Benzyloxycarbonyl-proline-prolinal (ZPP): Dual complementary roles for neutrophil inhibition

Biochemical and Biophysical Research Communications 517 (2019) 691e696

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Benzyloxycarbonyl-proline-prolinal (ZPP): Dual complementary roles for neutrophil inhibition D.W. Russell a, b, *, 1, M. Hardison a, b, 1, K.R. Genschmer a, b, T. Szul a, d, P.E. Bratcher f, M. Abdul Roda a, d, g, X. Xu a, d, e, L. Viera a, b, d, e, J.E. Blalock a, b, c, d, e, A. Gaggar a, b, c, d, e, h, 2, B.D. Noerager i, 2 a

Department of Medicine, Division of Pulmonary, Allergy, and Critical Care, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA Lung Health Center, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA Program in Protease and Matrix Biology, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA d Gregory Fleming James Cystic Fibrosis Research Center, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA e Department of Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA f Department of Pediatrics, National Jewish Health, Denver, CO, 80206, USA g Division of Pharmacology, Utrecht Institute for Pharmaceutical Science, Faculty of Science, Utrecht University, Utrecht, the Netherlands h Medical Service, Birmingham VA Medical Center Birmingham, AL, 35294, USA i University of Montevallo, Montevallo, AL, 35115, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2019 Accepted 28 July 2019 Available online 7 August 2019

Neutrophil influx and activation contributes to organ damage in several major lung diseases. This inflammatory influx is initiated and propagated by both classical chemokines such as interleukin-8 and by downstream mediators such as the collagen fragment cum neutrophil chemokine Pro-Gly-Pro (PGP), which share use of the ELR þ CXC receptor family. Benzyloxycarbonyl-proline-prolinal (ZPP) is known to suppress the PGP pathway via inhibition of prolyl endopeptidase (PE), the terminal enzyme in the generation of PGP from collagen. However, the structural homology of ZPP and PGP suggests that ZPP might also directly affect classical glutamate-leucine-arginine positive (ELRþ) CXC chemokine signaling. In this investigation, we confirm that ZPP inhibits PE in vitro, demonstrate that ZPP inhibits both ELR þ CXC and PGP-mediated chemotaxis in human and murine neutrophils, abrogates neutrophil influx induced by murine intratracheal challenge with LPS, and attenuates human neutrophil chemotaxis to sputum samples of human subjects with cystic fibrosis. Cumulatively, these data demonstrate that ZPP has dual, complementary inhibitory effects upon neutrophil chemokine/matrikine signaling which make it an attractive compound for clinical study of neutrophil inhibition in conditions (such as cystic fibrosis and chronic obstructive pulmonary disease) which evidence concurrent harmful increases of both chemokine and matrikine signaling. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Benzyloxycarbonyl-proline-prolinal ZPP CXCR COPD PGP Prolyl-endopeptidase

1. Introduction Neutrophils (PMNs) are an important component of the innate immune system. These immune system “first responders” are capable of phagocytosis of an invading pathogen, release of anti-

* Corresponding author. Department of Medicine, Division of Pulmonary, Allergy, and Critical Care, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA. E-mail address: [email protected] (D.W. Russell). 1 Co-first Authors. 2 Co-senior Authors.

microbial enzymes, and generation of an oxidative burst aimed at destroying a foreign organism [1]. Unfortunately, this repertoire of potent antimicrobial components can also lead to indiscriminate damage of host tissue during the process of clearing an inflammatory stimulus. In a number of important lung diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiolitis obliterans syndrome (BOS), PMN inflammation becomes chronic, leading to off-target extracellular matrix destruction and end-organ damage [2e5]. When there is dysregulated neutrophilic inflammation and more severe or sustained tissue injury occurs, PMNs change from effectors of normal homeostasis to mediators of pathology [6].

https://doi.org/10.1016/j.bbrc.2019.07.111 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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PMN recruitment classically occurs through a PMN-specific chemokine family typified by interleukin-8 (IL-8). IL-8 is a glutamate-leucine-arginine positive (ELRþ) CXC chemokine that acts via CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) in humans [5]. There have been attempts to blunt the effect of IL-8 in a clinical setting using a neutralizing monoclonal antibody (mAb), but with limited success [7]. The limited efficacy of IL-8 inhibition might in part relate the presence of an IL-8-independent CXCR signaling pathway. It is now known that traditional ELR þ CXC chemokines are not the exclusive mechanism by which PMNs may be recruited via CXCR activation to sites of damage. A collagen breakdown fragment, proline-glycine-proline (PGP), which bears structural and sequence similarities to an important functional motif in the ELR þ CXC chemokine family, is released from collagen in numerous chronic neutrophilic disorders [8e10]. Indeed, in animal models and ex vivo studies, PGP appears to contribute to PMN influx and chemotaxis as much as classical ELR þ CXC chemokines IL-8 and MIP-2 [2,4,8,11]. PGP, and its more potent amino-terminal acetylated form (AcPGP), are products of a protease cascade initiated by matrix-metalloproteases (MMPs) acting upon collagen to form oligopeptides, which are cleaved by a serine protease, prolyl endopeptidase (PE) [8,11]. PE was originally described as a processor of neuropeptides in the central nervous system [12e14]. More recently, it has been shown to play a key role outside the central nervous system. PE is the terminal enzyme in the generation of PGP from collagen, and has been shown to be active in a variety of chronic inflammatory lung diseases. All of the proteases necessary for PGP production, including PE, are found in PMNs and have been measured in clinical specimens from neutrophilic diseases [15,16]. Therefore, an initial PMN influx to collagen-rich tissues can be amplified by the generation of PGP in a feed-forward cycle. This PGP pathway therefore can operate independently of the classical ELR þ CXCR axis to sustain and/or propagate neutrophilic inflammation. Derangements of both the classical ELR þ CXCR pathway and PGP homeostasis have been linked to disease activity in COPD [8,17,18] and CF [19]. Therefore, therapeutic strategies aimed at effectively abrogating CXCR signaling in chronic neutrophilic disorders may require diminution of both the ELR þ CXCR and PGP pathways. Benzyloxycarbonyl-proline-prolinal (ZPP) was developed as a specific, noncompetitive inhibitor of PE [20e22], the terminal enzyme in PGP generation. In this report we describe and explain another property of ZPP, namely the inhibition of the receptor by which PGP and IL-8 exert biological activity on PMNs (CXCR1 and CXCR2). These dual inhibitory properties of ZPP may arise because of structural similarities between ZPP, PGP, and a binding pocket of CXCR1 and 2. We confirmed that ZPP is an effective inhibitor of PGP generation and were able to validate a potent, specific anti-CXCR effect of ZPP in vitro. We then validated potent inhibitory effects of ZPP on human PMN chemotaxis toward the sputum of subjects with cystic fibrosis, which is known to contain elevated levels of both PGP and IL-8. We conclude that ZPP offers a novel dual mechanism of PMN modulation with considerable therapeutic potential in diseases characterized by excessive PMN inflammation.

2.2. PE activity assay 100 ng PE was added to 1 mM N-Succinyl-Gly-Pro-paranitroanilide (Suc-GP-pNA) in 100 mL PBS with 10 mM bovine serum albumin with or without ZPP and reaction permitted overnight at 4  C. Substrate cleavage was then measured by production of paranitroanilide, read as absorbance at 410 nm. Electrospray ionization liquid chromatography/mass spectrometry/mass spectrometry for PGP detection. PGP and Ac-PGP were measured in all samples using an MDS Sciex (Applied Biosystems) API-4000 spectrometer equipped with a Shimadzu HPLC. HPLC was performed with a 2.1  150 mm Develosi C30 column (with buffer A: 0.1% formic acid and buffer B: acetonitrile plus 0.1% formic acid: 0e0.6 min 20% buffer B/80% buffer A, then increased over 0.6e5 min to 100% buffer B/0% buffer A. Background was then removed by flushing with 100% isopropanol plus 0.1% formic acid. Positive electrospray mass transitions were at 270e70 and 270e116 for PGP and 312e140 and 312e112 for Ac-PGP.

2.3. Neutrophil chemotaxis assays The chemokine of interest was placed in the bottom chamber of a 96 well 3 mm filter plate from Millipore in a volume of 150 ml. Sputum samples were diluted 1:6 in DMEM for all assays. The filter was placed on top and 2  105 human or murine neutrophils were loaded on top in 100 ml of DMEM with 0.5% BSA. The plates were incubated at 37  C in 5% CO2 for 1 h. The number of migrated cells were then determined using either photomicrographs taken with an Olympus IX70 or an LSRII flow cytometer (BD Biosciences, San Jose, CA). ZPP inhibition of chemotaxis was determined by preincubating the cells with ZPP for 45 min at 20  C with agitation. Following this, a customary chemotaxis assay was performed.

2.4. Human neutrophil isolation Whole blood was obtained from healthy volunteers by phlebotomy into heparinized vacuum phlebotomy vials. RBCs were allowed to sediment for 20 min at 20  C after 1:1 mixture with 5% dextran. Supernatant was eluted and centrifuged for 7 min at 1000g at 20  C. Residual RBCs were lysed by resuspending the pellet in ice-cold 0.2% saline for 30 s then rapidly bringing solution to isotonicity with 1.6% saline. The cell pellet was resuspended in filter-sterilized 0.9% saline solution equal to starting volume of whole blood and 10 mL of Ficoll gradient solution (GE Healthcare Bio-Sciences) was layered at the bottom of a 50 mL centrifuge tube. This was then centrifuged for 40 min at 1000g at 4  C. The pellet, now consisting of purified peripheral blood PMNs, was then resuspended in 1 mL filter-sterilized phosphate buffered saline (PBS) (pH 7.5 without CaCl2/MgCl2) and set on ice. A 1:10 dilution in Trypan blue dye was performed and cells counted; viability of >95% was confirmed for all experiments.

2. Materials and methods 2.5. Murine neutrophil isolation 2.1. Materials All antibodies were purchased from R & D Systems (Minneapolis, MN). Albumin from Bovine Serum, Cohn V Fraction was obtained from Sigma (St. Louis, MO). Z-Gly-Pro-pNA was synthesized by Chem Impex International. DMEM was purchased from Mediatech. Z-Pro-Prolinal was obtained from CalBiochem (Gibbstown, NY) and Enzo Life Sciences (Plymouth Meeting, PA).

Mice were sacrificed, the femur and the tibia from both hind legs were removed and freed of soft tissue attachments, and the extreme distal tip of each extremity was amputated to expose the marrow. HBSSeEDTA solution was forced through the bone with a syringe. After dispersing cell clumps, the cell suspension was centrifuged (400g, 10 min, 4  C) and resuspended in 1 ml PBS (pH 7.5 without CaCl2/MgCl2).

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2.6. In vivo administration of LPS and ZPP Balb/C mice (Jackson Laboratory, Bar Harbor ME) were kept in the UAB Animal Research Facility. Food and water were given ad libitum. All experimental procedures were in accordance with Institutional Animal Care and Use Committee regulations. LPS (10ng/mouse) was administered intratracheally in 50 ml of PBS. Mice were returned to the cage and sacrificed 24 h later by intraperitoneal (IP) injection of .2 ml Nembutal. ZPP was administered in 2% dioxane in PBS. ZPP was dosed at T-6, T ¼ 0 (this dose given in same aliquot as LPS), and Tþ6 h, with mice sacrificed and bronchoalveolar lavage performed at Tþ12 h. 2.7. Collection of mouse bronchalveolar lavage fluid Mice were euthanized with IP injection of Nembutal. A 10 cc syringe connected to a three way stop-cock was attached to a 22 G flexible catheter. The catheter was inserted into the proximal end of the trachea and the lungs were slowly perfused with 3  1 ml of PBS (room temperature). BAL was collected using an empty 10 cc syringe attached at the third spot on the stop-cock. Cell counts were determined using Trypan Blue staining and a hemocytometer. For further confirmation, cytospins were done of all samples and staining was performed using Trypan Blue (Sigma, St. Louis, MO). 2.8. PGP generation from intact collagen using neutrophil lysates 4  106 neutrophils/ml were lysed using 2x freeze-thaw cycles in PBS with 10 ng/ml Aprotinin (Sigma). 25 mg Type I collagen, 25 mg Type II collagen, with or without 0.25 mM ZPP, 150 ml lysates, 10 mM BSA, and 5 ml 10 mg/ml of bestatin (an inhibitor of the PGPdegrading enzyme leukotriene A4 hydrolase) was added every 6 h. After 24 h at 37  C, the samples were centrifuged, soluble fluid was recovered, washed on 10 kDa Millipore filters (Billerica, MA) and analyzed on ESI-LC-MS/MS. 2.9. Statistical analysis Statistics were performed using GraphPad Prism 5.0. One-way ANOVA with Tukey post-test were performed for comparison of 3 or more groups. Two-tailed student's T-tests were used for simple significance testing. Means are presented as standard error of measurement with error bars representing same. Statistical significance was inferred for p values < 0.05; p values are presented as: * <0.05, ** <0.01, *** <0.001, ****<0.0001. 3. Results AcPGP and ZPP are structurally similar. Computer modeling (Sybyl 7.0; Tripos, Inc.; Saint Louis, MO) of the neutrophil CXCR1 and 2 ligands Ac-PGP and the PE inhibitor ZPP demonstrated

Fig. 1. Three dimensional ball and stick structures of AcPGP and ZPP. Modeling was conducted with Sybyl 7.0 (Tripos, Inc.; Saint Louis, MO).

