Inhibition Of Porcine Pancreatic Elastase Research Articles

Chemical composition and antifungal activities of Ziziphora tenuir and Z. clinopodioides essential oils against dermatophytes

Summary Introduction: Ziziphora species are traditionally used for treatment of different infectious and non-infectious diseases as antiseptic agents. Objective: The aim of this study was to evaluate the chemical composition of Ziziphora clinopodioides and Z. tenuir essential oils and their antifungal effects againt five strains of dermatophytes. Methods: GC and GC-MS methods were used for essentional oils analize. The anti-elastase activities were determined by porcine pancreatic elastase assays. Results: 48 different compounds were identified in these two essential oils, which thymol, p-cymene, 1,8-cineole and γ-terpinene were their major components. The anti-dermatophyte activities of essential oils against dermatophytes showed that the essential oils (150 ppm) inhibited the mycelium growth, about 5–100%, which Z. clinopodioides essential oil had higher mycelium growth inhibition (28–100%) than that of Z. tenuir oil. The MIC and MFC values of essential oils were 0.01–1 µl/ml. 0.5 µl/ml essential oils inhibited porcine pancreatic elastase, dose-dependently. Conclusions: Due to the anti-dermatophyte and anti-elastase effects of Ziziphora sp., it can be considered as natural antifungal agent for more clinical and pre-clinical trials.

Kinetic and computational analysis of the reversible inhibition of porcine pancreatic elastase: a structural and mechanistic approach

Structural and mechanistic insights were revealed for the reversible inhibition of Porcine Pancreatic Elastase (PPE); the kinetics of uninhibited and inhibited hydrolysis of substrate Suc-AAA-pNA was analyzed thoroughly. Additionally, the interactions between PPE and its inhibitor were studied by computational techniques. The uninhibited hydrolysis of Suc-AAA-pNA by PPE proceeds through a virtual transition state, involving an inferior physical and another dominating chemical step, where two stabilized reactant states precede the predominant acyl-enzyme. Different kinds of bonding with the PPE-backbone residues, including those of the catalytic triad, were found during the MD simulation of 5 ns, as key interactions favoring a higher stabilization of the best ranked complex PPE-CF3C(O)-KA-NHPh-p-CF3. The proton inventories of the inhibited hydrolysis of Suc-AAA-pNA by PPE, were ruled out the existence of any virtual transition state and thus they argue for a different mode of catalysis involving a structurally disturbed PPE molecule. Thereafter, a novel inhibition mechanism was suggested.

Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases

Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides. Thus, identification of cell-free correlates that directly regulate the number of CD4(+) T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates. The number of stem cells that enter blood and are destined to become circulating CD4(+) T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLE(CS)) and the HLE(CS)-reactive active α(1)proteinase inhibitor (α(1)PI, α(1)antitrypsin, SerpinA1). In HIV-1 disease, α(1)PI is inactivated due to disease processes. In the early asymptomatic categories of HIV-1 disease, active α(1)PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α(1)PI (r(2)=0.93, p<0.0001, n=26) and inactive α(1)PI (r(2)=0.91, p<0.0001, n=26). Administration of α(1)PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α(1)PI participates in regulating the number of CD4(+) T cells in blood. With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α(1)PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α(1)PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α(1)PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA(3)NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α(1)PI in saliva. The resulting inhibition of PPE by active α(1)PI can be measured by adding the PPE substrate SA(3)NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α(1)PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable. Thus, active α(1)PI in saliva is calculated as a ratio to saliva protein content and is termed the α(1)PI Index. Results presented herein demonstrate that the α(1)PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

Lyngbyastatins 8–10, Elastase Inhibitors with Cyclic Depsipeptide Scaffolds Isolated from the Marine Cyanobacterium Lyngbya semiplena

Investigation of an extract from the marine cyanobacterium Lyngbya semiplena, collected in Tumon Bay, Guam, led to the identification of three new cyclodepsipeptides, lyngbyastatins 8–10 (1–3). The structures of 1–3 were determined by NMR, MS, ESIMS fragmentation and chemical degradation. Compounds 1–3 are closely related to lyngbyastatins 4–7. Like the latter compounds, we found 1–3 to inhibit porcine pancreatic elastase, with IC50 values of 123 nM, 210 nM and 120 nM, respectively.

