Thalidomide derivatives as nanomolar human neutrophil elastase inhibitors: Rational design, synthesis, antiproliferative activity and mechanism of action.

Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

Diversity oriented clicking delivers ß-substituted alkenyl sulfonyl fluorides as covalent human neutrophil elastase inhibitors.

Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented ß-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase.

Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

Base-catalysed 18F-labelling of trifluoromethyl ketones. Application to the synthesis of 18F-labelled neutrophil elastase inhibitors.

A new method for the fluorine-18 labelling of trifluoromethyl ketones has been developed. This method is based on the conversion of a-COCF3 functional group to a difluoro enol silyl ether followed by halogenation and fluorine-18 labelling. The utility of this new method was demonstrated by the synthesis of fluorine-18 labelled neutrophil elastase inhibitors, which are potentially useful for detection of inflammatory disorders.

The mutation of SPI51, a protease inhibitor of silkworm, resulted in the change of antifungal activity during domestication.

Domestication of silkworm has led to alterations in various gene expression patterns. For instance, many protease inhibitors were significantly downregulated in the domestic silkworm cocoon compared to its wild progenitor. Considering that SPI51 is the most abundant protease inhibitor in silkworm cocoons, herein, we compared the gene structures and sequences of SPI51 from B. mori (BmoSPI51) and B. mandarina (BmaSPI51). Comparing to the "RGGFR" active site in BmaSPI51, that of BmoPI51 is "KGSFP" and the C-terminal "YNTCECSCP" tail sequence is lost in the latter. To investigate the effect elicited by the active site and tail sequences on the function of SPI51, we expressed two mutated forms of BmoSPI51, namely, BmoSPI51 + tail and BmoSPI51M. BmoSPI51, BmoSPI51 + tail and BmoSPI51M were compared and found to have similar levels of inhibitory activity against trypsin. However, the BmoSPI51 + tail and BmoSPI51M proteins exhibited significantly stronger capacities to inhibit fungi growth, compared to BmoSPI51. We concluded that the specific amino acid sequence of the active site, as well as its the disulfide bond formed by C-terminal sequence in the BmaSPI51, represent the key factors responsible for its higher antifungal activity. This study provided new insights into the antifungal mechanisms elicited by protease inhibitors in the cocoons of silkworms.

The Three-Component Synthesis of 4-Sulfonyl-1,2,3-triazoles via a Sequential Aerobic Copper-Catalyzed Sulfonylation and Dimroth Cyclization.

4-Sulfonyl-1,2,3-triazole scaffolds possess promising bioactivities and applications as anion binders. However, these structures remain relatively unexplored and efficient synthetic procedures for their synthesis remain desirable. A practical room-temperature, aerobic copper-catalyzed three-component reaction of aromatic ketones, sodium sulfinates, and azides is reported. This procedure allows for facile access to 4-sulfonyl-1,5-disubstituted-1,2,3-triazoles in yields ranging from 34 to 89%. The reaction proceeds via a sequential aerobic copper(II)chloride-catalyzed oxidative sulfonylation and the Dimroth azide-enolate cycloaddition.

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

New 3-unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation.

Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine protease family and is involved in a variety of pathologies. Thus, compounds able to inhibit HNE represent promising therapeutics for the treatment of inflammatory diseases. Here, we report the further elaboration of our previously reported 3-methylisoxazolone derivatives, synthesizing a new series of 3-nor-derivatives bearing different substituents at the 4-phenyl ring. The most potent compounds 3a, 3g, and 3h, had IC50 values of 16, 11, and 18 nM, respectively. Molecular modeling studies and molecular dynamic (MD) simulations demonstrated no substantial differences between the 3-methylisoxazole derivatives previously tested and the corresponding 3-unsubstituted derivatives in the snapshot conformations sampled during the MD simulations, which is consistent with their similar levels of HNE inhibitory activity. Thus, we conclude that the isoxazolone scaffold is a good scaffold for developing HNE inhibitors, as it tolerates several modifications when adhering to basic scaffold requirements, and the resulting derivatives are quite potent HNE inhibitors.

Synthesis, biological evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE) inhibitors.

We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazolones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC50 values in the nanomolar range (20-70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded region about the site of the nucleophilic attack, which could explain its lowered activity. In addition molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions affording a favourable orientation for the nucleophilic attack.

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein.

Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.

Matrix metalloproteinase-7 induces homotypic tumor cell aggregation via proteolytic cleavage of the membrane-bound Kunitz-type inhibitor HAI-1.

