Design and synthesis of new orally active inhibitors of human neutrophil elastase

To identify new orally active inhibitors, further modification of 1 (ONO-6818) was performed. Peptidic derivatives 4b, 4c and 4n showed more potent inhibitory activity than nonpeptidic derivatives 3a-c. As a result, a series of peptidic inhibitors, 4a-s and 5a-v, were discovered. Among these N-aryl derivatives 5a-g, 5i, 5m and 5o-v showed oral activity. Their oral activity showed good correlation with their metabolic stability. Compounds 5h and 5j-l, which were extremely metabolically unstable in hamster plasma, did not show oral activity. Oral activity was considered to be determined by a combination of at least two factors: oral absorption and metabolic stability.     

Non-peptidic inhibitors of human neutrophil elastase: The design and synthesis of sulfonanilide-containing inhibitors

A novel series of pivaloyloxy benzene derivatives has been identified as potent and selective human neutrophil elastase (HNE) inhibitors. Convergent syntheses were developed in order to identify the inhibitors which are intravenously effective in an animal model. A compound of particular interest is the sulfonanilide-containing analogues. Structure-activity relationships are discussed. Structural requirements for metabolic stabilization are also discussed.     

Design, synthesis and evaluation of N-benzoylindazole derivatives and analogues as inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays an important role in tumour invasion and inflammation. A series of N-benzoylindazoles was synthesized and evaluated for their ability to inhibit HNE. We found that this scaffold is appropriate for HNE inhibitors and that the benzoyl fragment at position 1 is essential for activity. The most active compounds inhibited HNE activity with IC?? values in the submicromolar range. Furthermore, docking studies indicated that the geometry of an inhibitor within the binding site and energetics of Michaelis complex formation were key factors influencing the inhibitor's biological activity. Thus, N-benzoylindazole derivatives and their analogs represent novel structural templates that can be utilized for further development of efficacious HNE inhibitors.     

Flavonoids as inhibitors of human neutrophil elastase

Elastase is a proteolytic enzyme belonging to the family of hydrolases produced by human neutrophils, monocytes, macrophages, and endothelial cells. Human neutrophil elastase is known to play multiple roles in the human body, but an increase in its activity may cause a variety of diseases. Elastase inhibitors may prevent the development of psoriasis, chronic kidney disease, respiratory disorders (including COVID-19), immune disorders, and even cancers. Among polyphenolic compounds, some flavonoids and their derivatives, which are mostly found in herbal plants, have been revealed to influence elastase release and its action on human cells. This review focuses on elastase inhibitors that have been discovered from natural sources and are biochemically characterised as flavonoids. The inhibitory activity on elastase is a characteristic of flavonoid aglycones and their glycoside and methylated, acetylated and hydroxylated derivatives. The presented analysis of structure–activity relationship (SAR) enables the determination of the chemical groups responsible for evoking an inhibitory effect on elastase. Further study especially of the in vivo efficacy and safety of the described natural compounds is of interest in order to gain better understanding of their health-promoting potential.     

Simple phosphonic inhibitors of human neutrophil elastase

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val^P(OC₆H₄-4-COOMe)₂ displayed an apparent second-order inhibition value at 33,015 M⁻¹ s⁻¹.     

Cinnoline derivatives as human neutrophil elastase inhibitors

Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory activity but had lower potency compared to N-benzoylindazoles previously reported by us. On the other hand, they exhibited increased stability in aqueous solution. The most potent compound, 18a, had a good balance between HNE inhibitory activity (IC₅₀ value?=?56?nM) and chemical stability (t_1/2?=?114?min). Analysis of reaction kinetics revealed that these cinnoline derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking studies of the active products into the HNE binding site revealed two types of HNE inhibitors: molecules with cinnolin-4(1H)-one scaffold, which were attacked by the HNE Ser195 hydroxyl group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which is the point of attack of Ser195.     

Sesquiterpene lactones as inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) is a serine protease that has been implicated in the abnormal turnover of connective tissue proteins and has been described as an important pathogenic factor in several inflammatory diseases such as rheumatoid arthritis or cystic fibrosis. Here we investigated 17 sesquiterpene lactones (SLs) for their ability to inhibit human neutrophil elastase in an in vitro assay. Podachaenin was the most active compound with an IC(50) value of 7 microM. SLs do not covalently bind to the amino acids of the catalytic triad, thus differing from other elastase inhibitors with a lactone moiety. In contrast to most other biological activities of SLs HNE inhibition is not mediated by alpha,beta-unsaturated carbonyl functions. Ligand binding calculations using the X-ray structure of HNE and the program FlexX revealed structural elements which are a prerequisite for their inhibitory activity.     

Free radicals inactivate human neutrophil elastase and its inhibitors with comparable efficiency

Free radicals produced in a Fenton reaction (H202/Cu), modelling some xenobiotic and cell-mediated inflammatory affronts, efficiently inactivated the elastase-inhibitor eglin, but equally, human neutrophil elastase itself. Elastase activity was not regenerated from proteinase/inhibitor complexes during radical attack. Three different elastase inhibitors, eglin, secretory leukocyte proteinase inhibitor and alpha-1-proteinase inhibitor were all similarly sensitive to inactivation. Unlike certain oxidants which can selectively inactivate alpha-1-proteinase inhibitor, free radicals may influence comparably the availability of both proteinase inhibitors and their targets.     

Crystallization of human neutrophil elastase

Human neutrophil elastase was inactivated by methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. The modified enzyme was crystallized from 40 mM ammonium phosphate, pH 7.0 in the hexagonal space group P6(3) with unit cell parameters a = 74.53 A, b = 74.53 A, c = 70.88 A, alpha = beta = 90 degrees, gamma = 120 degrees. These crystals were resistant to radiation damage and diffracted beyond 1.84-A resolution. The asymmetric unit contained one 25,000-dalton monomer of human neutrophil elastase. Crystals were also grown from the enzyme modified with the analogous iodinated inactivator, p-iodoanilinosuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. These crystals proved to be isomorphous with those of methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane-modified human neutrophil elastase, and served as a single-site, heavy atom derivative for solving the tertiary structure of the enzyme.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Design, synthesis and stability of N-acyloxymethyl- and N-aminocarbonyloxymethyl-2-azetidinones as human leukocyte elastase inhibitors

A series of N-acyloxymethyl- and N-aminocarbonyloxymethyl derivatives of 2-azetidinones, 3, with different substituent patterns at the beta-lactam C-3 and C-4 positions, were designed as potential mechanism-based inhibitors for human leukocyte elastase and found to exhibit inhibitory potency and selectivity for the enzyme.     

Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Functionalized N-aryl azetidinones as novel mechanism-based inhibitors of neutrophil elastase

A functionalized N-aryl azetidinone has been shown to inactivate human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) by an enzyme-mediated process. The inactivation is characterized by the following kinetic constants at pH 8.0 and 37 degrees C: kinact = 0.035 s-1, KI = 1.2 x 10(-4) M for HLE, 0.08 s-1 and 2.7 x 10(-4) M for PPE, respectively. Two parent molecules devoid of the latent leaving group failed to inactivate HLE and PPE and behaved as substrates of these enzymes. A suicide mechanism is postulated involving the formation of an acyl-enzyme and the simultaneous unmasking of a latent quinonimmonium methide ion which irreversibly reacts with an active site nucleophile. Moreover, the inhibitor is still effective at inhibiting elastase preabsorbed onto elastin.     

Degradation of plasmodial antigens by human neutrophil elastase

Human neutrophil elastase (HNE) has been well-studied with respect to its role in pathologic states, but less is known about the physiologic functions of this important granulocyte enzyme. In the present study, we show that HNE can degrade the major circumsporozoite protein of the infective (sporozoite) stage of Plasmodium vivax malaria, and that this enzyme can also interfere with the cytoadherence of human E infected with Plasmodium falciparum (strain K+ FMG-FCR3) (IE). Cytoadherence reactions are not only blocked by treatment of IE with as little as 10 fg HNE/IE, but already adherent IE are also removed by the enzyme. Normal E surface Ag are not extensively destroyed by these doses of HNE. This suggests that the effect of HNE on cytoadherence is selective and probably due to degradation of the malarial Ag exported to the IE surface and responsible for the formation of "recognition knobs" upon which the cytoadherence reaction depends. This conclusion, in turn, was supported by the results of Western blot analysis showing that HNE degrades a high m.w. Ag found exclusively in membrane extracts of IE. Our results suggest that one physiologic role of HNE may be degradation of parasitic antigens during host defense against malaria.     