Tiglicamides A–C, cyclodepsipeptides from the marine cyanobacterium Lyngbya confervoides

The Floridian marine cyanobacterium Lyngbya confervoides afforded cyclodepsipeptides, termed tiglicamides A–C ( 1– 3), along with their previously reported analogues largamides A–C ( 4– 6), all of which possess an unusual tiglic acid moiety. Their structures were deduced by one- and two-dimensional NMR combined with mass spectrometry and the absolute configurations established by chiral HPLC and Marfey’s analysis of the degradation products. Compounds 1– 3 moderately inhibited porcine pancreatic elastase in vitro with IC 50 values from 2.14 to 7.28 μM. Compounds 1– 6 differ from each other by one amino acid residue within the cyclic core structure, suggesting an unusually relaxed substrate specificity of the nonribosomal peptide synthetase that is the putative biosynthetic enzyme responsible for the corresponding amino acid incorporation.

Azetidine-2,4-diones (4-Oxo-β-lactams) as Scaffolds for Designing Elastase Inhibitors

A new class of inhibitors 4-oxo-beta-lactams (azetidine-2,4-diones), containing the required structural elements for molecular recognition, inhibit porcine pancreatic elastase (PPE) but show a dramatically lower reactivity toward hydroxide compared with the analogous inhibitors 3-oxo-beta-sultams. Inhibition is the result of acylation of the active site serine and electron-withdrawing substituents at the N-(4-aryl) position in 3,3-diethyl- N-aryl derivatives increasing the rate of enzyme acylation and generating a Hammett rho-value of 0.65. Compared with a rho-value of 0.96 for the rates of alkaline hydrolysis of the same series, this is indicative of an earlier transition state for the enzyme-catalyzed reaction. Docking studies indicate favorable noncovalent interactions of the inhibitor with the enzyme. Compound 2i, the most potent inhibitor against PPE, emerged as a very potent HLE inhibitor, with a second-order rate for enzyme inactivation of approximately 5 x 10 (5) M (-1) s (-1).

Alpha-1-proteinase inhibitor is a heparin binding serpin: Molecular interactions with the Lys rich cluster of helix-F domain

Alpha-1-proteinase (α-1-PI) inhibitor is the major circulating serine protease inhibitor in humans. The porcine elastase and trypsin inhibitory activity of human and ovine α-1-PI is activated several fold in the presence of anti-coagulant heparin. The activation is allosteric and appears to be characterized by two steps of binding; a weak followed by a strong binding. The K ass for ovine and human α-1-PI inhibition of porcine pancreatic elastase was increased ∼45 fold and 38 fold respectively. Using a combinatorial approach of multiple sequence alignment, surface topology, chemical modification and tryptic peptide mapping to identify the sequence of the heparin bound peptide; we demonstrate that heparin binds to the lysyl rich region of the F-helix of α-1-PI, which differs from that of heparin-antithrombin (AT) interactions. Molecular docking prediction using the MEDock algorithm approximates the three positively charged lysines (K 154, K 155, K 174) of human α-1-PI in this interaction. This heparin α-1-PI interaction has been exploited to develop an affinity purification method, which can be used universally to obtain homogenous preparations of mammalian α-1-PIs useful for augmentation therapy. Collectively, all these findings imply that α-1-PI has a major role in regulating extra cellular protease activity and the physiological activator is heparin.