Matrix metalloproteinase-7 (MMP-7) plays important roles in tumor progression and metastasis. Our previous studies have demonstrated that MMP-7 binds to colon cancer cells via cell surface-bound cholesterol sulfate and induces significant cell aggregation by cleaving cell-surface protein(s). These aggregated cells exhibit a dramatically enhanced metastatic potential. However, the molecular mechanism inducing this cell-cell adhesion through the proteolytic action of MMP-7 remained to be clarified. Here, we explored MMP-7 substrates on the cell surface; the proteins on the cell surface were first biotinylated, and a labeled protein fragment specifically released from the cells after MMP-7 treatment was analyzed using LC-MS/MS. We found that hepatocyte growth factor activator inhibitor type 1 (HAI-1), a membrane-bound Kunitz-type serine protease inhibitor, is an MMP-7 substrate. We also found that the cell-bound MMP-7 cleaves HAI-1 mainly between Gly451 and Leu452 and thereby releases the extracellular region as soluble HAI-1 (sHAI-1). We further demonstrated that this sHAI-1 can induce cancer cell aggregation and determined that the HAI-1 region corresponding to amino acids 141-249, which does not include the serine protease inhibitor domain, has the cell aggregation-inducing activity. Interestingly, a cell-surface cholesterol sulfate-independent proteolytic action of MMP-7 is critical for the sHAI-1-mediated induction of cell aggregation, whereas cholesterol sulfate is needed for the MMP-7-catalyzed generation of sHAI-1. Considering that MMP-7-induced cancer cell aggregation is an important mechanism in cancer metastasis, we propose that sHAI-1 is an essential component of MMP-7-induced stimulation of cancer metastasis and may therefore represent a suitable target for antimetastatic therapeutic strategies.

Potential Anti-inflammatory Effects of the Fruits of Paulownia tomentosa.

As part of an ongoing search for new natural products from medicinal plants to treat respiratory disease, six new compounds, a dihydroflavonol (1) and five C-geranylated flavanones (3, 6, 8, 13, and 14), and 13 known compounds were isolated from mature fruits of Paulownia tomentosa. The structures of the new compounds were determined via interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ECD, and MS). In biological activity assays with human alveolar basal epithelial cells, the expression of TNF-α-induced proinflammatory cytokines (IL-8 and IL-6) was reduced significantly by the EtOAc fraction of a P. tomentosa extract as well as by the new compounds isolated from this fraction. Furthermore, the majority of the isolates (1-19 except 5-7) were found to inhibit human neutrophil elastase (HNE) activity, with IC50 values ranging from 2.4 ± 1.0 to 74.7 ± 8.5 µM. In kinetic enzymatic assays with the HNE substrate MeOSuc-AAPV-pNA, compound 17 exhibited the highest inhibitory activity (Ki = 3.2 µM) via noncompetitive inhibition. These findings suggest that the flavanone constituents of P. tomentosa fruits may be valuable for the development of new drug candidates to treat airway inflammation.

KLK14 interactions with HAI-1 and HAI-2 serine protease inhibitors: A molecular dynamics and relative free-energy calculations study.

Kallikrein 14 (KLK14) is a serine protease linked to several pathologies including prostate cancer and positively correlates with Gleason score. Though KLK14 functioning in cancer is poorly understood, it has been implicated in HGF/Met signaling, given that KLK14 proteolytically inhibits HGF activator-inhibitor 1 (HAI-1), which strongly inhibits pro-HGF activators, thereby contributing to tumor progression. In this work, KLK14 binding to either hepatocyte growth factor activator inhibitor type-1 (HAI-1) or type-2 (HAI-2) was essayed using homology modeling, molecular dynamic simulations and free-energy calculations through MM/PBSA and MM/GBSA. KLK14 was successfully modeled. Calculated free energies suggested higher binding affinity for the KLK14/HAI-1 interaction than for KLK14/HAI-2. This difference in binding affinity is largely explained by the higher stability of the hydrogen-bond networks in KLK14/HAI-1 along the simulation trajectory. A key arginine residue in both HAI-1 and HAI-2 is responsible for their interaction with the S1 pocket in KLK14. Additionally, MM/GBSA free-energy decomposition postulates that KLK14 Asp174 and Trp196 are hotspots for binding HAI-1 and HAI-2.

Biophysical insight into structure-function relation of Allium sativum Protease Inhibitor by thermal, chemical and pH-induced modulation using comprehensive spectroscopic analysis.

In this study, we have analyzed the structural and functional changes in the nature of Allium sativum Protease Inhibitor (ASPI) on undergoing various denaturation with variable range of pH, temperature and urea (at pH 8.2). ASPI being anti-tryptic in nature has native molecular mass of ∼15kDa. The conformational stability, functional parameters and their correlation were estimated under different conditions using circular dichroism, fluorescence and activity measurements. ASPI was found to fall in belongs to α+ß protein. It demonstrated structural and functional stability in the pH range 5.0-12.0 and up to70°C temperature. Further decrease in pH and increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were reversible and sigmoid. Tm (midpoint of denaturation), ΔCp (constant pressure heat capacity change) and ΔHm (van't Hoff enthalpy change at Tm were calculated to be 41.25±0.2°C, 1.3±0.07kcalmol-1K-1 and 61±2kcalmol-1 respectively for thermally denatured ASPI earlier. The reversibility of the protein was confirmed for both thermally and chemically denatured ASPI. The results obtained from trypsin inhibitory activity assay and structural studies are found to be in a significant correlation and hence established structure-function relationship of ASPI.

