Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for

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Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for Treatment of Diabetic Foot Ulcers in Humans and Discovery of a Potent and Selective Small-Molecule MMP-9 Inhibitor that Accelerates Healing Trung T. Nguyen, Derong Ding, William R. Wolter, Rocio L. Perez, Matthew M. Champion, Kiran V. Mahasenan, Dusan Hesek, Mijoon Lee, Valerie A. Schroeder, Jeffrey I. Jones, Elena Lastochkin, Margaret K. Rose, Charles E. Peterson, Mark A Suckow, Shahriar Mobashery, and Mayland Chang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01005 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Journal of Medicinal Chemistry

Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for Treatment of Diabetic Foot Ulcers in Humans and Discovery of a Potent and Selective Small-Molecule MMP-9 Inhibitor that Accelerates Healing Trung T. Nguyen,† Derong Ding,† William R. Wolter,‡ Rocio L. Pérez,† Matthew M. Champion,† Kiran V. Mahasenan,† Dusan Hesek,† Mijoon Lee,† Valerie A. Schroeder,‡ Jeffrey I. Jones,† Elena Lastochkin,† Margaret K. Rose,† Charles E. Peterson,¶ Mark A. Suckow,‡,§ Shahriar Mobashery,† and Mayland Chang†* †

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556

‡

Freimann Life Sciences Center and Department of Biological Sciences, University of Notre

Dame, Notre Dame, IN 46556 ¶

Center for Wound Healing, Elkhart General Hospital, Elkhart, IN 46514

§

Present address: Veterinary Population Medicine Department, College of Veterinary Medicine,

University of Minnesota, St. Paul, MN 55108

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure (R)- and (S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the (R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans, and that (R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.


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INTRODUCTION

The prevalence of diabetes has been increasing for the past several years, affecting 382 million people worldwide in 20131 and 30.3 million individuals in the United States in 2015.2 A common complication of diabetes is foot ulcers, which annually develop in 26.1 million people globally.3 The lifetime incidence of DFUs ranges from 19% to 34% of diabetic patients.3 A single FDA-approved drug, becaplermin, has been available since 1997, but it received a black box warning due to increased risk of cancer and death.4 Thus, it is seldom used as treatment for DFUs, while the standard-of-care is debridement, off-loading, and infection control. The lack of effective therapies for DFUs results in 108,000 lower-limb amputations every year in the United States.2 The one-year prognosis after amputation is poor and results in 44% mortality.5 The lack of treatment options for DFUs is in part due to poor understanding of the molecular basis of why DFUs do not heal. Matrix metalloproteinases (MMPs) play important roles in normal wound healing, as well as in the pathology of DFUs.6, 7 The challenge remained as to which MMP(s) among the 24 known human variants plays a role in the ailment, compounded by the fact that MMPs exist in three forms: the latent, the activated in complex with protein inhibitors TIMPs, and the activated uninhibited form. The first two are catalytically incompetent. By the use of an affinity resin that binds exclusively to the catalytically active MMPs, and in conjunction with proteomics analysis, we identified active MMP-8 and MMP-9 within diabetic mouse wounds.8 In a series of experiments we documented that MMP-8 promoted diabetic wound healing, whereas the upregulated activity of MMP-9 had a deleterious effect.8, 9 For these separate studies we used topical application of a selective MMP-9 inhibitor racemic ND-336 ((R,S)-ND-336, 51-fold selectivity), a selective inhibitor of MMP-8, MMP-9 knockout mice, and recombinant MMP-8 .


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We found that selective inhibition of MMP-9 or gene ablation of MMP-9 or topical exogenous application of MMP-8 accelerated diabetic wound healing,8, 9 while selective inhibition of MMP8 delayed wound healing in diabetic mice.8 These observations made MMP-9 an important target for inhibition with translational potential. We now validate the target MMP-9 by affinity resin and proteomics analysis of human diabetic wounds, where we identify the same active MMP-8 and MMP-9 found in diabetic mouse wounds, with higher levels of active MMP-9 in the more severe infected wounds, making MMP-9 an important target for inhibition for the treatment of DFUs. We synthesized and evaluated enantiomerically pure (R)- and (S)-ND-336 and report herein the discovery of a novel potent and selective inhibitor of MMP-9, (R)-ND-336, which accelerates wound healing in db/db mice more effectively than becaplermin and holds promise as recourse in treatment of DFUs.

RESULTS AND DISCUSSION

Validation of the Target MMP-9 in Human DFUs. Previous studies in human diabetic wounds have reported higher levels of MMP-9,10 which predict poor wound healing.11 However, these studies measured MMP-9 by gelatin zymography, a technique that does not differentiate between active MMP-9 and TIMP-complexed MMP-9, an inactive form of the proteinase. To address this challenge, we previously designed and synthesized an affinity resin12 (1, Figure 1a) that binds exclusively to the active forms of MMPs and not to the latent proMMPs or TIMP-complexed MMPs. We used this affinity resin coupled with proteomics and identified active MMP-8 and MMP-9 in wounds of diabetic mice.8 In order to demonstrate that the animal model resembles the human disease, we collected debridement tissue from 25 diabetic patients with chronic wounds—Wagner grade (WG)1-4, with 1-superficial ulcer to 4-partial foot gangrene—and


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cancer-free dermal tissue from 23 non-diabetic patients (control) who had undergone surgery for diagnosis of skin cancer (Table S1). We analyzed the samples with our affinity resin coupled with proteomics (Table S2) in three clusters: WG1-4 (all samples combined), WG1-2, and WG34. We identified the very same active MMP-8 and MMP-9 in diabetic human wounds, indicating that the mechanisms of pathology and repair are similar in humans and in mice. Human MMP-8 and MMP-9 share 99% homology (identity plus similarity) with their mouse orthologs.13, 14 Tenfold higher levels of active MMP-8 were observed in chronic wounds (in all three clusters) compared to control (p < 0.01, Figure 1b). Active MMP-9 was 7-, 20-, and 34-fold higher in WG1-2, WG1-4, and WG3-4 compared to control (p < 0.01, Figure 1c). Multivariate analysis using age, sex, and smoking status showed that WG correlated with active MMP-9 concentration with a correlation coefficient of 0.76. These increased levels for MMP-9 are indeed significant, considering that active MMP-9 is a catalyst/enzyme that contributes to the recalcitrance of wounds to heal. Since angiotensin II—a peptide hormone that induces fibroblast and keratinocyte migration for wound healing15—has been reported to induce MMP-9 expression in vascular smooth muscle by stimulating NF-κB activation,16 we measured the levels of NF-κB p65 in the tissues by ELISA. Levels of NF-κB p65 in chronic wounds (mean 187 ± 23 µg/mg tissue) were significantly upregulated compared to control (32 ± 4 µg/mg tissue, p = 0.0001) (Figure 1d), and correlated with WG with a correlation coefficient of 0.87. Concentrations of active MMP-9 correlated with those of NF-κB p65, with a correlation coefficient of 0.57 (Figure 1e). Concentrations of active MMP-9 and NF-κB p65 are highly correlated to WG, indicating that patients with more severe DFUs would be likely candidates to respond to selective MMP-9 inhibition therapy.


