharmaceuticalsPharmaceuticals [85]. 2019 20192019,, , 12 1212,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 7 7 7 of of of 20 20 20

Table 2. InhibitionTableTable 2. 2. Inhibition constantsInhibitionInhibition constants constantsconstants for the small-moleculefor forfor the thethe small-molecule small-moleculesmall-molecule inhibitors, inhi inhiinhibitors,bitors,bitors, showing showing showingshowing the the thethe progression progressionprogression to toto to selectivity. selectivity.selectivity. selectivity.

Ki (nM)KKii i (nM) (nM)(nM)

NameNameName SB-3CTSB-3CTSB-3CT ( (R,S (R,SR,SR,S)-ND-322)-ND-322)-ND-322)-ND-322 (((R,S(R,SR,S)-ND-336)-ND-336)-ND-336 (((RRR)-ND-336)-ND-336)-ND-336 MMP-9MMP-9 400 400 ± ± 15 15 870 870 ± ± 110 110 150 150 ± ± 10 10 19 19 ± ± 3 3 MMP-9 400 15 870 110 150 10 19 3 MMP-8MMP-8 2100 2100 ±± ± 400 400 2600 2600± ± ± 400 400 7700 7700 ± ± 100 100 8590 8590 ± ± 230 230 MMP-8SelectivitySelectivitySelectivity 2100 400 2600 400 7700 100 8590 230 ± ± ± ± Selectivity(((KKKii i MMP-8 MMP-8MMP-8/// 3.53.53.5 3.03.03.0 51 51 51 450 450 450 KKKii i MMP-9 MMP-9MMP-9))) 3.5 3.0 51 450 (Ki MMP-8/Ki MMP-9) MMP-9MMP-9 MMP-9 residenceresidenceresidenceresidence 13.413.413.413.4 31.4 31.4 31.4 47.4 47.4 47.4 47.4 ± ±± 4.4 4.44.4 300 300 300 ± ±± 1 111 time (min)timetimetime ± ± Reference(min)(min)(min) [82,83][67][86][48] ReferenceReferenceReference [82,83][82,83][82,83] [67] [67] [67] [86] [86] [86] [48] [48] [48]

In search ofInInIn water-soluble searchsearchsearch ofofof water-solublewater-solublewater-soluble selective selectiveselectiveselective MMP-9 MMP-9MMP-9 inhibitors,MMP-9 inhibitors,inhibitors,inhibitors, (R,S)-ND-322 (((R,SR,S)-ND-322)-ND-322)-ND-322 was waswas discoveredwas discovereddiscovereddiscovered with withwithwith a water aaa waterwater solubility solubility of of 4.9 4.9 mg/mL mg/mL [87], [87], inhibiting inhibiting MMP-9 MMP-9 in in a a slow-binding slow-binding manner manner with with a a K Kii i of ofof 870 870870 ± ±± 110 110110 solubility of 4.9 mg/mL [87], inhibiting MMP-9 in a slow-binding manner with a Ki of 870 110 nM nMnM andand inhibitinginhibiting MMP-8MMP-8 asas aa non-competitivenon-competitive inhibitorinhibitor withwith aa KKii i ofofof 260026002600 ±±± 400400400 nMnMnM (Table(Table(Table 2)2)±2) [67].[67].[67]. and inhibiting MMP-8 as a non-competitive inhibitor with a K of 2600 400 nM (Table2)[ 67]. TopicalTopical administrationadministration ofof ((R,SR,S)-ND-322)-ND-322)-ND-322 acceleratedacceleratedaccelerated woundwoundwound healinghealinghealingi ininin diabeticdiabeticdiabetic± micemicemice [67].[67].[67]. However,However,However, Topical administration(((R,SR,S)-ND-322)-ND-322)-ND-322 ofisisis ( R,Smetabolizedmetabolizedmetabolized)-ND-322 by acceleratedbyby NN-acetylation-acetylation-acetylation wound [87].[87].[87]. healing ReplacementReplacementReplacement in diabetic ofofof micethethethe anilineanilineaniline [67]. However, withwithwith (R,S)-ND-322methylaminophenylmethylaminophenyl is metabolized by toto N blockblock-acetylation metabolismmetabolism [87 byby]. ReplacementNN-acetylation-acetylation-acetylation resultedresultedresulted of the aniline ininin (((R,SR,S)-ND-336,)-ND-336,)-ND-336, with methylaminophenyl whichwhichwhich retainedretainedretained to block metabolismwaterwater solubilitysolubility by N ofof-acetylation 4.94.9 mg/mLmg/mL andand resulted withwith increasedincreased in (R,S potencypotency)-ND-336, ((KKii i ofofof which 150150150 ±±± 101010 retained nMnMnM forforfor MMP-9MMP-9MMP-9 water andandand solubility 770077007700 ±±± of 4.9 mg/mL and100100100 nM nM withnM for forfor increased MMP-8) MMP-8)MMP-8) and andand potency selectivity selectivityselectivity (K of(Table (Table(Table 150 2) 2)2) [86]. 10[86].[86]. nM ( ((R,SR,S for)-ND-336)-ND-336)-ND-336 MMP-9 showed showedshowed and 7700 better betterbetter efficacy 100efficacyefficacy nM in inin for accelerating acceleratingaccelerating MMP-8) and i ± ± selectivity (Tablediabeticdiabetic2)[ woundwound86]. ( healingR,Shealing)-ND-336 thanthan ((R,SR,S showed)-ND-322)-ND-322)-ND-322 better (Figures(Figures(Figures effi 3a3acacy3a andandand in 3b),3b),3b), accelerating whichwhichwhich waswaswas attributed diabeticattributedattributed wound tototo improvedimprovedimproved healing selectivityselectivityselectivity towardstowardstowards inhibitioninhibitioninhibition ofofof MMP-9MMP-9MMP-9 relativerelativerelative tototo MMP-8MMP-8MMP-8 (Figure(Figure(Figure 3c)3c)3c) [86].[86].[86]. than (R,S)-ND-322 (Figure3a,b), which was attributed to improved selectivity towards inhibition of MMP-9 relative to MMP-8 (Figure3c) [86]. The detrimental role of MMP-9 was confirmed with MMP-9 knockout mice treated with streptozotocin in order to induce diabetes. The diabetic MMP-9 knockout animals showed improved healing compared to streptozotocin-treated wild-type mice [86]. (R,S)-ND-336 was shown to have better efficacy than aclerastide (Figure4A–C) [ 49], an analog of angiotensin II that failed in phase III clinical trials for the treatment of DFUs. Aclerastide-treated wounds resulted in increased levels of active MMP-9 as demonstrated by with the batimastat affinity resin/proteomics method (Figure4D) and in-situ zymography (Figure4E). Aclerastide had previously shown efficacy in diabetic mice when administered immediately after wound infliction and when given for a shorter dosing regimen [71]. Aclerastide’s beneficial effect in promoting wound healing by inducing angiogenesis and migration of fibroblasts and keratinocytes [69,88] and its detrimental effect in delaying wound healing by increasing active MMP-9 cancelled each other, resulting in wound healing similar to that in vehicle-treated mice. This study found that expression of active MMP-9 likely contributed to aclerastide’s clinical failure in the treatment of DFUs [49].

Pharmaceuticals 2019, 12, x FOR PEER REVIEW 7 of 20

Ki (nM)

Name SB-3CT (R,S)-ND-322 (R,S)-ND-336 (R)-ND-336 MMP-9 400 ± 15 870 ± 110 150 ± 10 19 ± 3 MMP-8 2100 ± 400 2600 ± 400 7700 ± 100 8590 ± 230 Selectivity (Ki MMP-8/ 3.5 3.0 51 450 Ki MMP-9) MMP-9 residence 13.4 31.4 47.4 ± 4.4 300 ± 1 time (min) Reference [82,83] [67] [86] [48]

In search of water-soluble selective MMP-9 inhibitors, (R,S)-ND-322 was discovered with a water solubility of 4.9 mg/mL [87], inhibiting MMP-9 in a slow-binding manner with a Ki of 870 ± 110 nM and inhibiting MMP-8 as a non-competitive inhibitor with a Ki of 2600 ± 400 nM (Table 2) [67]. Topical administration of (R,S)-ND-322 accelerated wound healing in diabetic mice [67]. However, (R,S)-ND-322 is metabolized by N-acetylation [87]. Replacement of the aniline with methylaminophenyl to block metabolism by N-acetylation resulted in (R,S)-ND-336, which retained water solubility of 4.9 mg/mL and with increased potency (Ki of 150 ± 10 nM for MMP-9 and 7700 ± 100 nM for MMP-8) and selectivity (Table 2) [86]. (R,S)-ND-336 showed better efficacy in accelerating

diabeticPharmaceuticals wound2019 healing, 12, 79 than (R,S)-ND-322 (Figures 3a and 3b), which was attributed to improved8 of 21 selectivity towards inhibition of MMP-9 relative to MMP-8 (Figure 3c) [86].

FigureFigure 3. 3. SelectiveSelective MMP-9 MMP-9 inhibition inhibition accelerates accelerates diabetic diabetic wound wound healing. healing. (A (A) )Wound Wound healing healing after after treatmenttreatment with with 100 100 μµg/wound/dayg/wound/day of ((R,SR,S)-ND-336,)-ND-336, ((R,SR,S)-ND-322,)-ND-322, or or vehicle. vehicle. Mean Mean ±standard standard error error of ± ofthe the mean mean (SEM); (SEM);n n= =8 /8/groupgroup on on days days 7, 7, 10, 10, and and 14; 14; * *pp << 0.05, ** pp << 0.010.01 by by Mann–Whitney Mann–Whitney UU test.test. (B(B) )Representative Representative images images of of the the wounds. wounds. (C (C) )In-situ In-situ zymography zymography of of wounds wounds with with DQ-Gelatin DQ-Gelatin and and DQ-Collagen indicates that (R,S)-ND-336 inhibits MMP-9 but not MMP-8, while (R,S)-ND-322 inhibits MMP-9 and poorly inhibits MMP-8. Reproduced from Gao et al [86].

On the other hand, MMP-8 was shown to be beneficial to diabetic wound healing. Selective chemical inhibition of MMP-8 (Figure5A) delayed wound healing in diabetic mice (Figure5B,C) [ 67]. Confirmation of the beneficial role of MMP-8 was not feasible with MMP-8 knockout mice, as ablation of the MMP-8 gene results in significantly increased levels of MMP-9 [89]. Instead, diabetic mice were treated topically with recombinant MMP-8, which resulted in increased wound healing (Figure5D,E) [ 86]. Combination therapy with a selective MMP-9 inhibitor and topical application of MMP-8 further improved healing beyond either of the two individual treatments [86]. Given that MMP-9 is detrimental and MMP-8 is beneficial to healing of DFUs, the best strategy is to selectively inhibit MMP-9 without affecting MMP-8. To this effect, a search for more selective MMP-9 inhibitors led to the discovery of (R)-ND-336, which inhibits MMP-9 as a slow-binding inhibitor with a K of 19 3 nM and inhibits MMP-8 as a non-competitive inhibitor with a K of 8590 230 nM i ± i ± (Table2)[ 48]. (R)-ND-336 had superior efficacy compared to (R,S)-ND-336 (Figure6A,B) due to more potent and selective inhibition of MMP-9 (Figure6C,D). Pharmaceuticals 2019, 12, x FOR PEER REVIEW 8 of 20

DQ-Collagen indicates that (R,S)-ND-336 inhibits MMP-9 but not MMP-8, while (R,S)-ND-322 inhibits MMP-9 and poorly inhibits MMP-8. Reproduced from Gao et al [86].

The detrimental role of MMP-9 was confirmed with MMP-9 knockout mice treated with streptozotocin in order to induce diabetes. The diabetic MMP-9 knockout animals showed improved healing compared to streptozotocin-treated wild-type mice [86]. (R,S)-ND-336 was shown to have better efficacy than aclerastide (Figure 4A, 4B, and 4C) [49], an analog of angiotensin II that failed in phase III clinical trials for the treatment of DFUs. Aclerastide- treated wounds resulted in increased levels of active MMP-9 as demonstrated by with the batimastat affinity resin/proteomics method (Figure 4D) and in-situ zymography (Figure 4E). Aclerastide had previously shown efficacy in diabetic mice when administered immediately after wound infliction and when given for a shorter dosing regimen [71]. Aclerastide’s beneficial effect in promoting wound healing by inducing angiogenesis and migration of fibroblasts and keratinocytes [69,88] and its detrimental effect in delaying wound healing by increasing active MMP-9 cancelled each other, resulting in wound healing similar to that in vehicle-treated mice. This study found that expression Pharmaceuticals 2019, 12, 79 9 of 21 of active MMP-9 likely contributed to aclerastide’s clinical failure in the treatment of DFUs [49].

Figure 4. Aclerastide does not accelerate wound healing in diabetic mice when given at a clinically Figure 4. Aclerastide does not accelerate wound healing in diabetic mice when given at a clinically relevant dosing regimen. (A) Structure of aclerastide. Mice were treated topically 1 day after relevant dosing regimen. (A) Structure of aclerastide. Mice were treated topically 1 day after wound wound infliction with (R,S)-ND-336 or aclerastide at 100 µg/wound/day for 14 days or vehicle (water). infliction with (R,S)-ND-336 or aclerastide at 100 μg/wound/day for 14 days or vehicle (water). (B) (B) Wound closure measurements indicate that (R,S)-ND-336 accelerates wound healing faster than Wound closure measurements indicate that (R,S)-ND-336 accelerates wound healing faster than aclerastide; mean SEM, n = 12, 9, and 9 mice for (R,S)-ND-336 or aclerastide on days 7, 10, and ± 14, respectively; n = 13, 10, and 10 mice for vehicle on days 7, 10, and 14, respectively; ** p < 0.01, *** p < 0.001 by Mann–Whitney U two-tailed test. (C) Representative images of the wounds. (D) Increased active MMP-9 levels are observed in aclerastide-treated wounds by the batimastat affinity resin coupled with proteomics; mean S.D., n = 3 mice per group per time point, * p < 0.05 by Student’s ± t two-tailed test. (E) Aclerastide treatment increases the detrimental MMP-9 activity in diabetic wounds by in-situ zymography with DQ-Gelatin, whereas MMP-9 activity was inhibited in (R,S)-ND-336-treated wounds. Adapted from Nguyen et al. [49]. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 9 of 20

aclerastide; mean ± SEM, n = 12, 9, and 9 mice for (R,S)-ND-336 or aclerastide on days 7, 10, and 14, respectively; n = 13, 10, and 10 mice for vehicle on days 7, 10, and 14, respectively; **p < 0.01, ***p < 0.001 by Mann–Whitney U two-tailed test. (C) Representative images of the wounds. (D) Increased active MMP-9 levels are observed in aclerastide-treated wounds by the batimastat affinity resin coupled with proteomics; mean ± S.D., n = 3 mice per group per time point, *p < 0.05 by Student's t two-tailed test. (E) Aclerastide treatment increases the detrimental MMP-9 activity in diabetic wounds by in-situ zymography with DQ-Gelatin, whereas MMP-9 activity was inhibited in (R,S)-ND-336- treated wounds. Adapted from Nguyen et al. [49].

On the other hand, MMP-8 was shown to be beneficial to diabetic wound healing. Selective chemical inhibition of MMP-8 (Figure 5A) delayed wound healing in diabetic mice (Figures 5B and 5C) [67]. Confirmation of the beneficial role of MMP-8 was not feasible with MMP-8 knockout mice, as ablation of the MMP-8 gene results in significantly increased levels of MMP-9 [89]. Instead, diabetic mice were treated topically with recombinant MMP-8, which resulted in increased wound healing (Figures 5D and 5E) [86]. Combination therapy with a selective MMP-9 inhibitor and topical Pharmaceuticals 2019, 12, 79 10 of 21 application of MMP-8 further improved healing beyond either of the two individual treatments [86].

Figure 5. The role of MMP-8 in diabetic wound healing. (A) Structure of selective MMP-8 inhibitor. Figure(B,C) Topical 5. The treatmentrole of MMP-8 with selectivein diabetic MMP-8 wound inhibitor healing. (250 (Aµ) gStructure/wound/day) of selective delays healingMMP-8in inhibitor. diabetic (mice;B and mean C) TopicalSD, n treatment= 12, 6, and with 6 on selective days 7, 10,MMP-8 and 14, inhibitor respectively; (250 μ *g/wound/day)p < 0.05. (D,E) delays Topical healing treatment in ± diabeticwith exogenous mice; mean recombinant ± SD, n = MMP-8 12, 6, and (1 µ g6/ woundon days/day) 7, 10, accelerates and 14, respectively; diabetic wound *p < healing;0.05. (D meanand E) ± TopicalSEM, n =treatment20, 9, and with 9 mice exogenous on days 7,recombinant 10, and 14, respectively,MMP-8 (1 forμg/wound/day) the vehicle group; acceleratesn = 20, diabetic 10, and 10wound mice healing; on days mean 7, 10, ± and SEM, 14, n respectively, = 20, 9, and 9 for mice the on MMP-8 days 7, group; 10, and * p 14,< 0.05.respectively, Adapted for from the vehicle Gooyit group;et al. [67 n] = and 20, Gao10, and et al. 10 [86 mice]. on days 7, 10, and 14, respectively, for the MMP-8 group; *p < 0.05. Adapted from Gooyit et al. [67] and Gao et al. [86]. Becaplermin remains the only FDA-approved drug for the treatment of DFUs since it was approved in 1997.Given A side-by-side that MMP-9 comparison is detrimental of (Rand)-ND-336 MMP-8 and is beneficial becaplermin to healing demonstrated of DFUs, that the the best former strategy had issuperior to selectively efficacy inhibit early afterMMP-9 treatment without (Figure affecting7A,B), MMP-8. while To becaplermin this effect, accelerateda search for wound more selective healing relative to diabetic mice treated with vehicle at later days, paralleling what has been observed in clinical trials in which becaplermin differentiates from placebo in complete wound healing incidence after 10-week treatment [90]. The superior efficacy of (R)-ND-336 was attributed to complete inhibition of MMP-9 (Figure7C) [ 48]. Becaplermin poorly inhibits MMP-9 (17% inhibition at 4 µM) and indirectly decreases MMP-9 activity, but does not completely inhibit it. This was confirmed with the batimastat affinity resin, which resulted in 3-fold decrease in active MMP-9 levels compared to vehicle control on day 7 (Figure7C,D) [ 48]. This study showed that becaplermin reduces active MMP-9, and this may explain its efficacy. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 10 of 20

MMP-9 inhibitors led to the discovery of (R)-ND-336, which inhibits MMP-9 as a slow-binding inhibitor with a Ki of 19 ± 3 nM and inhibits MMP-8 as a non-competitive inhibitor with a Ki of 8590

±Pharmaceuticals 230 nM (Table2019 2), 12 [48]., 79 (R)-ND-336 had superior efficacy compared to (R,S)-ND-336 (Figures 6A11 and of 21 6B) due to more potent and selective inhibition of MMP-9 (Figures 6C and 6D).

FigureFigure 6. 6. (R(R)-ND-336)-ND-336 accelerates accelerates wound wound healing healing faster faster than than (R,S (R,S)-ND-336)-ND-336 in in diabetic diabetic mice. mice. Topical Topical treatmenttreatment with with (R (R)-,)-, or or (R,S (R,S)-ND-336)-ND-336 at at 50 50 μµg/wound/dayg/wound/day for for 14 14 days days or or vehicle vehicle (water). (water). (A (A) Wound) Wound healing;healing; mean mean ± SEM,SEM, n =n 7= mice/group/time7 mice/group/time point point for vehicle, for vehicle, (R,S)-ND-336, (R,S)-ND-336, n = 8/timen = 8 /pointtime pointfor (R for)- ± ND-336,(R)-ND-336, *p < 0.05, * p < **0.05,p < 0.01 ** p by< 0.01Mann by−Whitney Mann Whitney U two-tailed U two-tailed test. (B) Representative test. (B) Representative wound images. wound − (Cimages.) In-situ ( Czymography) In-situ zymography of the wounds of the with wounds DQ-gelatin with DQ-gelatin shows that shows (R)-ND-336 that (R)-ND-336 inhibits inhibitsMMP-9 MMP-9better thanbetter (R,S than)-ND-336; (R,S)-ND-336; n = 3 mice/group.n = 3 mice /Thegroup. bottom The row bottom shows row me showsrged mergedimages imageswith DAPI with nuclear DAPI nuclearDNA staining,DNA staining, 40× lens 40 (scalelens bars, (scale 50 bars,μm). 50(Dµ) m).In-situ (D) zymography In-situ zymography with DQ-collagen with DQ-collagen indicates indicates that (R,S that)- × and(R,S (R)-)-ND-336 and (R)-ND-336 do not inhibit do not MMP-8; inhibit MMP-8; n = 3 mice/group.n = 3 mice /Adaptedgroup. Adapted from Nguyen from Nguyen et al. [48]. et al. [48].

BecaplerminThese studies remains conclusively the only determined FDA-approved that MMP-9 drug hasfor the a detrimental treatment roleof DFUs in healing since ofit DFUswas approvedand that MMP-8in 1997. hasA side-b a beneficialy-side comparison one. This is of further (R)-ND-336 supported and bybecaplermin validation demonstrated of the target MMP-9 that the in formerdebridement had superior tissue efficacy from humans early after with treatment DFUs, where (Figures DFUs 7A were and stratified7B), while by becaplermin the Wagner accelerated grade (WG) woundof the ulcerhealing [48 relative]. MMP-8 to diabetic showed mice no correlation treated with with vehicle ulcer at grade later (Figuredays, paralleling8A), while what WG3-4 hasulcers been observedhave significantly in clinical higher trials concentrationsin which becaplermi of activen MMP-9differentiates than WG1-2 from (Figureplacebo8B), in whichcomplete in turn wound were healinghigher incidence than control after [48 10-week]. treatment [90]. The superior efficacy of (R)-ND-336 was attributed to complete inhibition of MMP-9 (Figure 7C) [48]. Becaplermin poorly inhibits MMP-9 (17% inhibition at 4 μM) and indirectly decreases MMP-9 activity, but does not completely inhibit it. This was confirmed with the batimastat affinity resin, which resulted in 3-fold decrease in active MMP-9 levels compared to vehicle control on day 7 (Figures 7C and 7D) [48]. This study showed that becaplermin reduces active MMP-9, and this may explain its efficacy.

Pharmaceuticals 2019, 12, 79 12 of 21 Pharmaceuticals 2019, 12, x FOR PEER REVIEW 11 of 20

Figure 7. (R)-ND-336 is superior to becaplermin in accelerating wound healing in diabetic mice. FigureMice were7. (R)-ND-336 treated topically is superior 1 day to becapler after woundmin in inflictionaccelerating (8 mm,wound Tegaderm-covered) healing in diabetic with mice. 5 Miceµg or were50µg /treatedwound /topicallyday of becaplermin 1 day after or (woundR)-ND-336, infliction respectively, (8 mm, or Tegaderm-covered) vehicle (water) for 14 with days. 5 (Aμg) Woundor 50 μmeasurementsg/wound/day of show becaplermin that (R)-ND-336 or (R)-ND-336, has better respectively, efficacy than or becaplermin, vehicle (water) mean for 14SEM; days.n (A=)11, Wound 8, and ± measurements8 for vehicle, n show= 11, that 7, and (R)-ND-336 7 for (R)-ND-336, has bettern efficacy= 12, 9, than and becaplermin, 9 for becaplermin mean on± SEM; days n 7, = 10,11, and8, and 14, 8respectively; for vehicle, n * =p 11,< 0.05, 7, and ** p7 neutrophil levels increase at the site to help fight infection. MMP-8, MMP-9, and reactiveThese oxygen studies species conclusively (ROS) aredetermined secreted bythat neutrophils. MMP-9 has ROSa detrimental function torole kill in bacteria, healing asof wellDFUs as andhelp that in formationMMP-8 has of a beneficial thrombus one. that protectsThis is further the wound supported preventing by validation further blood of the loss. target ROS MMP-9 activates in debridementNF-кB, which tissue upregulates from humans MMP-9 with and DFUs, is detrimental where DFUs to were DFU stratified healing [ 48by]. the On Wagner the other grade hand, (WG) the offunction the ulcer of [48]. MMP-8 MMP-8 is to showed repair damaged no correlation collagen with and ulcer in re-epithelializationgrade (Figure 8A), thatwhile is WG3-4 critical ulcers for the havewound significantly healing process higher [concentrations89]. of active MMP-9 than WG1-2 (Figure 8B), which in turn were higher than control [48]. Upon injury, neutrophil levels increase at the site to help fight infection. MMP-8, MMP-9, and reactive oxygen species (ROS) are secreted by neutrophils. ROS function to kill bacteria, as well as help in formation of a thrombus that protects the wound preventing further blood loss. ROS activates NF-кB, which upregulates MMP-9 and is detrimental to DFU healing [48]. On the other hand, the function of MMP-8 is to repair damaged collagen and in re-epithelialization that is critical for the wound healing process [89].

