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 22 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