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closely related structures (Fig. 1). Both molecules have a ring structure at the amino-terminus with a proline at the carboxyterminus. ZPP has a reactive aldehyde group on the C-terminus that forms a covalent bond with the serine in the catalytic triad of the active site of prolyl endopeptidase. The pronounced similarities between the molecules suggests structural orthology between an Ac-PGP binding pocket of CXCR1 and/or CXCR2 and the PE catalytic site which accommodates ZPP. ZPP inhibits prolyl endopeptidase activity. ZPP is known to be a potent PE inhibitor [20e22]. We also performed an ex vivo assay utilizing neutrophil lysates and collagen to generate PGP [9]. We repeated a dose curve of ZPP inhibiting PE activity using benzylocarbonyl pro-gly-paranitroanilide (ZGP-pNA), a PE substrate that generates para-nitroanilide upon cleavage. This confirmed that ZPP inhibits PE activity at nM concentrations (Fig. 2A). We then performed an ex vivo assay to assess the relevance of this inhibition to PMN-induced PGP generation [9]. Neutrophil lysates were incubated with intact Type I and Type II collagen and PGP generation was measured using electro-spray ionization liquid chromatography mass spectrometry/mass spectrometry (ESI-LC-MS/MS). We observed that PMN lysate is sufficient to produce PGP in the presence of intact collagen. In the presence of ZPP however, this ability to produce the chemotactic peptide was completely inhibited (Fig. 2B). These findings confirm that ZPP effectively prevents the production of PGP by PMNs through its inhibition of PE, the only known enzyme capable of liberating PGP from collagen fragments. ZPP inhibits in vitro murine and human PMN chemotaxis to ELR þ CXC chemokines. ZPP effectively inhibited the generation of PGP from intact collagen ex vivo. Given its structural similarities to AcPGP, we next assessed whether ZPP modulates neutrophil chemotaxis. To investigate this we performed transwell chemotaxis assays with neutrophils in the presence or absence of ZPP and ELR þ CXC chemokinetic ligands. ZPP completely inhibited murine and human neutrophil chemotaxis to ELR þ CXC chemokines (MIP2 and IL-8, respectively) in a dose-dependent manner, but did not affect chemotaxis to formyl-Methionine-Leucine-Phenylalanine (fMLP), another neutrophil chemoattractant that acts via a different receptor (Fig. 2C and D) [23,24]. ZPP is active in an in vivo model of neutrophilic inflammation. LPS (10 ng) was instilled in the trachea of Balb/C mice (T ¼ 0 h). LPS, a bacterial component, has been demonstrated to incite a rapid and robust neutrophil recruitment into the airways [25]. Inhaled LPS is known to induce PMN influx into the lung via both ELR þ CXC chemokine [26,27] and AcPGP signaling [8]. To measure the ability of ZPP to attenuate LPS-initiated pulmonary neutrophil influx, ZPP (100 mg/dose) was given intra-tracheally to treatment and control groups of mice 6 h prior to LPS administration, at time of LPS administration, and 6 h after LPS dosing. At T ¼ þ12 h, the mice were sacrificed and bronchoalveolar lavage was performed. Cells were pelleted and stained, then neutrophil and macrophage counts were determined and analyzed. LPS alone caused a >28-fold increase in the influx of neutrophils into the airways (Fig. 2E). However, when mice were pretreated with ZPP, there was a 30% decrease in the PMN burden. ZPP dose-dependently blocks neutrophil chemotaxis to CF sputum. Sputum from cystic fibrosis patients contains high levels of the neutrophil chemoattractants IL-8 and PGP [3,11,28]. Congruent with this, CF sputum is highly chemotactic to neutrophils ex vivo [29,30]. We utilized CF sputum as an ex vivo stimulus to study the potential effectiveness of ZPP as an inhibitor of a clinically significant source of neutrophil influx. IL-8 levels in the CF sputum tested were 2414 ± 1308 pg/mL. All tested sputum samples were highly chemotactic to neutrophils (Fig. 3A). ZPP (100 m g/mL), when preincubated with neutrophils, demonstrated inhibition of chemotaxis to sputum collected from CF patients. ZPP caused ~87%

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Fig. 2. 2A. ZPP dose dependently inhibits PE activity. ZPP was preincubated with PE for 5 minutes at varying concentrations before the addition of ZGP-pNA and measured by absorbance at 410 nm for production of para-nitroanilide. 2B. ZPP blocks PMN production of PGP from intact collagen. Neutrophil lysates were incubated for 24 h at 37  C with collagen with or without ZPP and the samples were passed through 10 kDa filters and analyzed via ESI-LCMS/MS. 2C. ZPP dose dependently and specifically inhibits ELR þ CXC chemokine mediated mouse neutrophil chemotaxis. ZPP or vehicle was preincubated with murine neutrophils for 45 min at 4  C prior to use in a chemotaxis assay. MIP-2 (10 ng/ml), AcPGP (100 mg/ml), or fMLP (10 mM), were placed in the bottom wells to incite PMN migration and the ratio of PMN migration between each condition and mean media control (chemotactic index) was calculated. Percentage inhibition was then quantified as: (1-[chemotactic index with inhibitor/chemotactic index without inhibitor])*100. 2D: ZPP dose dependently inhibits ELR þ CXC chemokine mediated human neutrophil chemotaxis. In the same manner as the mouse, IL-8 (20 ng/ml) and AcPGP (30 mg/ml) driven chemotaxis was measured in presence or absence of ZPP. 2E. ZPP prevents LPS driven neutrophil migration in vivo. ZPP (100mg/animal) was instilled intra-tracheally (IT) into 8e10 week old female Balb/C mice at T ¼ 6 h, T ¼ 0, and T ¼ 6 h, with 10 ng of LPS administered IT at T ¼ 0. Animals were sacrificed, bronchoalveolar lavage performed, cells pelleted from fluid, stained and counted.