Ecotoxicologically relevant cyclic peptides from cyanobacterial bloom (Planktothrix rubescens) - a threat to human and environmental health

Ecotoxicologically relevant cyclic peptides from cyanobacterial bloom (Planktothrix rubescens) - a threat to human and environmental healthBackground. The information of the overall production of major cyanobacterial cyclic peptides in a water body is essential for risk assessment and for the prediction of future development of the bloom. A procedure that gives a review of both toxic and non-hepatotoxic hydrophilic cyclic peptide production is important to evaluate the ecological conditions in the water environment and to predict the release of dangerous toxic and tumour promoting substances.Methods. The cyclic peptides were identified on the basis of their retention times, characteristic spectra, molecular masses and biological activity. The non-hepatotoxic cyclic peptides were characterised by their inhibition of porcine pancreatic elastase, while cytotoxicity to mammalian cells was tested with the MTT test on B16 cell line.Conclusions. The method presented gives a rapid, simultaneous assessment, preliminary identification and estimation of bioactive cyclic peptides. The synthesis of non-hepatotoxic cyclic peptides can mediate the release various toxic and otherwise biologically active substances that induce systemic genotoxicity in mammals.

The Bsmoc group as a novel scaffold for the design of irreversible inhibitors of cysteine proteases

Carbamate and ester derivatives of the 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) scaffold react readily with thiols via a Michael addition at rates not significantly affected by the nature of the carboxylic or carbamic acid leaving group. These Michael acceptors are irreversible inhibitors of the cysteine proteases papain and human liver cathepsin B, displaying first-order kinetics with respect to inhibitor concentration. In contrast, none of the Bsmoc derivatives inhibited porcine pancreatic elastase, a serine protease.

Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1.

We have isolated a polylactide or poly(L-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, epsilon-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA.

Structural and Functional Study of an Anemonia Elastase Inhibitor, a “Nonclassical” Kazal-Type Inhibitor fromAnemonia sulcata

Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.

Binding Structure of Elastase Inhibitor Scyptolin A

Natural bioactive compounds are of general interest to pharmaceutical research because they may be used as leads in drug development campaigns. Among them, scyptolin A and B from Scytonema hofmanni PCC 7110 are known to inhibit porcine pancreatic elastase, which in turn resembles the attractive drug target neutrophil elastase. The crystal structure of scyptolin A as bound to pancreatic elastase was solved at 2.8 Å resolution. The inhibitor occupies the most prominent subsites S1 through S4 of the elastase and prevents a hydrolytic attack by covering the active center with its rigid ring structure. The observed binding structure may help to design potent elastase inhibitors.

Synthesis and Evaluation of δ‐Lactams (Piperazones) as Elastase Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Structure of a hybrid squash inhibitor in complex with porcine pancreatic elastase at 1.8 Å resolution

The crystal structure of porcine pancreatic elastase in complex with a hybrid squash inhibitor (HEI-TOE I; 28 amino acids) has been determined to a resolution of 1.8 A. To construct the hybrid inhibitor, the trypsin-binding loop of the squash inhibitor from Ecballium elaterium was substituted by the sequence of a peptide that was derived from the third domain of the turkey ovomucoid inhibitor and was optimized to inhibit porcine pancreatic elastase. This modification of the squash inhibitor changed its specificity for trypsin to a specificity for porcine pancreatic elastase. Specific interactions of this hybrid inhibitor with porcine pancreatic elastase and the differences from the interactions of the ovomucoid inhibitor with human leukocyte elastase are discussed. The binding loop of the inhibitor adopts a 'canonical' conformation and the scissile bond Leu-Glu remains intact.

Synthesis and evaluation of δ-Lactams (Piperazones) as elastase inhibitors

A series of monocyclic δ-lactams (piperazones) was prepared and analysed as inhibitors of porcine pancreatic elastase and human neutrophil elastase.

X-ray Structure of a Serine Protease Acyl-Enzyme Complex at 0.95-Å Resolution

Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.