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Figure 1. Diabetic patients with chronic wounds show higher levels of active MMP-8, MMP-9, and NF-κB p65. (a) Structure of affinity resin. Quantification of (b) active MMP-8 and (c) active MMP-9 in chronic wounds of diabetic patients WG14 (n = 25) and in dermal samples from nondiabetic (control) patients (n = 23) using the affinity resin coupled with proteomics. Higher levels of active MMP-9 are observed in WG3-4 (n = 13) when compared to WG1-2 (n = 12). (d) NF-κB p65 levels as measured by ELISA. Diabetic patients with chronic wounds have a statistically significant increase in NF-κB p65 levels over control tissues. (e) Levels of active MMP-9 correlated to those of NF-κB p65. Data in (b) to (d) as mean ± SEM, **p < 0.01, ***p < 0.001 using Mann Whitney U two-tailed test.

Discovery of the Selective and Potent MMP-9 Inhibitor (R)-ND-336. SB-3CT, the prototype thiirane inhibitor, was designed as a mechanism-based inhibitor for MMP-2 and MMP-9, inhibiting these proteinases as slow-binding inhibitors with Ki values of 28 ± 7 nM and 400 ±.150 nM, respectively.17 This prototype inhibitor has been used in over 200 in vitro and in vivo


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studies.18 SB-3CT inhibits MMP-8 as a linear non-competitive inhibitor with Ki of 2100 ± 400 nM.18 A drawback of SB-3CT is its poor water solubility (2.3 µg/mL).19 In addition, SB-3CT is metabolized at the para-position of the terminal phenyl ring to generate p-hydroxy SB-3CT, a more active inhibitor of MMP-2 (Ki 6 ± 3 nM) and MMP-9 (Ki 160 ± 20 nM).20 In search of more potent, selective, and water-soluble MMP-9 inhibitors, we had previously designed and synthesized (R,S)-ND-336, which shows a selectivity of 51-fold towards inhibition of MMP-9 (Ki 150 ± 10 nM) compared to MMP-8 (Ki 7700 ± 100 nM) and accelerates wound healing in diabetic mice.9 As (R,S)-ND-336 showed efficacy, we synthesized (R)- and (S)-ND-336 as enantiomerically pure isomers (Scheme 1) and evaluated them for their respective MMP kinetic properties. (R)-ND-336 is a potent and selective slow-binding inhibitor of MMP-2, MMP-9, and MMP-14, with Ki values of 127 ± 1 nM, 19 ± 3 nM, and 119 ± 3 nM, respectively, and poorly inhibiting other MMPs (Ki >100,000 nM, Table 1). Since active MMP-2 and MMP-14 have not been detected in mouse8 or in human diabetic wounds (present work), (R)-ND-336 shows specificity for targeting active MMP-9 in treatment of diabetic wounds. The selectivity of (R)ND-336 in targeting MMP-9 is attributed to mechanism-based inhibition of this proteinase, which entails a reaction within the enzyme active site that opens up the thiirane ring to the corresponding thiolate that coordinates with zinc ion as a tight-binding inhibitor (Figure 2). As a result, (R)-ND-336 has a long residence time—the time the inhibitor is bound to MMP-9, calculated as 1/koff—of 300 ± 1 min (Table S3), which is actually 38- to 45-fold longer than those for the endogenous protein inhibitors TIMPs (7.9 min for MMP-9-TIMP1 and 6.7 min for MMP-9-TIMP2).21 We consider this observation remarkable, as TIMPs have evolved for inhibition of MMPs, but (R)-ND-336 is a designed small molecule. This long residence time, along with potency of inhibition, accounts for effective inhibition of MMP-9. In contrast, we


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observe only very poor linear non-competitive inhibition and short residence time (seconds) for MMP-8. On the other hand, (S)-ND-336 preferentially inhibits MMP-2 (Ki 37 ± 1 nM) and MMP-14 (Ki 53 ± 3 nM) over MMP-9 (Ki 190 ± 30 nM). The selectivity of (R)-ND-336, as measured by the ratio of Ki of MMP-8/Ki of MMP-9, is 450-fold, while that of (S)-ND-336 is 11fold, and racemic (R, S)-ND-336 has 51-fold selectivity (Table 1).

Scheme 1. Synthesis of (R)-ND-336 (2)

Figure 2. Mechanism of inhibition of (R)-ND-336. (a) (R)-ND-336 is a mechanism-based inhibitor of MMP-2, MMP-9, and MMP-14, where Glu-404 at the active site abstracts a proton alpha to sulfone, resulting in the corresponding thiolate that coordinates with zinc ion as a tightbinding inhibitor, for which the reversal occurs very slowly. (b) (R)-ND-336 inhibits MMP-2, MMP-9, and MMP-14 as a slow-binding inhibitor with long residence times. The compound is a poor non-competitive inhibitor for MMP-8, for which the residence time is very short.


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Table 1. Inhibition profiles for racemic (R,S), (R), and (S)-ND-336.

Enzyme

Inhibition type

MMP-1cat MMP-2 MMP-3cat MMP-7 MMP-8cat

Slow-binding Linear noncompetitive Slow-binding Slow-binding -

(R,S)-ND-336a 4% @ 100 µM 85 ± 1 nM 23% @ 100 µM 1% @ 100 µM 7,700 ± 100 nM

MMP-9cat 150 ± 10 nM MMP-14cat 120 ± 10 nM ADAM-9 31% @ 100 µM ADAM-10 14% @ 100 µM Selectivityc 51 a Reproduced from Gao et al.9 for ease of comparison. b Calculated from the ratio of koff/kon. c Calculated from Ki of MMP-8/ Ki of MMP-9.

Ki (nM) (R)-ND-336 22% @ 100 µM 127 ± 1 nMb 37% @ 100 µM 20% @ 100 µM 8590 ± 230 nM

(S)-ND-336 24% @ 100 µM 37 ± 1 nMb 22% @ 100 µM 15% @ 100 µM 2100 ± 56 nM

19 ± 3 nMb 119 ± 3 nMb 41% @ 100 µM 13% @ 100 µM 450

190 ± 30 nMb 53 ± 3 nMb 25% @ 100 µM 5% @ 100 µM 11

The structural basis for selectivity of (R)-ND-336 towards MMP-9 was also addressed by computational analysis (Figures 3a and 3b). We superimposed the X-ray structure for the catalytic domain of MMP-8 to that of MMP-9 docked with the (R)- and (S)-enantiomers. The two aromatic rings insert favorably into the S1′ pocket with the amide carbonyl oxygen of Ala417 hydrogen bonding to the p-aminomethyl group, which favorably positions the thiirane moiety to coordinate with the active-site zinc ion. The reason for 10-fold affinity difference between the two enantiomers could be attributed to the preferred orientation of the thiirane moiety to approach the zinc ion prior to coordination. The p-substituted position of the inhibitor clashes with Arg222 of MMP-8 (Figure 3c) indicating an unfavorable interaction. Although the side chain of arginine might possibly undergo motion to accommodate the aminomethyl moiety,


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a superimposition of 23 additional MMP-8 crystal structures (a total of 24) with various inhibitors (Figure 3c) suggests a conformational preference of Arg222 that will clash with the terminal aminomethyl moiety. The corresponding residue in MMP-9 is Thr426, which, due to its smaller size can better accommodate ND-336, hence, the selectivity for inhibition of MMP-9 over MMP-8. Figure 3. Computational analysis of (R)and (S)-ND-336 bound to MMP-8 and MMP-9. Stereo view of the docked binding pose of ND-336 to the MMP-9 catalytic domain. (a) The aromatic rings of (R)-ND-336 (purple for carbon in capped sticks) insert within the S1′ subsite of MMP-9 (green for carbon, loop, and translucent surface; PDB ID code 2OVX); Ala417 hydrogen bonds to the aminomethyl moiety. (b) The (S)enantiomer binds similarly. The two enantiomers differ in the orientation of the thiirane ring for interaction with the catalytic zinc ion (green sphere). The MMP-8 crystal structure (pink for carbon and for the loop; PDB ID code 2OY4) is superimposed to show Arg222 in place of Thr426 of MMP-9, which sterically disfavors complexation with ND-336. Hydrogen bonds are displayed as broken black lines. (c) Stereo view of the superimposition of 24 MMP-8 X-ray structures (differentially colored; see experimental section for PDB ID codes) to the MMP-9 (green translucent surface, except for the S1′ loop, zinc ion, the metalchelating histidine residues and the catalytic glutamate; the loop and amino-acid carbons are in green) in complex with docked RND-336 (purple for carbon in capped stick representation). The residue Arg222 in MMP-8 (labeled) shows a conformational preference that clashes with the aminomethyl moiety of the inhibitor and likely is the basis for the lack of observation of competitive inhibition for MMP-8.


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(R)-ND-336

Is

Not

Cytotoxic

and

Has

Minimal

Absorption

After

Topical

Administration. (R)-ND-336 was evaluated for cytotoxicity by the XTT assay. A favorable IC50 value of 143 ± 3 µM afforded a therapeutic index (IC50/Ki for MMP-9) ratio of 7530, indicating that the compound is not cytotoxic. (R)-ND-336 is also not mutagenic by the Ames test. The preferred route of administration for treatment of DFUs is topical. We evaluated the absorption of (R)-ND-336 through the wound by comparing plasma concentrations after topical administration, relative to those after intravenous dosing (Figure 4). Systemic exposure, as measured by the area-under-the-curve (AUC), was 1.3 µM.min after topical administration and 35 µM.min following intravenous dosing, for an AUCtop/AUCiv ratio of 3.7%, indicating minimal absorption after topical administration. The desirable low systemic exposure of (R)-ND336 after topical administration mitigates side effects, if any, including musculoskeletal syndrome, which was the most frequent side effect observed with broad-spectrum MMP inhibitors in clinical trials.22 Figure 4. Absorption of (R)-ND-336. Plasma concentrations in mice after single dose administration of (R)-ND-336 at 2 mg/kg. (R)ND-336 was applied topically to wounds of db/db mice (n = 3 mice/time point); a separate group of non-wounded mice (n = 3 mice/ time point) received (R)-ND-336 intravenously (iv) by tail-vein injection. Mean ± SD. Comparison of the area-under-the-curve (AUC) after topical administration (1.3 µM.min) to intravenous dosing (35 µM.min) gives a ratio of 3.7%, indicating minimal absorption after topical administration.

Efficacy of (R)-, (S)-, and (R,S)-ND-336 in Mouse Model of Diabetic Wound Healing. (R), (S)-, and (R,S)-ND-336 were evaluated in wound-inflicted db/db mice after topical treatment


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(Figures 5a and 5b). (R)-ND-336 had superior efficacy, better than racemic (R,S)-ND-336. (S)ND-336 exhibited no activity, comparable to vehicle. As the db/db mouse model produces splinted wounds that heal by re-epithelialization,23 the wounds were evaluated with hematoxylin and eosin (H&E) staining. (R)-ND-336-treated mice showed nearly complete re-epithelialization, whereas mice treated with vehicle, (R,S)-ND-336, or (S)-ND-336 showed partial reepithelialization (Figure 5c). The superior efficacy of (R)-ND-336 can be explained by its excellent selectivity towards inhibition of MMP-9—the detrimental proteinase—over MMP-8, the beneficial one. In vivo inhibition was ascertained by in-situ zymography using fluorescent DQ-collagen for MMP-8 activity and DQ-gelatin for MMP-9 activity, where either activity manifests as green fluorescence. Wounds treated with (R)-ND-336 show complete inhibition of MMP-9 (Figure 5d) and no discernable inhibition of MMP-8 (Figure 5e), whereas the results are mixed with (R,S)-ND-336 or (S)-ND-336 (Figures 5d and 5e), as their MMP-9/MMP-8 selectivity is lower.


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Figure 5. (R)-ND-336 accelerates wound healing faster than (R, S)- or (S)-ND-336 in db/db mice. Mice were given an 8-mm diameter full-thickness excision wound on the dorsal thorax. Mice were treated topically one day later with (S)-, (R)- or (R,S)-ND-336 at 50 µg/wound/day for 14 days or vehicle (water). (a) (R)-ND-336 accelerates wound healing faster than (R,S)-ND-336, (S)-ND-336, or vehicle; mean ± SEM, n = 7 mice/group/time point for vehicle, (R,S)-, and (S)ND-336; n = 8/time point for (R)-ND-336; * p < 0.05, ** p < 0.01 by Mann-Whitney U twotailed test. (b) Representative wound images. (c) Representative H&E staining on day 14, 10x lens. Re-epithelialization is shown by the black lines (scale bars at the upper right hand corner, 50 µm). (d) In-situ zymography of the wounds with DQ-gelatin shows (R)-ND-336 inhibits MMP-9 better than (R,S)- or (S)-ND-336; n = 3 mice/group. (e) In-situ zymography with DQcollagen shows (R)- and (R,S)-ND-336 do not inhibit MMP-8, while (S)-ND-336 inhibits MMP8 partially; n = 3 mice/group. The bottom row shows merged images with DAPI nuclear DNA staining, 40x lens (scale bars, 50 µm). (R)-ND-336 Has Better Efficacy than Becaplermin. A side-by-side comparison of (R)-ND336 and becaplermin was undertaken in wound-inflicted db/db mice after topical application.


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(R)-ND-336-treated wounds healed faster by day 7 compared to those treated with topical becaplermin (Figures 6a and 6b), while becaplermin-treated wounds healed faster than vehicle starting on day 10. This is consistent with results from clinical trials in which becaplermin starts differentiating from placebo in the incidence of complete healing after 10 weeks of treatment.24 Since we had identified MMP-9 as an impediment to wound healing and MMP-8 as beneficial, we investigated whether becaplermin had any effect on the two enzymatic activities. Becaplermin contains human platelet-derived growth factor (PDGF)-BB. We observed that human PDGF-BB inhibits MMP-9 poorly (17% inhibition at 4 µM). PDGF-D was shown recently to upregulate the expression of TIMP1, resulting in decrease of MMP-2 and MMP-9 activity.25 Next, we evaluated whether becaplermin affects MMP-9 and MMP-8 activity in vivo by analysis of the mouse wounds by in-situ zymography. While (R)-ND-336 completely inhibits active MMP-9, becaplermin partially decreases MMP-9 activity (Figure 6c), but does not completely suppress it. The superiority of (R)-ND-336 towards inhibition of the detrimental MMP-9 explains its enhanced efficacy. However, becaplermin did not affect MMP-8 levels by in-situ zymography (Figure 6c). Histological assessment of the wounds shows enhanced acceleration of re-epithelialization in (R)-ND-336-treated mice compared to vehicle or becaplermin groups (Figure 5d). Since reactive-oxygen species (ROS) has been shown to upregulate MMP-9,14, 26 we dosed the mice with luminol-based L-012 for in vivo imaging for ROS. Statistically significant decreases in ROS levels are observed in both becaplermin- and (R)ND-336-treated mice, when compared to vehicle on days 3 and 7 (Figures 6e and 6f). Finally, we analyzed the mouse wounds with our affinity resin coupled with proteomics. We found that becaplermin decreased active MMP-9 levels 3-fold compared to vehicle (Figure 6g), but did not affect active MMP-8 levels (Figure 6h). ROS are known to upregulate MMP-914, 26 via NF-κB


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regulation,16 which induces dermal inflammation.27 As indicated earlier for the human samples, we had found that levels of NF-κB p65 and active MMP-9 paralleled each other. We also examined the levels of NF-κB p65 in the diabetic mouse wounds by ELISA. NF-κB p65 levels were significantly reduced in the inflammation stage of wound healing on days 1 and 2 in becaplermin-treated mice and were comparable to those of (R)-ND-336 treatment. However, the effect of (R)-ND-336 on lowering NF-κB p65 levels was sustained and prolonged, whereas the effect of becaplermin was abrogated to the level of the control arm within the 7-day duration of the study (Figure 6i). Levels of NF-κB p65 over the course of 7 days expressed as AUC show that (R)-ND-336 is more efficacious at lowering NF-κB p65 (492 ± 103 µg/mg·day) when compared with vehicle (834 ± 121 µg/mg·day, p = 0.02) than becaplermin (648 ± 168 µg/mg·day, p = 0.19 between becaplermin and vehicle). We showed that both (R)-ND-336 and becaplermin decreased ROS and lowered NF-κB p65 levels; however, (R)-ND-336 more effectively lowered NF-κB p65, and this also explains its superior efficacy.


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Figure 6. (R)-ND-336 is superior to becaplermin in accelerating wound healing in db/db mice. Mice were treated topically one day after wound infliction (8 mm, Tegaderm-covered) with 5 µg or 50 µg/wound/day of becaplermin or (R)-ND-336, respectively, or vehicle (water) for 14 days. (a) Wound measurements show that (R)-ND-336 has better efficacy than becaplermin; mean ± SEM; n = 11, 8 and 8 for vehicle; n = 11, 7, and 7 for (R)-ND-336; n = 12, 9, and 9 for becaplermin on days 7, 10, and 14, respectively; * p < 0.05, ** p < 0.01 by Mann-Whitney U two-tailed test. (b) Representative wound images. (c) In-situ zymography shows that (R)-ND336 inhibits MMP-9 activity in vivo, while becaplermin decreases MMP-9 activity but does not completely inhibit it. In-situ zymography with DQ-collagen shows that (R)-ND-336 and becaplermin do not inhibit MMP-8. The bottom row shows merged images with nuclear DNA staining by DAPI. Images were taken with a 40x lens (scale bars, 50 µm). (d) H&E staining of representative wounds; day 7, 10x lens, re-epithelialization shown by black lines (scale bars, 50 µm). (e) In vivo imaging for ROS shows decrease in ROS levels in becaplermin- and (R)-ND336-treated mice. (f) ROS bioluminescent quantification indicates a statistically significant reduction in ROS in becaplermin- and (R)-ND-336-treated mice. (g) Analysis of the wounds with the affinity resin/proteomics indicates significant decrease in active MMP-9 in becaplermintreated animals (h) and no significant differences in the levels of active MMP-8 compared to vehicle-treated ones. (i) (R)-ND-336-treated mice show statistically significant reduction of NFκB p65. Mean ± SD, n = 3 mice/group/time point for (c)-(i); *p < 0.05, **p < 0.01 by Student’s t two-tailed test.

CONCLUSION Our work points to upregulation of active MMP-9 as a culprit in the pathology of DFUs and the involvement of active MMP-8 in promoting wound repair in diabetic mice.8, 9 As shown in the present study, the levels of MMP-9 in diabetic human wounds parallel those of NF-κB p65. The likely roles of MMP-8 and MMP-9 are illustrated in Figure 7. Neutrophils secrete MMP-8, MMP-9, and ROS; ROS triggers activation of NF-κB, which upregulates MMP-9, the detrimental proteinase. MMP-8 repairs damaged collagen and the extracellular matrix, assisting in healing. We validate the existence of the target MMP-9 in human DFUs and disclose the discovery of a new selective and potent inhibitor, (R)-ND-336, with unprecedented efficacy as a heretofore unexplored strategy in diabetic wound management. (R)-ND-336 shows superior efficacy to becaplermin in db/db mice and holds promise for the treatment of DFUs.


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Figure 7. Roles of MMP-8 and MMP-9 in diabetic wound healing. Upon injury, neutrophils are recruited to the injury site and secrete MMP-8, MMP-9, as well as ROS that kills bacteria and regulates thrombus formation. Increased ROS triggers activation of NF-κB, which stimulates the expression and upregulation of MMP-9, which is detrimental to diabetic wound healing. MMP-8 plays a beneficial role, repairing damaged collagen and the extracellular matrix, resulting in a healed wound. The best therapeutic approach for treatment of DFUs entails selective inhibition of the detrimental MMP-9 with the small-molecule (R)-ND-336, without affecting MMP-8 activity so that it can do its repair function.

EXPERIMENTAL SECTION Human Wound Samples. Debridement samples were obtained with informed consent from diabetic patients with chronic wounds (n = 25). Chronic wounds are defined as wounds that have existed longer than 30 days. Cancer-free dermal samples from non-diabetic patients were obtained with informed consent from patients undergoing Mohs surgery for diagnosis of skin


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cancer. Collection of human tissue was approved by the Institutional Review Boards at the University of Notre Dame, Elkhart General Hospital, and Beacon Memorial Hospital. Samples were immediately frozen after collection and stored at -80 ºC until analysis. Demographics of patient samples and human biospecimen are detailed in Table S1. Affinity Resin. The affinity resin (1, Figure 1a) was synthesized in 14 synthetic steps following a previously reported method.12 Analysis of Wound Samples with Affinity Resin and Proteomics. Processing of the tissue samples (n = 3 mice/group/time point), incubation with the affinity resin, and identification of active MMPs were done as reported.28 For quantification, three peptides for each MMP were custom-synthesized (GenScript, Piscataway, NJ). The synthesized peptides used for quantifying MMPs in mouse wounds are the same as previously reported28 and those for quantification of human MMPs are shown in Table S2. Quantification in the unknown wound samples was performed using the calibration curves in control human plasma or in control mouse tissue relative to internal standard and three peptides per MMP, with three transitions as qualifier to identify the protein and three transitions as quantifier to quantify the MMP. Proteomic analysis was repeated 3 times. Measurement of NF-κB p65 by ELISA. Tissues were homogenized in cold lysis buffer containing EDTA-free protease inhibitor cocktail (Pierce, Thermo Fisher Scientific, Hanover Park, IL). Total protein concentrations of collected homogenates were analyzed by the BCA assay.29 The levels of NF-κB p65 in the samples were measured with a NF-κB p65 ELISA kit, following the manufacturer’s instructions (Abcam, Cambridge, MA), and expressed in µg per mg of tissue. The area under the curve (AUC) was calculated in GraphPad Prism 5 using the linear trapezoid rule. Multivariate analysis was performed using the multivariate modeling module of


19


JMP 11.00 (SAS, Cary, NC). Control tissue was assigned as WG 0. Samples were analyzed in duplicates. Syntheses of Enantiomerically Pure (R)-ND-336 and (S)-ND-336. All chemicals, reagents, and solvents were used directly as purchased without further purification. Analytical thin-layer chromatography was performed on silica gel 60 F254. For column chromatography, silica gel 60, 230−400 mesh, 40−63 µm was used. 1H NMR and

13

C NMR spectra were recorded on Bruker

AVANCE III HD 400 or Bruker AVANCE III HD 500 spectrometers (Bruker Daltonik, Bremen, Germany) and operating at 1H resonance frequencies of 400.13 and 500.13 MHz, respectively. Chemical shifts are referenced to the residual/deuterated solvent (e.g., for CDCl3, δ = 7.26 and 77.16 ppm for 1H and 13C NMR, respectively) and reported in parts per million (ppm, δ) relative to tetramethylsilane (TMS, δ = 0.00 ppm). Coupling constants (J) are reported in Hz, and the splitting abbreviations used are: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad. Highresolution mass spectra were measured using a Bruker micrOTOF/Q2 mass spectrometer in electrospray ionization (ESI). (R)-4-((3-Chloro-2-hydroxypropyl)thio)phenol ((R)-4). A 250-mL flask was charged with 4-hydroxylthiophenol (3) (6.81 g, 54.05 mmol) and K2CO3 (7.46 g, 54.05 mmol) in 120 mL ethanol at ice-water temperature. (R)-Epichlorohydrin (5.00 g, 54.05 mmol) was added dropwise to the above solution. The resulting mixture was brought to room temperature within 15 min and was stirred for 8 h, at which time the starting material was consumed. The reaction mixture was filtered through a thin layer of silica gel and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica-gel chromatography (hexanes/ethyl acetate 2:1) to yield compound (R)-4 as a colorless viscous oil (11.31 g, 96%). The enantiomeric ratio of the title product was determined by chiral HPLC analysis (Figures S1a, S1b, and S1c). The


20

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HPLC instrument consisted of a PerkinElmer series 200 autosampler, series 200 UV/vis detector, series 200 pump, using TotalChrom Navigator software (PerkinElmer, Shelton, CT USA). The HPLC conditions consisted of isocratic elution on a chiral PAK AD-H column (Daicel Corp., Japan, 5 µm, 250 × 4.6 mm i.d.), at a flow rate of 1 mL/min with 75% hexane and 25% i-PrOH. Effluent was detected at 238 nm. A 15-µL aliquot of the diluted sample in 75% hexane and 25% i-PrOH was analyzed by HPLC: (R)-4 (99.6%, Rt = 6.52 min), (S)-4 (0.40%, Rt = 8.99 min); 99.2 % ee. 1H NMR (500 MHz, CDCl3) δ 2.98-2.94 (m, 1H), 3.07-3.03 (m, 1H), 3.69-3.62 (m, 2H), 3.87-3.83 (m, 1H), 6.80-6.77 (m, 2H), 7.36-7.26 (m, 2H);

13

C NMR (125 MHz, CDCl3) δ 40.4,

48.0, 69.7, 116.7, 124.6, 134.4, 155.9; HRMS [M + H]+ calcd for C9H12ClO2S 219.0168; found 219.0241. (R)-1-Chloro-3-((4-(4-(hydroxymethyl)phenoxy)phenyl)thio)propan-2-ol ((R)-5). A 250mL round-bottomed flask was charged with (R)-4 (4.36 g, 20 mmol), 4-(hydroxymethyl)phenyl boronic acid (6.08 g, 40 mmol), Cu(OAc)2 (3.71 g, 20.4 mmol), Et3N (10.1 g, 100 mmol) and 0.65 g activated 4-Å molecular-sieve powder in 120 mL of dichloromethane and the mixture was stirred under an oxygen balloon. The reaction was stopped after 26 h and the solution was filtered through a thin layer of celite. The filtrate was concentrated to dryness under reduced pressure and the residue was purified by silica-gel chromatograph (hexanes/ethyl acetate 6:1 to 4:1 to 2:1) to provide (R)-5 as a colorless oil (1.34 g, 21 %). 1H NMR (500 MHz, CDCl3) δ 2.36 (br, 1H), 3.03-2.99 (m, 1H), 3.12-3.08 (m, 1H), 3.70-3.64 (m, 2H), 3.91-3.88 (m, 1H), 4.67 (s, 2H), 6.95-6.93 (m, 2H), 7.01-6.99 (m, 2H), 7.38-7.34 (m, 2H), 7.40-7.39 (m, 2H);

13

C NMR

(125 MHz, CDCl3) δ 39.8, 48.2, 65.0, 69.7, 119.5, 128.2, 129.0, 133.3, 136.5, 156.3, 157.3; HRMS [M + Na]+ calcd for C16H17ClNaO3S 347.0479 ; found 347.0514.


21


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(S)-(4-(4-((Oxiran-2-ylmethyl)thio)phenoxy)phenyl)methanol ((S)-6). Compound (R)-5 (0.24 g, 0.74 mmol) was dissolved in 9 mL MeOH/CH3CN (1:2, v/v) at ice-water temperature, K2CO3 (0.20 g, 1.49 mmol) was added to the above solution and the reaction mixture was allowed to warm up to the room temperature and maintained for 2 h. The solution was concentrated to dryness under reduced pressure. The residue was taken up in 3 mL of dichloromethane, and the solution was purified by silica-gel chromatograph (hexanes/ethyl acetate 4:1 to 2.5:1) to give (S)-6 as a colorless viscous oil (0.15 g, 69%).

1

H NMR (500 MHz,

CDCl3) δ 1.82 (br, 1H), 2.49-2.48 (m, 1H), 2.79-2.77 (m, 1H), 2.89-2.85 (m, 1H), 3.11-3.08 (m, 1H), 3.17-3.14 (m, 1H), 4.67 (d, J = 3.95 Hz, 2H), 6.95-6.92 (m, 2H), 7.01-6.99 (m, 2H), 7.367.33 (m, 2H), 7.44-7.41 (m, 2H);

13

C NMR (125 MHz, CDCl3) δ 38.1, 47.6, 51.3, 65.0, 119.3,

119.4, 128.9, 129.0, 133.7, 136.5, 156.4, 157.2; HRMS [M + Na]+, calcd for C16H16ClNaO3S, 311.0712; found 311.0717. (S)-di-tert-Butyl 4-(4-((oxiran-2-ylmethyl)sulfonyl)phenoxy)benzyl-iminodicarboxylate ((S)-8). PBu3 (0.51 mL, 2.04 mmol) was added slowly dropwise to a solution of (S)-6 (0.39 g, 1.36 mmol), NH(Boc)2 (0.44 g, 2.04 mmol) and 1,1´-(azodicarbonyl)dipiperidine (0.51 g, 2.04 mmol) in 15 mL toluene at ice-water temperature. Subsequently, this reaction mixture was allowed to warm up to room temperature and stirring was continued at room temperature for 6 h. The suspension was filtered and the filtrate was concentrated to dryness in vacuo to give an oil for product (S)-7, which was used in the next step without further purification. The oil was dissolved in 10 mL dichloromethane and the solution was cooled to ice-water temperature. 3Chloroperoxybenzoic acid (m-CPBA, 0.47 g, 2.72 mmol) was added to the solution in one portion and the resulting mixture was stirred at ice-water temperature for 30 min. The reaction mixture was filtered and the filtrate was diluted with approximately 30 mL of ethyl acetate. The


22

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solution was washed with 10% aqueous sodium thiosulfate, followed by saturated sodium bicarbonate and brine. The organic layer was dried over anhydrous sodium sulfate and was filtered. The filtrate was concentrated under vacuum to dryness. The residue was dissolved in 3 mL of dichloromethane and the sample was purified by silica-gel chromatography (hexanes/ethyl acetate 8:1 to 4:1) to provide (S)-8 as a colorless oil (0.29 g, 41% over two steps). 1H NMR (500 MHz, CDCl3) δ 1.48 (s, 18H), 2.48-2.47 (m, 1H), 2.83-2.81 (m, 1H), 3.34-3.25 (m, 3H), 4.78 (s, 2H), 7.07-7.01 (m, 4H), 7.36 (d, J = 8.55 Hz, 2H), 7.88-7.85 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 28.2, 46.0, 46.1, 49.0, 59.9, 82.9, 117.7, 120.6, 129.6, 130.7, 132.6, 135.8, 152.8, 153.9, 163.1; HRMS [M + Na]+, calcd for C26H33NNaO8S 542.1819; found 542.1813. (R)-di-tert-Butyl 4-(4-((thiiran-2-ylmethyl)sulfonyl)phenoxy)benzyl-iminodicarboxylate ((R)-9). Thiourea (0.093 g, 1.22 mmol) was added to a solution of compound (S)-8 (0.32 g, 0.61 mmol) in 8 mL methanol at room temperature. The mixture was stirred for 24 h, at which time the reaction was completed by TLC monitoring. The solvent was removed in vacuo and the residue was dissolved in 3 mL dichloromethane, which was purified by silica-gel chromatography (hexanes/ethyl acetate 8:1 to 6:1) to afford (R)-9 as a colorless oil (0.23 g, 70%). The enantiomeric ratio of the title product was determined by chiral HPLC analysis (Figures S1d, S1e, and S1f). The HPLC conditions were described earlier in the preparation of compound (R)-4 using i-propanol/hexane (3:97, v/v) as the mobile phase. (R)-9 (99.3%, Rt = 37.0 min), (S)-9 (0.7%, Rt = 40.4 min); 98.6% ee. 1H NMR (500 MHz, CDCl3) δ 1.48 (s, 18H), 2.16-2.15 (m, 1H), 2.54-2.52 (m, 1H), 3.08-3.03 (m, 1H), 3.19-3.14 (m, 1H), 3.53-3.50 (m, 1H), 4.78 (s, 2H), 7.08-7.02 (m, 4H), 7.37-7.35 (m, 2H), 7.86-7.84 (m, 2H);

13

C NMR(125 MHz,

CDCl3) δ 24.4, 26.3, 28.2, 49.0, 62.9, 82.9, 117.8, 120.5, 129.6, 130.9, 132.1, 135.9, 152.8, 154.0, 163.2; HRMS [M + Na]+, calcd for C26H33NNaO7S2 558.1590 ; found 558.1578.


23


(R)-ND-336:

Page 24 of 38

(R)-(4-(4-((Thiiran-2-ylmethyl)sulfonyl)phenoxy)phenyl)methanamine

hydrochloride). A solution of HCl in 1,4–dioxane (4 M, 3.1 mL, 12.4 mmol) was added to a solution of (R)-9 (0.22 g, 0.41 mmol) in 20 mL of dichloromethane/ethyl acetate (1:1, v/v). The reaction mixture was stirred at room temperature for 24 h, at which time the starting material had been consumed. The solvent was evaporated in vacuo and the residue was triturated with ethyl acetate to provide (R)-ND-336 as a white solid (0.13 g, 89.0%). 1H NMR (500 MHz, MeOD-d4)

δ 2.14-2.13 (m, 1H), 2.52 -2.51 (m, 1H), 3.06-3.04 (m, 1H), 3.55-3.45 (m, 2H), 4.15 (s, 2H), 7.22-7.15 (m, 4H), 7.56-7.54 (m, 2H), 7.94-7.91 (m, 2H);

13

C NMR(125 MHz, MeOD-d4) δ

22.8, 25.7, 42.5, 61.9, 118.1, 120.6, 129.9, 131.0, 131.2, 133.0, 156.2, 162.4; HRMS [M + H – NH2]+, calcd for C16H15O3S2 319.0462 ; found 319.0457. (S)-ND-336:

(S)-(4-(4-((Thiiran-2-ylmethyl)sulfonyl)phenoxy)phenyl)methanamine

hydrochloride. It was synthesized according to the same procedure as for (R)-ND-336. The enantiomeric purities were: 99.6% ee for (S)-4, 97.9% ee for (S)-9. The 1H, 13C NMR and mass spectra were identical to those of (R)-ND-336. 1H NMR (500 MHz, MeOD-d4) δ 2.13-2.15 (m, 1H), 2.51-2.52 (m, 1H), 3.04-3.07 (m, 1H), 3.45-3.55 (m, 2H), 4.15 (s, 2H), 7.16-7.21 (m, 4H), 7.54-7.57 (m, 2H), 7.92-7.94 (m, 2H); 13C NMR (125 MHz, MeOD-d4) δ 22.9, 25.8, 42.5, 61.9, 118.1, 120.6, 130.0, 131.0, 131.2, 133.1, 156.2, 162.5.; HRMS [M + H – NH2]+, calcd for C16H15O3S2 319.0457; found 319.0455. Compound purity. The purity of compounds (R,S)-, (R)-, and (S)-ND-336 was 96.8%, 97.1%, and 96.6%, respectively, as determined by UPLC with UV detection. A Waters Acquity UPLC system (Waters Corporation, Milford, MA, USA) equipped with a binary solvent manager, an autosampler, a column heater, and a photodiode array detector was used. The analyses of compounds were performed on a Kinetex C18 column (2.6 µm, 2.1 µm x 100 mm,


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Phenomenex, Torrance, CA, USA) at a column temperature of 48 °C. The mobile phase consisted of elution at 0.4 mL/min with 90% A/10% B for 2 min, followed by a 8-min linear gradient to 10% A/90% B, then 2 min with 90% A/10% B (A = water containing 0.1% formic acid, B = acetonitrile containing 0.1% formic acid). The effluent was monitored by UV detection at 260 nm and the area of the peaks was integrated. MMP kinetics of (R)- and (S)-ND-336. Human recombinant active MMP-2 and MMP-7, and the catalytic domains of MMP-3 and MMP-14 were purchased from EMD Chemicals, Inc. (Burlington, MA); human recombinant catalytic domains of MMP-1, MMP-8, and MMP-9 were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY); human recombinant active ADAM9 and ADAM10 were purchased from R&D Systems (Minneapolis, MN). Fluorogenic substrates MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 (for MMP-2, MMP-7, MMP-9, and MMP-14) and MOCAc-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 (for MMP3) were purchased from Peptides International (Louisville, KY); Mca-Lys-Pro-Leu-Gly-LeuDpa-Ala-Arg-NH2 (for MMP-1, MMP-8, and ADAM10) and Mca-Pro-Leu-Ala-Gln-Ala-ValDpa-Arg-Ser-Ser-Ser-Arg-NH2 (for ADAM9) were purchased from R&D Systems. Human recombinant PDGF-BB was purchased from TONBO Biosciences (San Diego, CA). The Km values used for MMP-2, MMP-9, and MMP-14 with the fluorogenic substrate MOCAc-Pro-LeuGly-Leu-A2pr(Dnp)-Ala-Arg-NH2 were calculated before the inhibition studies and were similar to the previous published values.30 Inhibitor stock solutions (10 mM) were prepared freshly in DMSO before enzyme-inhibition assays. We followed the same methodology for enzymeinhibition studies as reported before by Ikejiri et al.31 Enzyme-inhibition studies were carried out using a Cary Eclipse fluorescence spectrophotometer (Varian, Agilent Technologies, Santa


25


Page 26 of 38

Clara, CA). Racemic (R,S)-ND-336, (R)-ND-336 and (S)-ND-336 were stable in the buffers used in the kinetic assays. The experiments were done in duplicates and repeated three times. Computational Analysis. The X-ray crystallographic structure coordinates of MMP-9 (PDB ID: 2OVX), and MMP-8 (PDB ID codes: 2OY4, 1A85, 1A86, 1BZS, 1I73, 1I76, 1JAN, 1JAO, 1JAP, 1JAQ, 1JH1, 1JJ9, 1KBC, 1MMB, 1ZP5, 1ZS0, 1ZVX, 2OY2, 2OY4, 3DNG, 3DPE, 3DPF, 3TT4, and 4QKZ) were obtained from the Protein Data Bank (www.rcsb.org/pdb). Protein coordinates were prepared and energy-minimized with Protein Preparation Wizard module of Maestro program (Schrödinger, LLC, Cambridge, MA) using OPLS2005 force field. The ligand coordinates were built based on our previous calculations.30,

32

The (R) and (S)

stereoisomers of ND-336 were flexibly docked to the active site of human MMP-9 catalytic domain using the Glide program33 (version 6.7, Schrödinger, LLC) and scored with standard precision method. Absorption of (R)-ND-366 after topical administration. The extent of absorption of (R)ND-366 was determined by comparing the plasma concentrations after topical application of (R)ND-366 to those after intravenous administration. Female mice (C57BLKS/6J, 8 weeks, 18-25 g, same background as diabetic mice; Jackson Laboratory (Bar Harbor, ME), n = 3 mice per time point, 7 time points) were given an 8 mm full-thickness wound on the dorsal thorax on day 0. The next day, the wounds were treated topically with a single 50 µg dose of (R)-ND-336 (100 µL of 0.5 mg/mL in water, equivalent to 2 mg/kg). Mice (n = 3 per time point) were sacrificed at 5, 15, 30, 60, 90, 120, and 240 min. Terminal blood (heparin) was collected by cardiac puncture and centrifuged to obtain plasma. Plasma was analyzed for concentrations of (R)-ND-336 by UPLC with MRM.


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A second study was conducted in uninjured C57BLKS/6J mice (n = 3 mice/time point, 7 time points). Mice received a single dose of 2 mg/kg (equivalent to 50 ng, 100 µL of 0.5 mg/mL in water) intravenously by tail-vein injection. Mice (n = 3/time point) were sacrificed at 5, 15, 30, 60, 90, 120, and 240 min; terminal blood was collected by cardiac puncture and processed as described for the topical administration. A 60-µL aliquot of plasma was mixed with 120 µL of internal standard32 in cold acetonitrile to a final concentration of 1.5 µM. The mixture was centrifuged at 10,000g for 15 min. The supernatant was analyzed by reversedphase UPLC with (+)ESI-MRM of the transitions 319→182 for (R)-ND-336 and 300→93 for the internal standard. Calibration curves of (R)-ND-336 were prepared by fortification of blank mouse plasma with (R)-ND-336 at concentrations up to 20 µM. Samples and calibration standards were analyzed on a Kinetex C18 column (2.6 µm, 2.1 µm x 100 mm, Phenomenex, Torrance, CA). Quantification was performed using peak-area ratios relative to the internal standard and linear-regression parameters calculated from the calibration curves. The area-underthe curve (AUC) after topical and intravenous administration was calculated. The percent absorption was determined from the ratio of AUCtopical/AUCintravenous. Compounds. (R,S)-ND-336 was synthesized as previously described.9 Becaplermin (Regranex™) was purchased from a local retail pharmacy as a gel containing 0.01% PGDF in carboxymethylcellulose. Assay Interference Compounds. (R)-, (S)-, and (R,S)-ND-336 were screened against the list of pan-assay interference compounds34 and using the filter http://www.cbligand.org/PAINS/. No flags were reported.


27


Animal Studies. All animal studies were conducted with approval and oversight by the Institutional Animal Care and Use Committee at the University of Notre Dame. All animal studies included a vehicle control group and were repeated at least 2 times, combining the data for the separate studies. Full-thickness Excision Animal Model. Female db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, 8-weeks old, ~40 g body weight) were purchased from Jackson Laboratory. Animals were fed 5001 Laboratory Rodent Diet (LabDiet, St. Louis, MO) and were given water ad libitum. The mice were housed in polycarbonate cages containing corncob bedding and maintained at 72 ± 2 ºF with a light/dark cycle of 12/12 h. Mice were shaved in the dorsal area and anesthetized with isoflurane. A single 8-mm diameter full-thickness excisional wound was created on the dorsal thorax using a biopsy punch (Miltex, Plainsboro, NJ) and it was covered with Tegaderm dressing (3M Company, St. Paul, MN). Topical treatments were administered under the Tegaderm dressing using a syringe on the following day (day 1). The dressings were changed on days 7 and 10 after photographs of the wounds were taken. At specific time points, mice were sacrificed, and wounds were harvested and immediately frozen in liquid nitrogen, followed by storage at 80 ºC. Wound Measurements. Following isofluorane anesthesia, the wounds were photographed using a Nikon D5300 camera (Nikon, Inc., Melville, NY), which was mounted on a tripod at a fixed distance. A ruler was included in the photographic frame. Calculation of wound areas was done using NIH ImageJ software (version 1.51). Wound healing was calculated as percent change in wound area relative to day 0. Wound measurement analyses were performed separately by two experimenters, one of whom was blinded.


28

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Histological Evaluation and In-situ Zymography. Fresh wound tissue was harvested, embedded in optimum cutting temperature compound, and cryosectioned at a thickness of 12-µm for H&E staining and at 8-µm and for in-situ zymography. Re-epithelialization was assessed morphologically35 on a Nikon Eclipse 90i Fluorescent Microscope (Nikon Instruments Inc., Melville, NY). In-situ zymography was performed as described.36 H&E and in-situ zymography were assessed on n = 3 mice/group/time point. In Vivo Imaging for ROS. Mice were anesthetized by inhalation with 2% isoflurane on days 1, 2, 3 and 7 post-wound infliction. The mice (n = 3/group/time point) were injected intraperitoneally with 200 µL of L-01237 (Wako Chemicals, Richmond, VA) dissolved in PBS at 5 mg/mL. Images were acquired immediately with a Xenogen IVIS Lumina instrument (Caliper Life Sciences, Waltham, MA), controlled by Living Image software (v 3.0), at 5-min intervals. The images were analyzed by ImageJ software 1.51 version. (R)-, (S)-, and (R,S)-ND-336 Wound Healing Study. This study included four groups: n = 7 mice/group for vehicle, (R,S)-, and (S)-ND-336, n = 8 mice for (R)-ND-336. (R)-, (S)-, and (R,S)ND-336 were dissolved in water at a concentration of 0.5 mg/mL; the vehicle consisted of distilled sterile water. The solutions were sterile-filtered and stored at 4 ºC. The drug solutions were prepared freshly every 2 days and warmed to room temperature before dosing. Mice were administered 100 µL of (R)-, (S), (R,S)-ND-336, or vehicle solutions topically once a day for 14 days. The dose was equivalent to 50 µg/wound/day. Wounds were measured on days 0, 7, 10, and 14. On day 14, the remaining mice were sacrificed. Becaplermin and (R)-ND-336 Wound Healing Study. This study consisted of three groups: n = 11 mice/group for vehicle and (R)-ND-336, and n = 12 mice for becaplermin. (R)ND-336 was dissolved in water at a concentration of 1.0 mg/mL; the vehicle consisted of


29


Page 30 of 38

distilled sterile water. The solutions were sterile-filtered and stored at 4 ºC. The drug solutions were prepared freshly every 2 days and warmed to room temperature before dosing. Mice were administered 50 µL of (R)-ND-336, becaplermin gel, or vehicle topically once a day for 14 days. The (R)-ND-336 dose was equivalent to 50 µg/wound/day; the becaplermin dose was equivalent to 5 µg/wound/day. Wounds were measured on days 0, 7, 10, and 14. On day 7, n = 3 mice per group were euthanized for H&E and in-situ zymography, with the remaining mice were sacrificed on day 14. Statistics. Data for n ≥ 7 were expressed as mean ± SEM and for n < 7 as mean ± SD. Data were analyzed for statistical significance using the Mann Whitney U test for n ≥ 7 with twotailed hypothesis or the Student’s t test for n < 7 with two-tailed hypothesis. ASSOCIATED CONTENT Supporting

Information.

Sample

size,

data

inclusion/exclusion

criteria,

replicates,

randomization, blinding, demographics of human wound samples, human biospecimens description, peptides used for proteomic quantification of active MMP-8 and MMP-9 in human wounds, enantiomeric excess determination of chiral intermediates, kinetic parameters, XTT assay, bacterial reverse mutation assay. The Supporting Information file is available free of charge on the ACS Publication website at DOI: AUTHOR INFORMATION Corresponding Author *Mayland Chang: Phone: (574) 631-2965; Email: [email protected] ORCID


30

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Mayland Chang: 0000-0002-4333-3775 Present Address §

Present address: Veterinary Population Medicine Department, College of Veterinary Medicine,

University of Minnesota, St. Paul, MN 55108 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare that US patents 9,604,957 and 9,951,035 B2 have been issued for (R)-ND336. ACKNOWLEDGMENTS We thank the patients who donated their tissue samples used in this work. We thank Sarah Chapman for the preparation of wound tissue sections and H&E staining, and Dr. Luiz Pantalena at Beacon Medical Group and Toni Page-Mayberry at the Harper Cancer Research Institute Tissue Biorepository at the University of Notre Dame for collection of the dermal tissue from uninjured non-diabetic patients. TTN is a Ruth L. Kirschtein National Research Service Award Fellow of the Chemistry-Biochemistry-Biology Interface Program at the University of Notre Dame, supported by training grant T32 GM075762 from the National Institutes of Health. This work was supported by the American Diabetes Association Pathway to Stop Diabetes grant 1-15ACN-06. ABBREVIATIONS


31


ADAM, a disintegrin and metalloproteinase; AUC, area-under-the-curve; BCA, bicinchoninic acid; Boc, t-Butoxycarbonyl; m-CPBA, meta-chloroperbenzoic acid; DFU, diabetic foot ulcer; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; H&E, hematoxylin and eosin; HRMS, high resolution mass spectrometry; MMP, matrix metalloproteinase; MOCAc, (7-methoxycoumarin-4-yl)acetyl; MRM, multiple reaction monitoring; NMR, nuclear magnetic resonance; MS, mass spectrometry; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PBS, phosphate buffered saline; OCT, optimal cutting temperature; ROS, reactive oxygen species; SD, standard deviation; SEM, standard error of the mean; TIMP, tissue inhibitor of metalloproteinase; TLC, thin-layer chromatography; TUNEL, terminal deoxynucleotidyl transferase-mediated DUTP-nick- end labeling; UPLC, ultra-performance liquid chromatography; XTT, (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide). REFERENCES (1)

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Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for

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