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• W2808067178 abstract "Cystic fibrosis (CF) is a multifactorial disease in which dysfunction of protease–antiprotease balance plays a key role. The current CF therapy relies on dornase α, hypertonic saline, and antibiotics and does not address the high neutrophil elastase (NE) activity observed in the lung and sputum of CF patients. Our hypothesis is that variants of heparin, which potently inhibit NE but are not anticoagulant, would help restore the protease–antiprotease balance in CF. To realize this concept, we studied molecular principles governing the effectiveness of different heparins, especially 2-O,3-O-desulfated heparin (ODSH), in the presence of sputum components and therapeutic agents. Using sputa from CF patients and an NE activity assay, we found that heparins are ineffective if used in the absence of dornase. This is true even when mucolytics, such as DTT or N-acetylcysteine, were used. Computational modeling suggested that ODSH and DNA compete for binding to an overlapping allosteric site on NE, which reduces the anti-NE potential of ODSH. NE inhibition of both DNA and ODSH is chain length–dependent, but ODSH chains exhibit higher potency per unit residue length. Likewise, ODSH chains exhibit higher NE inhibition potential compared with DNA chains in the presence of saline. These studies suggest fundamental differences in DNA and ODSH recognition and inhibition of NE despite engaging overlapping sites and offer unique insights into molecular principles that could be used in developing antiprotease agents in the presence of current treatments, such as dornase and hypertonic saline. Cystic fibrosis (CF) is a multifactorial disease in which dysfunction of protease–antiprotease balance plays a key role. The current CF therapy relies on dornase α, hypertonic saline, and antibiotics and does not address the high neutrophil elastase (NE) activity observed in the lung and sputum of CF patients. Our hypothesis is that variants of heparin, which potently inhibit NE but are not anticoagulant, would help restore the protease–antiprotease balance in CF. To realize this concept, we studied molecular principles governing the effectiveness of different heparins, especially 2-O,3-O-desulfated heparin (ODSH), in the presence of sputum components and therapeutic agents. Using sputa from CF patients and an NE activity assay, we found that heparins are ineffective if used in the absence of dornase. This is true even when mucolytics, such as DTT or N-acetylcysteine, were used. Computational modeling suggested that ODSH and DNA compete for binding to an overlapping allosteric site on NE, which reduces the anti-NE potential of ODSH. NE inhibition of both DNA and ODSH is chain length–dependent, but ODSH chains exhibit higher potency per unit residue length. Likewise, ODSH chains exhibit higher NE inhibition potential compared with DNA chains in the presence of saline. These studies suggest fundamental differences in DNA and ODSH recognition and inhibition of NE despite engaging overlapping sites and offer unique insights into molecular principles that could be used in developing antiprotease agents in the presence of current treatments, such as dornase and hypertonic saline. Dysregulation in mutant CFTR 4The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisCVLScombinatorial virtual library screeningNACN-acetyl-l-cysteineNEneutrophil elastasefNEfluoresceinylated NENETneutrophil extracellular trapNSPneutrophil serine proteasesODSH2-O,3-O-desulfated heparinRMSDroot mean square deviationUFHunfractionated heparinILinterleukinHShypertonic saline. anion conductance results in mucus accumulation, which promotes bacterial infections, thereby initiating a cascade of events, including failure of innate immunity to clear infections and release of inflammatory mediators that cause lung injury and ultimately respiratory failure. Although improvements in CF therapy over the past 2 decades have increased life expectancy to about 41 years (1.Ramsey B.W. Welsh M.J. AJRCCM: 100-year anniversary: progress along the pathway of discovery leading to treatment and cure of cystic fibrosis.Am. J. Respir. Crit. Care Med. 2017; 195 (28459323): 1092-109910.1164/rccm.201702-0266EDCrossref PubMed Scopus (18) Google Scholar), much remains to be accomplished. cystic fibrosis transmembrane conductance regulator cystic fibrosis combinatorial virtual library screening N-acetyl-l-cysteine neutrophil elastase fluoresceinylated NE neutrophil extracellular trap neutrophil serine proteases 2-O,3-O-desulfated heparin root mean square deviation unfractionated heparin interleukin hypertonic saline. The CF therapy currently recommended by the United States Cystic Fibrosis Foundation includes inhaled dornase α to reduce sputum viscosity, antibiotics to kill bacteria, inhaled 7% saline to enhance sputum hydration, and immunomodulatory therapies (ibuprofen and azithromycin) to decrease lung injury (1.Ramsey B.W. Welsh M.J. AJRCCM: 100-year anniversary: progress along the pathway of discovery leading to treatment and cure of cystic fibrosis.Am. J. Respir. Crit. Care Med. 2017; 195 (28459323): 1092-109910.1164/rccm.201702-0266EDCrossref PubMed Scopus (18) Google Scholar). Other agents recommended for use include ivacaftor for patients with class 3 CFTR mutations and Orkambi for patients homozygous for phe508del to rescue CFTR function (2.Paranjape S.M. Mogayzel Jr., P.J. Cystic fibrosis in the era of precision medicine.Paediatr. Respir. Rev. 2018; 25 (28372929): 64-72Crossref PubMed Scopus (22) Google Scholar). It is instructive to note that for resolving cystic fibrosis, no agent has been approved for clinical use to inhibit neutrophil serine proteases (NSPs), a major cause of airway injury in CF. Innate immunity in CF is mediated to a large extent by polymorphonuclear neutrophils. These are attracted to sites of bacterial infection in CF by IL-8 (CXCL8) and, following activation, release inflammatory mediators including NSPs, such as elastase, proteinase 3, and cathepsin G, and release DNA and reactive oxygen species (3.Twigg M.S. Brockbank S. Lowry P. FitzGerald S.P. Taggart C. Weldon S. The role of serine proteases and antiproteases in the cystic fibrosis lung.Mediators Inflamm. 2015; 2015 (26185359): 293053Crossref PubMed Scopus (73) Google Scholar). However, in the CF airway milieu, neutrophils release extracellular traps (NETs) (4.Gray R.D. McCullagh B.N. McCray P.B. NETs and CF lung disease: current status and future prospects.Antibiotics. 2015; 4 (27025615): 62-7510.3390/antibiotics4010062Crossref Scopus (35) Google Scholar), a primary source of extracellular DNA polymers and NE in the CF airway. The load of NE in the airway overwhelms the antiprotease activities provided by α1-proteinase inhibitor and serine leukoprotease inhibitor. An attractive, yet unrealized, pathway to control lung injury in CF is re-establishment of protease–antiprotease balance through inhibition of NE (5.Voynow J.A. Fischer B.M. Zheng S. Proteases and cystic fibrosis.Int. J. Biochem. Cell Biol. 2008; 40 (18395488): 1238-124510.1016/j.biocel.2008.03.003Crossref PubMed Scopus (149) Google Scholar). NE activates other proteases, such as matrix metalloprotease-9, degrades antiproteases, such as tissue inhibitor of metalloproteases, up-regulates neutrophilic chemokines, such as IL-8 and tumor necrosis factor α, and thereby augments inflammation (6.Taggart C.C. Greene C.M. Carroll T.P. O'Neill S.J. McElvaney N.G. Elastolytic proteases: inflammation resolution and dysregulation in chronic infective lung disease.Am. J. Respir. Crit. Care Med. 2005; 171 (15695494): 1070-107610.1164/rccm.200407-881PPCrossref PubMed Scopus (87) Google Scholar). Additionally, NE impairs ciliary motility; up-regulates mucin expression and secretion; causes epithelial injury, apoptosis, and senescence; and blocks epithelial repair (5.Voynow J.A. Fischer B.M. Zheng S. Proteases and cystic fibrosis.Int. J. Biochem. Cell Biol. 2008; 40 (18395488): 1238-124510.1016/j.biocel.2008.03.003Crossref PubMed Scopus (149) Google Scholar). As such, NE is a well-established biomarker of lung disease progression in patients with CF (7.Sly P.D. Gangell C.L. Chen L. Ware R.S. Ranganathan S. Mott L.S. Murray C.P. Stick S.M. AREST CF InvestigatorsRisk factors for bronchiectasis in children with cystic fibrosis.N. Engl. J. Med. 2013; 368 (23692169): 1963-197010.1056/NEJMoa1301725Crossref PubMed Scopus (412) Google Scholar, 8.Sagel S.D. Wagner B.D. Anthony M.M. Emmett P. Zemanick E.T. Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis.Am. J. Respir. Crit. Care Med. 2012; 186 (22904182): 857-86510.1164/rccm.201203-0507OCCrossref PubMed Scopus (183) Google Scholar), yet there are no approved therapies that block NE activity for patients with CF. Although many different types of inhibitors of NE have been reported (9.Kelly E. Greene C.M. McElvaney N.G. Targeting neutrophil elastase in cystic fibrosis.Expert Opin. Ther. Targets. 2008; 12 (18208364): 145-15710.1517/14728222.12.2.145Crossref PubMed Scopus (69) Google Scholar), an agent that exhibits considerable promise is heparin. In addition to potently inhibiting NE (1–20 nm), heparin inhibits cathepsin G, another neutrophil serine protease present in high concentrations in the CF airway (10.Rao N.V. Argyle B. Xu X. Reynolds P.R. Walenga J.M. Prechel M. Prestwich G.D. MacArthur R.B. Walters B.B. Hoidal J.R. Kennedy T.P. Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.Am. J. Physiol. Cell Physiol. 2010; 299 (20375277): C97-C11010.1152/ajpcell.00009.2010Crossref PubMed Scopus (111) Google Scholar). Additionally, heparin has other anti-inflammatory activities, including inhibiting activation of the receptor for advanced glycation end-products and blocking the release of its ligand, high mobility group box 1; inhibiting binding to L-selectin and P-selectin; and inhibiting NF-κB (10.Rao N.V. Argyle B. Xu X. Reynolds P.R. Walenga J.M. Prechel M. Prestwich G.D. MacArthur R.B. Walters B.B. Hoidal J.R. Kennedy T.P. Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.Am. J. Physiol. Cell Physiol. 2010; 299 (20375277): C97-C11010.1152/ajpcell.00009.2010Crossref PubMed Scopus (111) Google Scholar). Heparin also binds to pro-inflammatory chemokines, such as IL-8 and IL-6 (10.Rao N.V. Argyle B. Xu X. Reynolds P.R. Walenga J.M. Prechel M. Prestwich G.D. MacArthur R.B. Walters B.B. Hoidal J.R. Kennedy T.P. Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.Am. J. Physiol. Cell Physiol. 2010; 299 (20375277): C97-C11010.1152/ajpcell.00009.2010Crossref PubMed Scopus (111) Google Scholar), which implies that soluble heparin administration may also reduce neutrophil chemotaxis by providing competitive sites. However, heparin's high anticoagulant activity may limit its value as a therapy. We have proposed 2-O,3-O-desulfated heparin (ODSH), a modified heparin displaying potent anti-inflammatory and antiprotease activity with minimal anticoagulant effect, as an anti-NE agent for use in CF (10.Rao N.V. Argyle B. Xu X. Reynolds P.R. Walenga J.M. Prechel M. Prestwich G.D. MacArthur R.B. Walters B.B. Hoidal J.R. Kennedy T.P. Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.Am. J. Physiol. Cell Physiol. 2010; 299 (20375277): C97-C11010.1152/ajpcell.00009.2010Crossref PubMed Scopus (111) Google Scholar, 11.Griffin K.L. Fischer B.M. Kummarapurugu A.B. Zheng S. Kennedy T.P. Rao N.V. Foster W.M. Voynow J.A. 2-O,3-O-desulfated heparin inhibits neutrophil elastase-induced HMGB-1 secretion and airway inflammation.Am. J. Respir. Cell Mol. Biol. 2014; 50 (24325600): 684-68910.1165/rcmb.2013-0338RCCrossref PubMed Scopus (36) Google Scholar). ODSH blocks airway neutrophilic inflammation in mice administered NE intratracheally (11.Griffin K.L. Fischer B.M. Kummarapurugu A.B. Zheng S. Kennedy T.P. Rao N.V. Foster W.M. Voynow J.A. 2-O,3-O-desulfated heparin inhibits neutrophil elastase-induced HMGB-1 secretion and airway inflammation.Am. J. Respir. Cell Mol. Biol. 2014; 50 (24325600): 684-68910.1165/rcmb.2013-0338RCCrossref PubMed Scopus (36) Google Scholar), and in animals with Pseudomonas aeruginosa pneumonia, ODSH recovers impaired macrophage function, promotes bacterial clearance, and improves survival (12.Sharma L. Wu J. Patel V. Sitapara R. Rao N.V. Kennedy T.P. Mantell L.L. Partially-desulfated heparin improves survival in Pseudomonas pneumonia by enhancing bacterial clearance and ameliorating lung injury.J. Immunotoxicol. 2014; 11 (24099632): 260-26710.3109/1547691X.2013.839587Crossref PubMed Scopus (19) Google Scholar). To understand the molecular principles of ODSH action in CF-related samples, we studied its inhibition of NE activity in CF sputum. This work shows that NE is not inhibited by ODSH or heparin if only mucolytics are used, including N-acetylcysteine (NAC), Sputolysin (0.1% DTT), or hypertonic saline. More importantly, this work reveals that either ODSH or heparin inhibits NE in native CF sputum pretreated with dornase. The molecular basis for this favorable combinational outcome involves fundamental mechanistic biochemistry and leads to a putative therapy for treatment of CF that can be of value irrespective of the patient's genetic status. Heparin is a polysulfated polysaccharide that is clinically used as an anti-coagulant and also has potent antiprotease activity against serine proteases, including NE (13.Spencer J.L. Stone P.J. Nugent M.A. New insights into the inhibition of human neutrophil elastase by heparin.Biochemistry. 2006; 45 (16866356): 9104-912010.1021/bi060338rCrossref PubMed Scopus (44) Google Scholar) and cathepsin G, and furthermore, heparin is not degraded by exposure to proteases. However, heparin anticoagulant activity may potentially increase the risk for hemoptysis in CF. Therefore, we evaluated the anti-NE activity of a modified heparin, ODSH, that has minimal anti-coagulant activity. We first determined the potency of ODSH in inhibiting NE by the S1384 hydrolysis assay and measured an IC50 of 13.6 nm (Fig. 1A), which was consistent with its reported antiprotease activity (10.Rao N.V. Argyle B. Xu X. Reynolds P.R. Walenga J.M. Prechel M. Prestwich G.D. MacArthur R.B. Walters B.B. Hoidal J.R. Kennedy T.P. Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.Am. J. Physiol. Cell Physiol. 2010; 299 (20375277): C97-C11010.1152/ajpcell.00009.2010Crossref PubMed Scopus (111) Google Scholar). We then solubilized CF sputum with normal saline (1:1, w/v) to test whether ODSH could inhibit NE in the typical airway milieu of CF patients. Unexpectedly, we found that ODSH did not reduce NE activity at all in CF sputum at concentrations as high as 10 μm, which is ∼735-fold higher than its IC50 in pH 7.4 buffer (Fig. 1B). The observation that ODSH did not inhibit NE in solubilized CF sputum suggests that it is not able to effectively bind to NE, perhaps due to competition for binding sites. At least two major anionic polymers, DNA and mucin polymers, are present at high concentrations in CF sputum and potentially bind to and inhibit NE (14.Chernick W.S. Barbero G.J. Composition of tracheobronchial secretions in cystic fibrosis of the pancreas and bronchiectasis.Pediatrics. 1959; 24 (13809672): 739-745Crossref PubMed Google Scholar, 15.Henderson A.G. Ehre C. Button B. Abdullah L.H. Cai L.H. Leigh M.W. DeMaria G.C. Matsui H. Donaldson S.H. Davis C.W. Sheehan J.K. Boucher R.C. Kesimer M. Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure.J. Clin. Invest. 2014; 124 (24892808): 3047-306010.1172/JCI73469Crossref PubMed Scopus (212) Google Scholar, 16.Xia B. Royall J.A. Damera G. Sachdev G.P. Cummings R.D. Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis.Glycobiology. 2005; 15 (15994837): 747-77510.1093/glycob/cwi061Crossref PubMed Scopus (134) Google Scholar, 17.Nadziejko C. Finkelstein I. Inhibition of neutrophil elastase by mucus glycoprotein.Am. J. Respir. Cell Mol. Biol. 1994; 11 (7912511): 103-10710.1165/ajrcmb.11.1.7912511Crossref PubMed Scopus (32) Google Scholar, 18.Belorgey D. Bieth J.G. DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity.FEBS Lett. 1995; 361 (7698335): 265-26810.1016/0014-5793(95)00173-7Crossref PubMed Scopus (34) Google Scholar). To determine the potential NE-binding activity of these polymers, we first chemically or enzymatically degraded de-identified CF sputum samples to prepare sputum supernatants. For chemical degradation, we used either NAC (19.Voynow J.A. Mascarenhas M. Kelly A. Scanlin T.F. Cystic fibrosis.in: Grippi M.A. Elias J.A. Fishman J.A. Kotloff R.M. Pack A.I. Senior R.M. Siegel M.D. Fishman's Pulmonary Disesases and Disorders. 5th Ed. McGraw-Hill Education, New York2015: 757-778Google Scholar) or DTT (8.Sagel S.D. Wagner B.D. Anthony M.M. Emmett P. Zemanick E.T. Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis.Am. J. Respir. Crit. Care Med. 2012; 186 (22904182): 857-86510.1164/rccm.201203-0507OCCrossref PubMed Scopus (183) Google Scholar), typical reducing agents that degrade disulfide bonds. For enzymatic degradation, we treated sputum with bovine DNase-I. For all three agents, appropriate concentration ranges were chosen that matched either pharmacologic concentrations or research reports (8.Sagel S.D. Wagner B.D. Anthony M.M. Emmett P. Zemanick E.T. Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis.Am. J. Respir. Crit. Care Med. 2012; 186 (22904182): 857-86510.1164/rccm.201203-0507OCCrossref PubMed Scopus (183) Google Scholar, 19.Voynow J.A. Mascarenhas M. Kelly A. Scanlin T.F. Cystic fibrosis.in: Grippi M.A. Elias J.A. Fishman J.A. Kotloff R.M. Pack A.I. Senior R.M. Siegel M.D. Fishman's Pulmonary Disesases and Disorders. 5th Ed. McGraw-Hill Education, New York2015: 757-778Google Scholar). Following sputum supernatant preparation, NE activity was measured using the S1384 hydrolysis assay, as described under “Experimental procedures.” Interestingly, Sputolysin as well as DNase-I significantly increased NE activity of treated sputum supernatants (Fig. 2). However, although Sputolysin dose-dependently enhanced NE activity, its effect was much less than the nearly 300% increase in NE activity with DNase-I. We then studied whether ODSH could decrease NE activity, measured using a substrate hydrolysis assay, in mucolytic-pretreated CF sputum. We found that ODSH only inhibited NE activity following DNase-I treatment and had no effect on sputum degraded with NAC or Sputolysin (Fig. 3). These results imply that disulfide bond reduction does not help ODSH to bind to NE. On the contrary, enzymatic degradation of polymeric DNA, to presumably smaller strands, allowed ODSH to bind to NE and reduce its catalytic activity. Nevertheless, the concentration at which ODSH started to inhibit NE in sputum is much higher than in pH 7.4 buffer. A plausible explanation for this is the possibility of competition between ODSH and DNA, both negatively charged polymers, for binding to NE. This explanation is also in line with results with disulfide-reducing agents, which are not expected to significantly reduce electrostatically bound DNA from NE. The evidence that DNase I is needed for ODSH to exhibit its anti-NE activity suggested a critical point. Polymeric DNA chains impeded ODSH from binding to NE, whereas oligomeric DNA sequences appeared to do so less effectively. We then asked whether heparins exhibit chain length–dependent inhibition of NE. Fig. 4 shows NE activity in CF sputum with and without DNase-I pretreatment as well as with and without heparins of different chain lengths (Table S1) at a fixed concentration of 100 μm each. The best inhibitor was found to be unfractionated heparin (UFH; Mr 15,000), which was followed by ODSH (Mr 10,000) and low-molecular weight heparin (Mr 4,500). Fondaparinux (Mr 1,772) had no anti-NE activity in DNase-I–treated CF sputum. These results indicate that a minimum saccharide length is critical for heparins to bind to and inhibit NE. It is known that heparin and DNA each inhibit NE (18.Belorgey D. Bieth J.G. DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity.FEBS Lett. 1995; 361 (7698335): 265-26810.1016/0014-5793(95)00173-7Crossref PubMed Scopus (34) Google Scholar, 20.Redini F. Tixier J.M. Petitou M. Choay J. Robert L. Hornebeck W. Inhibition of leucocyte elastase by heparin and its derivatives.Biochem. J. 1988; 252 (3415672): 515-51910.1042/bj2520515Crossref PubMed Scopus (111) Google Scholar). Nearly all studies have employed full-length polymeric DNA and heparin preparations. Considering that heparin chains of different chain lengths inhibited NE differently, it is possible that DNA chains also exhibit a similar chain length dependence. Thus, we studied three DNA variants (6-, 12-, and 24-mer), which are homogeneous variants of unique chain lengths (Table S2). With regard to heparins, we studied three clinically used variants, including UFH (∼50-mer), ODSH (∼35-mer), and enoxaparin (low-molecular weight heparin; ∼15-mer), which are heterogeneous preparations containing ∼30% variation in chain length on either side of the mean. We also studied fondaparinux, a clinically used anticoagulant, which has a defined structure and chain length (5-mer). The dose dependence of NE inhibition by each inhibitor is shown in Fig. 5. Whereas corresponding longer chains of each class display almost equal IC50 and inhibition efficacy, significant differences were observed for smaller chains (Table 1). In fact, the 6-mer DNA displayed essentially no inhibition, whereas 5-mer fondaparinux displayed a reasonable IC50 of 1.2 μm and an efficacy of nearly 70%. Although the number of residues for corresponding DNA and heparin chains may be similar, the linear molecular sizes are not expected to be identical. For example, computational modeling shows that the molecular length of a 6-mer DNA in its extended conformation was ∼42 Å, whereas it was ∼22 Å for fondaparinux (not shown). This implies that the formal negative charge density of the two polymers is ∼0.25 and ∼0.40 negative charges per Å. Thus, a priori, the electrostatic interaction of NE with heparins is expected to be stronger than that with DNA of equal chain length.Table 1Inhibition of neutrophil elastase by DNA oligomers and heparinsCorresponding chain lengthsIC50InhibitionDNA chainHeparin chainDNA chainHeparin chainnm%%50 ± 10-merNA7.5 ± 0.4NA95 ± 224-mer or 35 ± 8-mer10.5 ± 1.213.6 ± 0.587 ± 291 ± 112-mer or 15 ± 5-mer22.3 ± 1.325.4 ± 2.2101 ± 588 ± 36-mer or 5-mer>10,0001,178 ± 260ND71 ± 6 Open table in a new tab Hypertonic saline (7% (w/v) NaCl) is clinically used to aid mucus clearance in CF patients. Considering that NE interactions are electrostatically mediated, we reasoned that DNA and heparin inhibition of NE might be adversely impacted in the presence of salt. To test the comparative inhibition potential of the two classes of inhibitors, we studied 24-mer DNA and 35-mer heparin (ODSH). Fig. 6 shows the dose dependence of DNA and ODSH inhibition of NE at varying Na+ levels (62.5–362.5 mm or 0.36–2.1% (w/v)). The results show that as Na+ concentration increased from 62.5 to 162.5 mm, the IC50 of 24-mer DNA increased ∼6-fold (Table 2). A more striking effect was noted with respect to inhibition efficacy (ΔY), which decreased from 87 to 35%. At 262.5 mm Na+, the 24-mer DNA was essentially noninhibitory. In contrast, ODSH displayed a significantly different profile. At 162.5 mm Na+, the 35-mer heparin was only 2-fold less active than its potency at 62.5 mm Na+. More importantly, ODSH retained almost full NE inhibition efficacy (85%) at this high Na+ level (0.94% (w/v)). This efficacy was reduced to 52% at 262.5 mm Na+ and almost negligible at 362.5 mm Na+ (Table 2).Table 2Salt dependence of NE inhibition by 24-mer DNA and 35-mer ODSH[Na+]IC50InhibitionDNA chainHeparin chainDNA chainHeparin chainmm (% w/v)nm%%62.5 (0.36)10.5 ± 1.214.1 ± 0.987 ± 291 ± 6112.5 (0.65)10.1 ± 2.013.5 ± 1.181 ± 690 ± 5162.5 (0.94)66.6 ± 24.926.7 ± 2.235 ± 585 ± 4262.5 (1.5)>5,000261 ± 33ND52 ± 4362.5 (2.1)ND>50,000NDND Open table in a new tab These results suggest an atomistic level difference in the nature of interactions of DNA and heparin with NE. Both depend on electrostatic forces as Na+ reduces DNA and ODSH inhibition potency. For comparable chain lengths, DNA lost its NE inhibition potential faster in salt-containing buffers compared with ODSH. This implies that ODSH is a better inhibitor of NE than DNA at comparable chain lengths. More importantly, ODSH appeared to exhibit reasonable NE inhibition at Na+ levels reaching 1.5% (w/v), which is likely to be reached in the lung of a CF patient when inhaling 7.0% (w/v) hypertonic saline. The above results show that heparin was effective only in the presence of DNase-I, which presumably depolymerizes polymeric DNA into smaller chains liberating bound NE for subsequent inhibition by heparins, including ODSH. A simple model that explains this phenomenon is that ODSH and DNA bind to an overlapping site on NE, thereby competing with each other. Because a huge amount of DNA, most probably in the form of NETs, was present in the sputum of CF patients, ODSH was unable to compete, resulting in its failure to inhibit NE in sputum. To assess whether DNA and heparins compete for an overlapping site of binding on NE, we used computational molecular modeling. We used combinatorial virtual library screening (CVLS) to identify sites of heparin binding on proteins (21.Zheng S. Kummarapurugu A.B. Afosah D.K. Sankaranarayanan N.V. Boothello R.S. Desai U.R. Kennedy T. Voynow J.A. 2-O,3-O desulfated heparin blocks high mobility group box 1 release by inhibition of p300 acetyltransferase activity.Am. J. Respir. Cell Mol. Biol. 2017; 56 (27585400): 90-9810.1165/rcmb.2016-0069OCCrossref PubMed Scopus (21) Google Scholar, 22.Sankaranarayanan N.V. Desai U.R. Toward a robust computational screening strategy for identifying glycosaminoglycan sequences that display high specificity for target proteins.Glycobiology. 2014; 24 (25049239): 1323-133310.1093/glycob/cwu077Crossref PubMed Scopus (33) Google Scholar, 23.Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera: a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25 (15264254): 1605-161210.1002/jcc.20084Crossref PubMed Scopus (27922) Google Scholar). The algorithm predicts the binding site residues as well as the most preferred heparin sequence for the target site. We built a library of 1,728 sequences, which represent the most common hexameric chains likely to be present in a sample of ODSH. Application of the CVLS algorithm showed that many sequences bound NE with high GOLD score and high consistency, suggesting a potentially high-affinity interaction (Table S3). Calculations using Chimera (23.Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera: a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25 (15264254): 1605-161210.1002/jcc.20084Crossref PubMed Scopus (27922) Google Scholar) showed that three of these sequences were predicted to bind to NE at a site encompassed by Gln-34, Leu-35, Arg-36, Gly-38, His-40, Phe-41, Cys-42, and Arg-147 (Fig. 7). This implies that many of the 19 arginines on the NE surface are predicted to be uninvolved in binding to ODSH. More importantly, the site of ODSH binding did not include NE's active site residues (Fig. 7), which are located ∼6 Å away from the heparin chain. To assess whether longer ODSH sequences would bind to NE and interact, the most common octa- and decasaccharide sequences were studied using our CVLS approach. The results showed that the preferred binding orientations of both octa- and decasaccharide sequences were similar to that of the hexasaccharide sequences bound to NE (see Fig. S1). To assess the nature of DNA-NE interactions, we used a HADDOCK-based docking and scoring strategy (24.Karaca E. Melquiond A.S. de Vries S.J. Kastritis P.L. Bonvin A.M. Building macromolecular assemblies by information-driven docking: introducing the HADDOCK multibody docking server.Mol. Cell. Proteomics. 2010; 9 (20305088): 1784-179410.1074/mcp.M000051-MCP201Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), which is routinely used to determine the site of DNA binding on proteins (25.van Dijk M. van Dijk A.D. Hsu V. Boelens R. Bonvin A.M. Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility.Nucleic Acids Res. 2006; 34 (16820531): 3317-332510.1093/nar/gkl412Crossref PubMed Scopus (151) Google Scholar). HADDOCK study of 6-, 12-, and 24-mer DNA showed that that the two latter oligomers bound avidly to NE (see Table S4), whereas the 6-mer was ineffective. Both 12- and 24-mer bound to NE with a root mean square deviation (RMSD) of <2.5 Å from the overall lowest energy structure, which supports the inhibition results discussed above. Chimera analysis (23.Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng" @default.
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• W2808067178 title "Molecular principles for heparin oligosaccharide–based inhibition of neutrophil elastase in cystic fibrosis" @default.
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