Reduced protease inhibitory capacity in patients with abdominal aortic aneurysms is reversed with surgical repair

Background: Abdominal aortic aneurysm (AAA) disease is a complex degenerative process that is associated with elevated proteolytic activity. This increased proteolytic activity may be linked to an imbalance in the protease regulatory mechanisms. We hypothesize that reduced AAA plasma inhibitory capacity is related to increased elastolytic activity observed in patients with AAA, which promotes matrix degradation in the aortic wall and AAA expansion. Methods: Preoperative plasma from patients with AAA (n = 86; mean age, 75 ± 8 years) and healthy controls (n = 30; mean age, 72 ± 10 years) was assayed for inhibitory capacity. Postoperative plasma inhibitory capacity was evaluated with comparison of the activity of porcine pancreatic elastase on succinylated elastin substrate, in the presence and absence of AAA and control plasma. Results: AAA plasma showed a significantly reduced capacity to inhibit porcine pancreatic elastase (71.31% ± 1.47%) compared with control plasma (85.52% ± 1.70%; P <.001). Within the AAA group, the inhibitory capacity increased with AAA diameter (P <.05). In addition, surgical repair of AAA resulted in a significant increase in plasma inhibitory capacity (79.83% ± 1.86%) as compared with preoperative levels (63.98% ± 2.69%; P <.001). Of note, the postoperative plasma inhibitory capacity is not significantly different than that of control. Conclusion: AAA plasma has a significantly reduced inhibitory capacity, which varies with aortic diameter and returns to control levels after surgical repair of the AAA. This suggests that protease regulation is a dynamic balance that changes with AAA progression and may play an important role in AAA progression. (J Vasc Surg 2002;35:792-7.)

Scyptolin A and B, cyclic depsipeptides from axenic cultures of Scytonema hofmanni PCC 7110

Two novel cyclic depsipeptides were isolated from axenic cultures of the terrestrial cyanobacterium Scytonema hofmanni PCC 7110 and designated scyptolin A and B. Amino acid analyses in context with mass and 1H/ 13C NMR spectroscopies revealed a composition typical for heterologous cyanopeptolins but containing the uncommon residue 3′-chloro- N-methyl-Tyr (cmTyr) and a unique sidechain. Scyptolin A and B both consist of the N-acylated peptide But 1-Ala 2-Thr 3-Thr 4-Leu 5-Ahp 6 (3-amino-6-hydroxy-2-oxo-1-piperidine)-Thr 7-cmTyr 8-Val 9, which forms a 19-membered ring by esterification of the carboxyl of Val 9 with the hydroxyl of Thr 4. In scyptolin B, the hydroxyl of the Thr 3 residue is additionally esterified with N-butyroyl-Ala. Both scyptolin A and B exhibit selective inhibition of porcine pancreatic elastase in vitro with IC 50 values of 3.1 μg/ml.

Inhibition of serine proteases by anti-inflammatory triterpenoids.

The lupane triterpenoid lupeol, the ursane triterpenoid alpha-amyrin and esters of these compounds are present in the bark of roots of Alstonia boonei (Apocynaceae) and have anti-inflammatory properties. alpha-Amyrin is a competitive inhibitor of bovine trypsin and chymotrypsin (Ki values 29 microM and 18 microM, respectively). Lupeol linoleate, lupeol palmitate and alpha-amyrin linoleate are non-competitive inhibitors of trypsin (Ki values 7 microM, 10 microM and 16 microM, respectively). alpha-Amyrin linoleate is also a non-competitive inhibitor of chymotrypsin (Ki value 28 microM). Lupeol is a competitive inhibitor of both trypsin and chymotrypsin (Ki values 22 and 8 microM, respectively). alpha-Amyrin palmitate is a potent non-competitive inhibitor of chymotrypsin (Ki 6 microM). Lupeol, alpha-amyrin and the palmitic and linoleic acid esters of these compounds are ineffective or very weak as inhibitors of porcine pancreatic elastase and of Lucilia cuprina and Helicoverpa punctigera leucine aminopeptidases. These hydrophobic triterpenoids represent further examples of anti-inflammatory triterpenoids that are PKA inhibitors as well as being selective protease inhibitors.