Design and study of peptide-based inhibitors of amylin cytotoxicity

The incidence of type II diabetes is on the increase each year and the World Health Organisation (WHO) predicts there to be over 360 million diabetic patients worldwide by the year 2030. Deposits consisting mainly of a small protein, called islet amyloid polypeptide (amylin), which aggregates into oligo-/polymeric beta sheet structures is responsible for cytoxicity to the pancreatic β-cells, thus inhibition of this process has been explored as a potential prevention or treatment. N-Methylated and non N-methylated peptides spanning the length of amylin_1–37 were synthesised and evaluated for their inhibition of full length amylin mediated cytoxicity to RIN-5F cells. The non N-methylated peptides were very effective in inhibiting the cytotoxicity while the N-methylated peptides were not. Both the N-methylated and non N-methylated versions of the 29-34 region were equally effective.     

Effects of structure on inhibitory activity in a series of mechanism-based inhibitors of human neutrophil elastase

A structurally-diverse series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-one 1,1 dioxide scaffold were synthesized and used to probe the S′ subsites of human neutrophil elastase (HNE) and neutrophil proteinase 3 (Pr 3). Several compounds are potent inhibitors of HNE but devoid of inhibitory activity toward Pr 3, suggesting that the S′ subsites of HNE exhibit significant plasticity and can, unlike Pr 3, tolerate various large hydrophobic groups. The results provide a promising framework for the design of highly selective inhibitors of the two enzymes.     

Development of a highly water-soluble peptide-based human neutrophil elastase inhibitor; AE-3763 for treatment of acute organ injury

A series of peptide-based transition-state human neutrophil elastase (HNE) inhibitors with N-terminal acidic moieties were synthesized and their inhibitory activity against HNE was evaluated both in vitro and in vivo. Our results show that compounds containing cyclic amide bridged acidic moieties at the N-terminal have not only improved water solubility but also high in vivo potency. Among these compounds, AE-3763 showed remarkable efficacy in hamster models of elastase-induced lung hemorrhage and lipopolysaccharide (LPS)-induced lung injury as well as in a mouse model of LPS/galactosamine-induced acute multiple organ dysfunctions. The water solubility of AE-3763 (>1000 mg/ml in H₂O) was also far superior to that of any of the other compounds synthesized. Thus, it is believed that AE-3763 would be useful for treatment of HNE-associated respiratory disorders, such as acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and acute exacerbation of chronic obstructive pulmonary disease (COPD).     

Design and synthesis of photoactivatable aryl diketo acid-containing HIV-1 integrase inhibitors as potential affinity probes

Aryl diketo acids (ADKs) represent an important new class of HIV-1 integrase (IN) inhibitors. In order to facilitate examination of the structural basis underlying IN?ADK interaction, biphenyl ketone and phenyl azide photophores were incorporated into ADK structures. Of particular note is the novel dual utilization of azide and phenyketone moieties for both enzyme recognition and for crosslinking. The resulting analogues maintained low micromolar inhibitory potency against IN in recombinant in vitro assays. These potential HIV-1 integrase photoaffinity labels may provide useful tools for studying enzyme interactions of the ADK inhibitor class.     

Asymmetric synthesis and affinity of potent sialyltransferase inhibitors based on transition-state analogues

Inhibitors that are structurally related to the transition-state model of the proposed SN1-type mechanism of sialyl transfer, exhibit particularly high binding affinities to alpha(2-6)sialyltransferases. Furthermore, replacing the neuraminyl residue with a simple aryl or hetaryl ring and substituting the carboxylate group for a phosphonate moiety, improves both binding affinity and synthetic accessibility. Herein we report on the synthesis and inhibition of a wide range of novel, potent transition-state analogue based alpha(2-6)sialyltransferase inhibitors comprising a planar anomeric carbon, an increased distance between the anomeric carbon and the CMP leaving group, and at least two negative charges. We also present a short, efficient asymmetric synthesis of the most promising benzyl inhibitors, providing rapid access to large quantities of highly potent, stereochemically-pure (>96% de) inhibitors for further biological investigation (e.g.(R)-3b, Ki = 70 nM).     

Design and synthesis of potent carboxylic acid DGAT1 inhibitors with high cell permeability

A series of potent carboxylic acid DGAT1 inhibitors with high permeability were developed from a virtual screening hit. Strategies were employed to reduce Pgp substrate recognition and increase passive permeability, resulting in the discovery of a series showing good inhibition of cellular triglyceride synthesis. The mutagenic potential of prospective metabolites was evaluated in the Ames assay, and one aniline was shown to be devoid of mutagenicity.