Pharmaceuticals 2019, 12, 79 13 of 21 Pharmaceuticals 2019, 12, x FOR PEER REVIEW 12 of 20

Active MMP-8 Active MMP-9 ABActive MMP-8 Active MMP-9 ** ** ** ** ** ** **

FigureFigure 8. 8.Validation Validation of theof the target target MMP-9 MMP-9 in debridement in debridemen tissuet tissue from from human human patients patients withdiabetic with diabetic foot ulcersfoot (DFUs).ulcers (DFUs). Measurements Measurements of (A) active of (A MMP-8) active andMMP-8 (B) active and ( MMP-9B) active in MMP-9 human DFUsin human stratified DFUs by the Wagner grade (WG) using the batimastat affinity resin coupled with proteomics; mean SEM: stratified by the Wagner grade (WG) using the batimastat affinity resin coupled with proteomics;± * p < 0.05, ** p < 0.01 by Mann Whitney U two-tailed test. Adapted from Nguyen at al. [48]. mean ± SEM: *p < 0.05, **p <− 0.01 by Mann−Whitney U two-tailed test. Adapted from Nguyen at al. 5. Targeting[48]. MMP-9 with Therapeutics

5. TargetingSelective inhibitionMMP-9 with of Therapeutics MMP-9 (gelatinase B) is a challenge because of the shared substrate specificity and homology of MMPs [91,92], especially with MMP-2 (gelatinase A). Even in the face of Selective inhibition of MMP-9 (gelatinase B) is a challenge because of the shared substrate this challenge, the failure of the early broad-spectrum MMPIs demonstrated the need for selective specificity and homology of MMPs [91,92], especially with MMP-2 (gelatinase A). Even in the face of MMPIs. The confirmed beneficial role of MMP-8 makes selective inhibition of MMP-9 all the more this challenge, the failure of the early broad-spectrum MMPIs demonstrated the need for selective important for the development of a therapeutic for the treatment of DFUs. However, as active MMP-2 MMPIs. The confirmed beneficial role of MMP-8 makes selective inhibition of MMP-9 all the more is not present in diabetic wounds [67], this is not an insurmountable challenge. There is still a strong important for the development of a therapeutic for the treatment of DFUs. However, as active MMP- effort being made by the community to overcome these challenges and to deliver a selective MMP-9 2 is not present in diabetic wounds [67], this is not an insurmountable challenge. There is still a strong inhibitor [93]. effort being made by the community to overcome these challenges and to deliver a selective MMP-9 5.1.inhibitor Small-Molecule [93]. Inhibitors

5.1.One Small-Molecule of the promising Inhibitors stories for a therapeutic MMPI is the development of (R)-ND-336, a member of the thiirane class of MMP-9 inhibitors. Based on the Ki values, the racemate (R,S)-ND-336 has 51-foldOne selectivity of the towardspromising MMP-9 stories compared for a therapeutic to MMP-8 MMPI [86]. Asis (theR,S )-ND-336development accelerated of (R)-ND-336, diabetic a woundmember healing of the inthiirane mice, class (R)- andof MMP-9 (S)-ND-336 inhibitors. were Based synthesized on the K asi values, enantiomerically the racemate pure (R,S isomers.)-ND-336 (Rhas)-ND-336 51-fold was selectivity shown to towards be a 10-fold MMP-9 more compared potent inhibitor to MMP-8 of MMP-9 [86]. As than (R,S (S)-ND-336 [accelerated48], with 450-folddiabetic selectivity wound healing towards in MMP-9 mice, relative(R)- and to (S MMP-8)-ND-336 compared were synthesized to 11-fold selectivity as enantiomerically for (S)-ND-336 pure isomers. (R)-ND-336 was shown to be a 10-fold more potent inhibitor of MMP-9 than (S)-ND-336 [48], (Table2). Selectivity based on Ki values is further enhanced, as (R)-ND-336 inhibits MMP-9 as a mechanism-basedwith 450-fold selectivity inhibitor, towards undergoing MMP-9 ring-opening relative to MMP-8 at the activecompared site to 11-fold produce selectivity a thiolate for that (S)- coordinatesND-336 (Table with 2). the Selectivity active-site based zinc for on whichKi values the is reversal further is enhanced, extremely as slow (R)-ND-336 (Figure9 ),inhibits imparting MMP-9 a longas a residence mechanism-based time for MMP-9 inhibitor, of 300 undergoing1 min and ring-openi further selectivityng at the active when site compared to produce to the a fastthiolatek for that ± off MMP-8coordinates (residence with timethe active-site for MMP-8 zinc<1 s)for [48 which]. (R)-ND-336 the reversal has is superior extremely effi cacyslow than(Figure becaplermin, 9), imparting the a onlylong FDA-approved residence time therapeutic for MMP-9 for of the300 treatment ± 1 min and of DFUs, further indicating selectivity its when promise compared as a therapeutic to the fast for koff thefor treatment MMP-8 (residence of DFUs. time for MMP-8 <1 s) [48]. (R)-ND-336 has superior efficacy than becaplermin, the only FDA-approved therapeutic for the treatment of DFUs, indicating its promise as a therapeutic for the treatment of DFUs.

Pharmaceuticals 2019, 12, 79 14 of 21 Pharmaceuticals 2019, 12, x FOR PEER REVIEW 13 of 20

A

B

Figure 9.9. MechanismMechanism of of inhibition inhibition of of (R )-ND-336.(R)-ND-336. (A )((AR))-ND-336 (R)-ND-336 inhibits inhibits MMP-9 MMP-9 as a mechanism-basedas a mechanism- basedinhibitor, inhibitor, where Glu-404where Glu-404 at the active at th sitee active abstracts site a abstracts proton α toa sulfone,proton α resulting to sulfone, in the resulting corresponding in the correspondingthiolate that coordinates thiolate that with coordinates zinc ion as awith tight-binding zinc ion as inhibitor, a tight-binding for which inhibitor, the reversal for occurs which very the reversalslowly. (occursB)(R)-ND-336 very slowly. inhibits (B) ( MMP-9R)-ND-336 as ainhibits slow-binding MMP-9 inhibitoras a slow-b withinding a long inhibitor residence with time a long of residence300 1 min time. The of compound300 ± 1 min. is aThe poor compound non-competitive is a poor inhibitor non-competitive for MMP-8, inhibitor for which for the MMP-8, residence for ± whichtime is the very residence short. Adapted time is very from short. Nguyen Adapted et al. [ 48from]. Nguyen et al. [48].

5.2. Antibody-Based Inhibitors One of the possible avenues to address the challengechallenge of selectivity for inhibitors is the use of antibodies. A number number of of inhibitory inhibitory antibodies antibodies have have been been developed developed to to target target MMPs, MMPs, such such as as GS-5745, GS-5745, which targets MMP-9.MMP-9. GS-5745 GS-5745 shows shows a a dual-faceted method of inhibition for MMP-9, sterically hindering both access to the active site (IC for MMP-9cat = 1.3 0.1 nM) and the ability of the hindering both access to the active site (IC50 for50 MMP-9cat = 1.3 ± 0.1 nM)± and the ability of the zymogen tozymogen be cleaved to be to cleaved the active to the form active by MMP-3 form by [94]. MMP-3 In addition, [94]. In addition, it reports it being reports well-tolerated being well-tolerated during µ phaseduring I clinical phase I trials clinical [95,96]. trials Other [95,96 antibodies,]. Other antibodies, such as SDS3 such (K asi MMP-9 SDS3 (=K 1i MMP-9μM) [97]= and1 REGA-3G12M) [97] and [98,99],REGA-3G12 have [been98,99 reported], have been as MMP-9 reported inhibitors. as MMP-9 Ho inhibitors.wever, all three However, of these all threehave ofonly these been have applied only towardbeen applied cancers, toward and not cancers, wound and healing. not wound The clinical healing. potential The clinical of these potential inhibitors of thesefor wound inhibitors healing, for woundin the context healing, of inthe the wealth context of ofknowledge the wealth about of knowledge the importance about theof MMP-9 importance [42], ofis MMP-9a promising [42], but is a promisingunexplored but territory. unexplored territory.

5.3. Advanced Wound Dressings 5.3. Advanced Wound Dressings As dressings areare commonlycommonly employed employed in in the the clinic clinic to to help help with with wound wound healing, healing, both both to controlto control the woundthe wound environment environment and manageand manage exudate exudate [100], [100], they arethey an are ideal an vehicleideal vehicle for altering for altering the MMP the profile. MMP Thereprofile. has There been has interest been in interest tethering in antethering MMPI toan aMMPI wound to dressing, a wound though dressing this, though currently this involves currently the involvesuse of nonspecific the use of inhibitors, nonspecific such inhi asbitors, bisphosphonates such as bisphosphonates [101]. Localization [101]. ofLocalization the inhibitor of to the the inhibitor wound tosite the and wound the use site of and compounds the use of with compounds a known tolerancewith a known has been tolerance proposed has been to minimize proposed the to risk minimize of side theeffects risk [ 101of side]. Another effects [101]. option Anot alongher this option path along is a wound this path dressing is a wo composedund dressing of typecomposed I atelocollagen of type I atelocollagenfunctionalized functionalized with 4-vinylbenzyl with chloride, 4-vinylbenzyl a compound chloride, that a was compou hypothesizednd that towas complex hypothesized with active to complexMMPs in with wound active exudates MMPs [in102 wound]. Support exudates for this [102]. hypothesis Support wasfor this demonstrated hypothesis bywas its demonstrated reduction of byMMP-9 its reduction activity unlikeof MMP-9 standard-of-care activity unlike dressings. standard-of-care As a result, dressings. the use ofAs the a result, dressing the on use a mouseof the dressingmodel of on diabetic a mouse wounds model resulted of diabetic in improved wounds re healingsulted whenin improved compared healing to a commercialwhen compared adhesive to a commercialcontrol [102]. adhesive While this control methodology [102]. While is interesting, this methodology the use of a broad-spectrumis interesting, the inhibitor use of rather a broad- than spectruma selective inhibitor MMP-9 inhibitorrather than is ofa selective concern inMMP-9 the context inhibitor of the is of reduced concern wound in the healingcontext of observed the reduced with woundthe use ofhealing the broad-spectrum observed with MMPIthe use GM of the 6001 broad-spectrum in humans [41 ].MMPI GM 6001 in humans [41].