The crystal structure of a multidomain protease inhibitor (HAI-1) reveals the mechanism of its auto-inhibition.

Hepatocyte growth factor activator inhibitor 1 (HAI-1) is a membrane-bound multidomain protein essential to the integrity of the basement membrane during placental development and is also important in maintaining postnatal homeostasis in many tissues. HAI-1 is a Kunitz-type serine protease inhibitor, and soluble fragments of HAI-1 with variable lengths have been identified in vivo The full-length extracellular portion of HAI-1 (sHAI-1) shows weaker inhibitory activity toward target proteases than the smaller fragments, suggesting auto-inhibition of HAI-1. However, this possible regulatory mechanism has not yet been evaluated. Here, we solved the crystal structure of sHAI-1 and determined the solution structure by small-angle X-ray scattering. These structural analyses revealed that, despite the presence of long linkers, sHAI-1 exists in a compact conformation in which sHAI-1 active sites in Kunitz domain 1 are sterically blocked by neighboring structural elements. We also found that in the presence of target proteases, sHAI-1 adopts an extended conformation that disables the auto-inhibition effect. Our results also reveal the roles of non-inhibitory domains of this multidomain protein and explain the low activity of the full-length protein. The structural insights gained here improve our understanding of the regulation of HAI-1 inhibitory activities and point to new approaches for better controlling these activities.

Recombinant hepatocyte growth factor activator inhibitor 1: expression in Drosophila S2 cells, purification and crystallization.

Hepatocyte growth factor activator inhibitor 1 (HAI-1) is a multi-domain membrane-associated protease inhibitor that potently inhibits a variety of serine proteases such as hepatocyte growth factor activator and matriptase. Different truncates of HAI-1 show varying potencies for inhibition of target proteases, suggesting that the domain organization of HAI-1 plays a critical role in its function. Here, the soluble full-length extracellular part of HAI-1 (sHAI-1) was expressed using the Drosophila S2 insect-cell expression system. Diffraction-quality crystals of sHAI-1 were produced using ammonium sulfate as precipitant. The crystal diffracted to 3.8 Šresolution and belonged to space group P41212, with unit-cell parameters a = b = 95.42, c = 124.50 Å. The asymmetric unit contains one sHAI-1 molecule.

Phileucin - A Cyclic Dipeptide Similar to Phevalin (Aureusimine B) from Streptomyces coelicolor M1146.

Overexpression of a putative type III polyketide synthase (PKSIII) from the marine myxobacterium Enhygromyxa salina SWB007 in Streptoinyces coelicolor MI 146 led to the accumulation of a novel monoketopiperazine consisting of phenylalanine and isoleucine. This compound was named phileucin and shows high structural similarity to phevalin (aureusimine B). The protease inhibiting activity was tested against human cathepsin L, human leukocyte elastase; bovine trypsin and bovine chymotrypsin. In contrast to phevalin, no protease inhibition was observed.

Human neutrophil elastase inhibitory potential of flavonoids from Campylotropis hirtella and their kinetics.

Campylotropis hirtella is used as a food supplement in the subtropical region of China. In an intensive hunt for human neutrophil elastase inhibitors, we isolated eight flavonoids from C. hirtella three of which (1-3) emerged to be elastase inhibitors. Geranylated flavonoids (1-3) displayed significant inhibitory activity with IC50s between 8.5 and 30.8 µM. The most striking example was geranylated isofavanone 3 that inhibited elastase significantly (IC50 = 30.8 µM) but its parent compound (dalbergioidin) and isoflavone analog (5) were inactive (IC50 > 200 µM). Compounds (1-3) displayed different kinetic mechanisms (noncompetitive, competitive, and mixed type, respectively) that were dependent upon the parent skeleton. The competitive inhibitor, isoflavan-3-ol-4-one 2 manifested an inhibition of isomerization profile for elastase with kinetic parameters K5 = 0.0386 M-1S-1, K6 = 0.0244 µM-1S-1 and Kiapp = 16.3427 µM. The specific identification of metabolites was accomplished by LC-DAD-ESI/MS that was also used to analyze abundance of active components (1-3) within the plant.

Synthesis and Pharmacological Evaluation of Indole Derivatives as Deaza Analogues of Potent Human Neutrophil Elastase Inhibitors.

Preclinical Research A number of N-benzoylindoles were designed and synthesized as deaza analogs of previously reported potent and selective HNE inhibitors with an indazole scaffold. The new compounds containing substituents and functions that were most active in the previous series were active in the micromolar range (the most potent had IC50 = 3.8 µM) or inactive. These results demonstrated the importance of N-2 in the indazole nucleus. Docking studies performed on several compounds containing the same substituents but with an indole or an indazole scaffold, respectively, highlight interesting aspects concerning the molecule orientation and H-bonding interactions, which could help to explain the lower activity of this new series. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc.