Integration of HPLC-ESI-MS/MS and NMR Techniques for Characterizing Monoterpene Indole Alkaloids in Leaves and Stems of Psychotria densicostata and Their Potential as Inhibitors of Human Neutrophil Elastase

ABSTRACTIntroduction: Human neutrophil elastase (HNE) is involved in a variety of serious chronic diseases, especially cardiopulmonary pathologies. For this reason, the regulation of HNE activity represents a promising therapeutic approach, which is evident by the development of a number of new and selective HNE inhibitors, both in the academic and pharmaceutical environments.Areas covered: The present review analyzes and summarizes the patent literature regarding human neutrophil elastase inhibitors for the treatment of cardiopulmonary diseases over 2014–2018.Expert opinion: HNE is an interesting and defined target to treat various inflammatory diseases, including a number of cardiopulmonary pathologies. The research in this field is quite active, and a number of HNE inhibitors are currently in various stages of clinical development. In addition, new opportunities for HNE inhibitor development stem from recent studies demonstrating the involvement of HNE in many other inflammatory pathologies, including rheumatoid arthritis, inflammatory bowel disease, skin diseases, and cancer. Furthermore, the development of dual HNE/proteinase 3 inhibitors is being pursued as an innovative approach for the treatment of neutrophilic inflammatory diseases. Thus, these new developments will likely stimulate new and increased interest in this important therapeutic target and for the development of novel and selective HNE inhibitors.

Bovine lens alpha-crystallin inhibited both porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE), but not in the same manner. PPE was immediately inhibited with a stoichiometry of 10 moles of PPE inhibited per mole of alpha-crystallin. The inhibition was markedly decreased by the addition of even low levels of salts. The inhibition was transient, as PPE activity returned to normal with a t1/2 of 30 min even in low salt. HNE required a short preincubation to show maximum inhibition with a stoichiometry of approximately one mole of HNE inhibited per mole of alpha-crystallin. The inhibition of HNE was only slightly decreased by the addition of 0.1 M salt, and HNE activity returned slowly exhibiting a t1/2 of 30 hrs under these conditions. The inhibition of each enzyme by alpha-crystallin was evaluated by Dixon plots giving Ki values of 1.5 nM for PPE and 0.25 nM for HNE. DFP-trypsin was able to compete with PPE for binding to alpha-crystallin and cause the release of PPE already bound to alpha-crystallin. The inhibition of HNE, however, was unaffected by the addition of DFP-trypsin. A mixture of HNE and alpha-crystallin in 0.1 M NaCl was incubated at 25 degrees C for 6 hours. Aliquots showed a slow, continuous cleavage of the alpha-crystallin subunits by SDS-PAGE, but little or no increase in HNE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Ventilator-Associated Pneumonia Is Characterized by Excessive Release of Neutrophil Proteases in the Lung

Human neutrophil elastase (HNE) is an enzyme that plays a key role in the body's inflammatory response. It has been linked to several diseases such as chronic obstructive pulmonary disease (COPD), emphysema, and cystic fibrosis. As potential treatments for these diseases, HNE inhibitors are of great interest. Metabolites derived from plants, particularly terpenoids such as β-caryophyllene found in black pepper and other plants, and geraniol present in several essential oils, are recognized as significant sources of inhibitors for HNE. Because of their ability to inhibit HNE, terpenoids are considered promising candidates for developing novel therapies to treat inflammatory conditions such as COPD and emphysema. Furthermore, nature can serve as an excellent designer, and it may offer a safer drug candidate for inhibiting HNE production and activity in the future. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses were searched to get relevant and up-to-date literature on terpenoids as human neutrophil elastase inhibitors. This review focuses on the isolation, chemical diversity, and inhibition of human neutrophil elastase (HNE) of various terpenoids reported from natural sources up to 2022. A total of 251 compounds from various terpenoids classes have been reported. Further, it also provides a summary of HNE inhibitors and includes a thorough discussion on the structure-activity relationship.

1,5,6,7-Tetrahydro-4H-indazol-4-ones as human neutrophil elastase (HNE) inhibitors

Human neutrophil elastase (HNE) is a potent serine protease belonging to the chymotrypsin family and is involved in a variety of pathologies affecting the respiratory system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here the synthesis of new thiazol-2-(3H)-ones as an elaboration of potent HNE inhibitors with an isoxazol-5-(2H)-one scaffold that we recently identified. Two-dimensional NMR spectroscopic techniques and tandem mass spectrometry allowed us to correctly assign the structure of the final compounds arising from both tautomers of the thiazol-2-(3H)-one nucleus (N-3 of the thiazol-2-(3H)-one and 3-OH of the thiazole). All new compounds were tested as HNE inhibitors, and no activity was found at the highest concentration used (40 µM), demonstrating that the thiazol-2-(3H)-one is not a good scaffold for HNE inhibitors. Molecular modelling experiments indicate that the low-energy pose might limit the nucleophilic attack on the endocyclic carbonyl group of the thiazolone-based compounds by HNE catalytic Ser195, in contrast to isoxazol-5-(2H)-one analogues.

MDL 201,404YA (alternatively, CE-1037), a potent selective inhibitor of human neutrophil elastase (HNE), was tested both intratracheally and intravenously for its activity at inhibiting HNE. MDL 201,404YA given either i.t. or i.v. was effective in preventing the pulmonary hemorrhage which occurred after intratracheal instillation of HNE. Additional studies with MDL 201,404YA suggested that this compound remained active in the lung environment for at least 6 hours. Further evaluation of MDL 201,404YA suggested that it could inhibit the ongoing proteolysis caused by HNE when given 15 minutes after the initial HNE challenge. These data suggest that MDL 201,404YA may be effective therapeutically in treating conditions which result from an imbalance between elastase and its endogenous inhibitor alpha-1-proteinase inhibitor (alpha 1-PI).

Background: Excess activity of neutrophil elastase (NE) in the lung of COPD patients results in the degradation of extracellular matrix (ECM) proteins and an increased activation of other proteases, leading to the destruction of alveolar septa and, ultimately, emphysema development. Direct inhibition of NE is expected to reduce lung parenchyma destruction, emphysema formation and progression in patients with COPD. Aim: We investigated the in vitro and in vivo profile of the novel NE inhibitor BI 1323495. Methods: Inhibition of human NE and the related proteases cathepsin G and proteinase 3 was determined in enzymatic assays using purified enzymes. In addition, inhibition of NE in plasma from zymosan-stimulated human whole blood was measured. An acute lung injury model in mice was used to monitor the in vivo inhibition of NE in the lung by BI 1323495. Lung injury was induced by intratracheal application of 25 µg human NE and haemoglobin concentration as a read-out for lung injury, and neutrophil counts as a read-out for inflammation were measured in bronchioalveolar lavage fluid after 4 hours. Results: BI 1323495 blocked NE in vitro with an IC50 of 0.4 nM and demonstrated a > 4000-fold selectivity versus related proteases cathepsin G and proteinase 3. BI 1323495 fully inhibited the activity of NE released by zymosan-stimulated human neutrophils with a mean IC50 of 1 nM. Treatment of mice with an oral formulation of BI 1323495 attenuated lung damage and inflammation induced by intratracheal instillation of human recombinant NE with an ED50 of 1.9 mg/kg. Conclusions: BI 1323495 is a very potent and highly selective inhibitor of human NE with in vivo efficacy.

Alveolar architecture is spared during most pneumococcal pneumonias, despite the presence in pneumonic exudate of many neutrophils containing a potent elastase. We explored the possibility that pneumococci might contain an inhibitor of this enzyme. We found that pneumococcal extracts prepared by sonication or by lysis with sodium deoxycholate contained 2 different inhibitors of human neutrophil elastase. Both inhibitors were specific for neutrophil elastase and did not affect pancreatic elastase or trypsin. Inhibitor I was partly purified by affinity chromatography and preparative acrylamide gel electrophoresis and shown to be a negatively charged, low molecular weight substance that inhibited competitively (Lineweaver-Burk analysis). Inhibition depended on ionic interaction with the cationic enzyme and could be blocked by 0.15 M NaCl. For this reason, the first agent seemed unlikely to play an important role in modulating neutrophil elastase activity in inflammatory exudates and was not studied further. The second agent (Inhibitor II) eluted in the high molecular weight fraction during Sephacryl S-300 chromatography. Gradient SDS-polyacrylamide gel electrophoresis of partly purified Inhibitor II revealed an apparent molecular weight of 140,000 daltons. This agent inhibited noncompetitively and remained active in the presence of 0.15 M NaCl. Prolonged incubation with TPCK-trypsin resulted in cleavage of Inhibitor II into smaller fragments, which could be further dissociated by reduction with dithiothreitol. Inactivation of neutrophil elastase with N-acetyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone prevented complex formation between this enzyme and Inhibitor II, suggesting that an unblocked binding pocket in neutrophil elastase is required for its complexation to the noncompetitive pneumococcal inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

ICI 186,756 is a representative of a new chemical class of synthetic inhibitors of human neutrophil elastase (HNE). This compound demonstrated competitive inhibition of HNE with a Ki of 3.6 +/- 0.8 x 10(-9) mol/L. The selectivity of ICI 186,756 for HNE versus a variety of enzymes ranged from a minimum of 870-fold to greater than 640,000. The compound effectively inhibited hydrolysis of bovine ligamentum nuchae elastin by HNE. Pretreatment of hamsters with ICI 186,756 up to 2 h before intratracheal administration of HNE inhibited enzyme-induced increases in lung weight, total lavageable red cells, and total lavageable white cells measured 24 h after HNE administration. In contrast, similar lung effects produced by intratracheal administration of porcine pancreatic elastase (PPE) were not inhibited by ICI 186,756. Treatment of hamsters with 43 mumol/kg (sc) of ICI 186,756 for 14 or 28 days modulated the increases in alveolar diameter produced by both PPE and HNE, respectively. It is concluded that ICI 186,756 is a potent, competitive, and selective inhibitor of HNE that may be useful in understanding the role of this enzyme in animal models of various diseases. Furthermore, the maintenance or progression of emphysema-like lesions induced in hamsters by PPE do not appear to be due to the persistence of that enzyme within the lung.

ICI 200,880 and its close structural analog, ICI 200,355, are representatives of a new chemical class of inhibitors of human neutrophil elastase (HNE). Both compounds are substituted tripeptide ketones, which demonstrated competitive kinetics versus HNE, with identical Ki values of 5.0 x 10(-10) M. The selectivity of ICI 200,880 for HNE versus a variety of enzymes ranged from 150-fold [relative to porcine pancreatic elastase (PPE)] to greater than 360,000-fold in favor of HNE. The compound effectively inhibited HNE-hydrolysis of bovine ligamentum nuchae elastin. In pharmacokinetic studies, ICI 200,880 and ICI 200,355 displayed long retention times when administered directly to the lung and were rapidly eliminated after intravenous administration. Pretreatment of hamsters with either inhibitor before intratracheal administration of HNE produced dose- and time-dependent inhibition of enzyme-induced increases in lung weight, total lavageable red cells, and total lavageable white cells. Aerosol administration of ICI 200,880 produced similar results. Subcutaneous administration of either 50 or 100 mumol/kg (twice/day) of ICI 200,880 for 14 or 28 days prevented the time-dependent increase in alveolar diameter produced by a single intratracheal dose of PPE when compound dosing was initiated 24 h after the enzyme. Treatment of hamsters with the same protocol and doses of ICI 200,880 for 8 wk prevented the destructive lesion induced by a single intratracheal dose of HNE. It is concluded that ICI 200,880 and ICI 200,355 have biochemical, pharmacokinetic, and pharmacologic profiles that make them useful therapeutic agents for understanding the role of HNE in various diseases. ICI 200,880 is presently being evaluated in humans.

Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9

1. TEI-8362, 4-(N-(3-((3-carboxypropyl)amino)-8-methyl-1-oxo-4-azaisochromen-6- yl)carbamoyl)-4-((phenylmethoxy)carbonylamino)butanoic acid (C26H28N4O9) is a novel inhibitor of human neutrophil elastase (HNE). We evaluated its pharmacological profile in vitro and in vivo. 2. TEI-8362 demonstrated potent inhibition of HNE with a Ki value of 1.38 x 10(-9) M. Its selectivity for HNE among a variety of proteases ranged from 163 fold to 68,000 fold in favour of HNE. 3. The pulmonary haemorrhage that occurred after i.t. instillation of HNE to hamsters was inhibited by either i.t., i.v., or inhalant administration of TEI-8362. 4. Intratracheal administration of lipopolysaccharide induced pulmonary neutrophilia. Twenty-four hours after lipopolysaccharide administration, the additional treatment with formyl-methionyl-leucyl-phenylalanine resulted in a specific neutrophil-dependent acute lung injury. In this model, lung injury was significantly attenuated by i.t., i.v., or inhalant administration of TEI-8362. 5. These pharmacological actions of TEI-8362 suggest that this drug has therapeutic value in the treatment of destructive lung diseases due to neutrophils.

Described herein is a modern approach to the rapid preparation and evaluation of compounds as potential back-up drug candidates. GW311616A, 1, a derivative of pyrrolidine trans-lactams, has previously been described as a potent, orally active inhibitor of human neutrophil elastase (HNE) for the treatment of respiratory disease. These properties made it a suitable candidate for development. Described here is the discovery of three further derivatives of pyrrolidine trans-lactams, which fulfill the criteria required for back-up candidates 28, 29, and 32. These include increased activity in inhibiting HNE in human whole blood (HWB) and comparable pharmacokinetic properties, in particular clearance, in two species. To provide a rapid assessment of clearance, cassette dosing in dog was used. Modern array techniques, including the synthesis of mixtures, were used to synthesize compounds rapidly. Having selected three potential compounds as back-up candidates, they were prepared as single enantiomers and profiled in in vitro and in vivo assays and evaluated pharmacokinetically in rat and dog. These compounds are highly potent and selective HNE inhibitors, with a prolonged pharmacodynamic action. Pharmacokinetically, these compounds are comparable with 1 while they are more potent in HWB. Compound 28, however, has a higher clearance. One of these compounds, 32, was cocrystallized with HNE, and features of this structure are described and compared with the cocrystal structure of 1 in porcine pancreatic elastase.

In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE). Kinetic analyses were completed using in vitro assays with synthetic and insoluble elastin substrates in the presence of HNE and various heparin preparations (14-15 kDa; 17-19 kDa), heparin-derived oligosaccharides (4-22 saccharides), and chemically modified heparins (2-O-, 6-O-, O-, and N-desulfated). Results showed that heparin inhibits HNE by a tight-binding, hyperbolic, competitive mechanism, contrary to previous reports in the literature. A minimum length of at least 12-14 saccharides is required for inhibition, after which inhibitory activity increases with chain length (or molecular mass). Although all N- and O-sulfate groups contribute to inhibition, 2-O-sulfate groups are less critical than either N- or 6-O-sulfate groups, indicating that inhibitory activity is dependent upon the heparin fine structure. Molecular-docking simulations support the kinetic results and provide a plausible model for the size requirement, whereby positively charged, clamp-like regions at the ends of the interdomain crevice (elastase fold) are used by heparin to bridge the active site and inhibit activity.