5.4. RNA-Based Therapies

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Another option for targeting MMP-9 is to interfere with expression at the gene level. RNA 5.4. RNA-Based Therapies inhibitors are a promising method of treatment for many diseases [103], but face the challenge of deliveringAnother the small option interfering for targeting RNA (siRNA) MMP-9 in is a to safe interfere and efficient with expression manner [104]. at the One gene of the level. possible RNA toolsinhibitors for delivering are a promising the siRNA method is cationic of treatment star-shaped for polymers many diseases based [on103 cyclodextrins], but face the (CDs) challenge [105– of 107],delivering as they the have small been interfering shown RNA to (siRNA)have low in atoxicity safe and [108]. efficient After manner a study [104 ].reporting One of the on possible the overexpressiontools for delivering of MMP-9 the siRNA relative iscationic to TIMP-1 star-shaped in a rat model polymers of diabetic based on wounds cyclodextrins [109], the (CDs) design [105 and–107 ], efficacyas they of have β-CD-(D been3) shown7/MMP-9-siRNA to have low was toxicity reported. [108 It]. was After demon a studystrated reporting to be oneffectively the overexpression taken up byof fibroblast MMP-9 cells, relative reducing to TIMP-1 MMP-9 in agene rat modelexpression, of diabetic and to woundssignificantly [109 ],improve the design wound and healing efficacy in of a ratβ-CD-(D model3) 7of/MMP-9-siRNA diabetic ulcers was [110]. reported. A follow-up It was demonstratedstudy looked toat betoxicity effectively in the taken liver up and by kidney, fibroblast findingcells, reducingonly a transient MMP-9 effect gene for expression, the naked and siRNA to significantly and none in improve the β-CD-(D wound3)7/MMP-9-siRNA healing in a rat [111]. model Moreof diabetic recently, ulcers there [ 110was]. a A report follow-up of microRNA(m study lookediRNA)-129 at toxicity and in miR-335 the liver as and an kidney, inhibitor finding of MMP-9 only a expressiontransient einff ecta diabetic for the nakedmouse siRNA model, and which none re insults the βin-CD-(D a commensurate3)7/MMP-9-siRNA improvement [111]. More in wound recently, healingthere was[112]. a report of microRNA(miRNA)-129 and miR-335 as an inhibitor of MMP-9 expression in a diabetic mouse model, which results in a commensurate improvement in wound healing [112]. 6. Conclusion and Future Perspectives 6. Conclusion and Future Perspectives The initial failure of broad-spectrum MMPIs halted interest in the development of MMPIs as therapeutics.The initial However, failure as of the broad-spectrum importance of MMPIstargeting halted MMP-9 interest in thein treatment the development of DFUs has of MMPIsbecomeas evident,therapeutics. a generation However, of selective as the importance MMP-9 inhibito of targetingrs has MMP-9 emerged. in theThere treatment are multiple of DFUs avenues has become for selectiveevident, inhibitor a generation design, of selectivefrom small-molecule MMP-9 inhibitors inhibitors has emerged.that selectively There target are multiple active MMP-9 avenues to for RNAselective inhibitors inhibitor that design,aim to block from small-moleculeexpression of MMP-9 inhibitors at the that gene selectively level. target active MMP-9 to RNA inhibitorsAs chronic that aimwounds to block remain expression a complex of MMP-9 challeng at thee, genethe development level. of a safe and effective treatmentAs chronicoption woundsis of outmost remain importance. a complexchallenge, While translation the development from laboratory of a safe andto clinic effective takes treatment time, resources,option is and of outmost effort (Figure importance. 10), there While is huge translation promise from shown laboratory by the tocurrent clinic takeswave time,of therapeutics resources, in and development.effort (Figure 10), there is huge promise shown by the current wave of therapeutics in development.

FigureFigure 10. 10. SchematicSchematic showing showing the the progress progress of oftranslationa translationall work work to tobring bring a selective a selective MMP-9 MMP-9 inhibitor inhibitor towardstowards the the clinic clinic for for treatment treatment of ofDFUs. DFUs.

Author Contributions: Author Contributions: J.I.J., T.T.N., and M.C. wrote the manuscript. Z.P. performed Author Contributions: Author Contributions: J.I.J., T.T.N., and M.C. wrote the manuscript. Z.P. performed recoveryrecovery of ofrepresentative representative ac activetive MMPs MMPs and and ADAMs ADAMs by by the the ba batimastattimastat affinity affinity resin. resin. Funding:Funding: J.I.J.J.I.J. is isa aRuth Ruth L. L. Kirschtein Kirschtein NationalNational Research Research Service Service Award Award Fellow Fellow ofthe of the Chemistry-Biochemistry-Biology Chemistry-Biochemistry- BiologyInterface Interface Program Program at the University at the University of Notre of Dame, Notre supported Dame, supported by training by granttraining T32 grant GM075762 T32 GM075762 from the National from theInstitutes National of Institutes Health. Thisof Health. work wasThis supportedwork was bysupported the American by the Diabetes American Association Diabetes Association Pathway to StopPathway Diabetes to Grant 1-15-ACN-06. Stop Diabetes Grant 1-15-ACN-06. Conflicts of Interest: The authors declare the following competing financial interests: U.S. patents 9,604,957 and Conflicts10,253,013 of Interest: and European The authors patent declare 3107905 the have following been issued competing for (R)-ND-336. financial interests: U.S. patents 9,604,957 and 10,253,013 and European patent 3107905 have been issued for (R)-ND-336. References References 1. Cooper, S. The biology of the skin. J. R. Soc. Med. 2002, 95, 109. [CrossRef] 1. Cooper, S. The biology of the skin. J. R. Soc. Med. 2002, 95, 109–109.

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