(Fig. 3B) inhibition of neutrophil chemotaxis. This near-complete neutralization is similar to that observed in the previous experiments with IL-8 and PGP and is all the more impressive considering the variety of neutrophil chemokines in CF sputum, including leukotriene B4 and fMLP present in CF sputum [31,32]. These data indicate that ZPP is effective not only against isolated neutrophil chemokines, but also in neutralization of PMN chemotaxis via disease specimens. 4. Discussion The major finding of this work is that the compound ZPP is able to function in dual complementary roles as a neutrophil chemokine receptor antagonist by not only reducing the production of the CXCR1/2 ligand PGP but also through a direct inhibitory effect upon the receptor. Chronic neutrophilic inflammation is a hallmark of a number of major pulmonary diseases. The prolonged increase in proteolytic enzymes, and reactive oxygen species seen with

persistent neutrophilia causes deleterious remodeling of the pulmonary extracellular matrix (ECM) [33,34]. In many of these disorders, a decline in lung function has been shown to correlate with the level of neutrophil influx, neutrophil chemokines, and neutrophil-derived proteases present in clinical samples from diseased patients [35e38]. To our knowledge, this is the first known compound capable of acting as both an inhibitor of the generation of an inflammatory ligand and as an antagonist for that ligand's receptor. As such, ZPP would seem to have significant therapeutic potential in chronic PMN-predominant inflammatory disorders in which excesses of PGP and IL-8 signaling co-occur (e.g., COPD and CF) [35]. Using the paradigm we describe here, the rational design of therapeutics that inhibit an enzyme that produces the receptor ligand and the receptor binding site itself allows for identification of single compounds with action upon multiple levels within a pathway. More specifically, our findings provide proof of principle for a potent suppression of pulmonary neutrophilic inflammation

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References

Fig. 3. 3A: ZPP dose dependently inhibits neutrophil chemotaxis to the sputum of n ¼ 4 cystic fibrosis patients. Chemotactic index was measured as in 2  C. for each sample and indexed to DMEM. 3B: PMNs were preincubated at 20  C for 30 min with either vehicle or ZPP prior to chemotaxis assay.

derived from the properties of ZPP upon multiple levels of ELR þ CXCR signaling. This might be therapeutically useful in chronic neutrophilic disorders such as COPD and CF. Because of the structural similarities between the PE and CXC receptor ligands ZPP and AcPGP, respectively (Fig. 1), we suspect that the dual inhibitory activity of ZPP is due to similarity between the catalytic site of PE and the active site of CXCR1/2. Therefore, other inhibitors of PE might have CXCR-inhibitory activity analogous to that of ZPP. At least one known PE inhibitor is already in clinical use for other indications. Valproic acid (VPA) is a catalytic site inhibitor of PE with good bioavailability used to treat a variety of psychiatric conditions such as seizure disorders and bipolar disorder [39,40]. However, whether VPA inhibits CXCR remains untested. In conclusion, we have demonstrated that ZPP plays dual, complementary roles to effect potent inhibition of neutrophil influx in vitro and in vivo. This finding has therapeutic implications for disorders characterized by excessive neutrophilic inflammation in the lung and elsewhere. Also, we explore the intriguing concept of a single compound capable of blocking the generation of a neutrophil chemokine receptor ligand while also directly blocking the receptor itself. This concept could facilitate rational drug design for development of more potent agents to inhibit pathways for which multilevel blockade of feed-forward regulatory derangements are known to be advantageous, such as with the renin-angiotensin system in cardiovascular disease.

Funding This work was supported by NIH/NHLBI: T32HL105346-07 to DWR, R35HL135710 to JEB, and R01HL126596 to JEB and AG; as well as the Netherlands Organization for Scientific Research: Grant 017.008.029 to MAR

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.111.

Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.111.

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