In this present investigation, thiazolylcoumarin derivatives (5a-5k) were synthesized from thiosemicarbazide, ethyl acetoacetate, and naphthaldehyde through a multistep route. The formation of thiazolylcoumarin derivatives with bioactive scaffolds was confirmed through nuclear magnetic resonance spectroscopy. A solvatochromic study of synthesized thiazolylcoumarin derivatives was carried out using ultraviolet-visible methods for dimethylformamide (DMF), ethyl acetate, and ethanol solvents. The redox behaviour of as-synthesized thiazolylcoumarin derivatives (5a-5k) was examined in dimethyl sulphoxide by conducting an electrochemical study. Fluorescence properties of thiazolylcoumarin derivatives were studied in DMF, ethanol, and ethyl acetate to visualize the solvent effect on the emitting ability of thiazolylcoumarin derivatives.

The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to SDS-PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the Glu-342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.

Serpins have a large external peptide loop known as the reactive loop. Part of the reactive loop functions as the primary recognition site for target proteases; however, the complete role of the reactive loop in determining serpin specificity is unclear. In the current study, we investigated the reactive loop region that could potentially interact with the extended binding site of target proteases; the P6-P3' region. We utilized a reactive loop switching strategy to determine the extent to which the inhibitory activity of alpha-1-protease inhibitor (PI) against human neutrophil elastase (HNE) could be transferred to alpha-1-antichymotrypsin (ACT), a serpin that does not inhibit HNE. A series of ACT-PI chimeras were constructed in which segments of increasing length taken from the P6-P3' region of PI replaced the corresponding residues of ACT. The effectiveness of each chimera as an inhibitor of HNE was assessed by measuring (1) the rate of inhibitory complex formation and (2) the rate of complex breakdown (complex stability). Although all the ACT-PI chimeras were fully functional against chymotrypsin-like proteases, the series of chimeras showed no consistent progress toward the production of an inhibitor with the inhibitory properties of PI. The most rapid complex formation and most stable complexes were observed for chimeras with the P3-P1 residues of PI, whereas extending the replacement region to the P6 residue resulted in a considerable decrease in both inhibitory parameters. In order to study two additional features of the PI reactive loop that may play a role in the presentation of the P6-P3' region to HNE, we constructed variants that contained a P4' proline and deleted the P6'-P9' residues. Changes on the prime side appeared to have little effect on rates of inhibition or complex stability. Overall, even the most effective chimeras demonstrated an inhibition rate constant at least 60-fold less than that observed for PI inhibition of HNE and the most long lived chimera-HNE complexes broke down more rapidly than PI-HNE complexes. These results indicate that residues in the reactive loop region predicted to contact a specific target protease cannot fully transfer inhibitory activity from one serpin to another, suggesting that specific reactive loop-serpin body and serpin body-protease body interactions play a significant role in determining serpin inhibitory activity against target proteases.

Inhibition of human chymase by the serpins alpha(1)-antichymotrypsin (ACT) and alpha(1)-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin, The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction, As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions, In this study the effect of pH on the inhibition rate constant (k(inh)) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition, Reactions were evaluated over a pH range to correlate k(inh) and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity, The results show that the effects of pH on SI and k(?inh), differ for each inhibitor, On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in k(inh) whereas the chymase PI reaction revealed an increase in SI and a marked decrease in k(inh). On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in k(inh) whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in k(inh). Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for k(cat)/K-m and k(cat) and decreases for K-m. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on k(inh) and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase, The constancy of k(inh) for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity, Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.

The inhibition of human chymase, a chymotrypsin-like proteinase stored in mast cell granules, by secretory leukocyte proteinase inhibitor (SLPI) is investigated in this study. SLPI is a serine proteinase inhibitor present in human mucus secretions and tissues. It binds heparin, a highly sulfated glycosaminoglycan also found in mast cell secretory granules, and this interaction increases its effectiveness as an inhibitor of neutrophil elastase. Analysis of the chymase–SLPI interaction by equilibrium and kinetic methods indicates that the inhibition of chymase results from the reversible formation of a stable 1:1 enzyme–inhibitor complex. The dissociation equilibrium constant (Ki) determined in reactions containing 0.18Mor 1.0MNaCl (pH 8.0, 25°C) was 5 × 10−8and 2 × 10−8M, respectively. Addition of heparin to the low-salt reaction decreased theKiapproximately 10-fold to a value of 3 × 10−9M, making SLPI a more effective inhibitor of human chymase. The decrease was due primarily to an approximately 10-fold increase in the association rate constant (kass) from 2 × 104to 3 × 105M−1s−1. The magnitudes of the rate and dissociation equilibrium constants indicate that SLPI has the potential to be a good chymase inhibitorin vivo,especially if chymase and heparin are released from mast cell granules simultaneously. The enhanced interaction in the presence of heparin supports the importance of this glycosaminoglycan to the inhibitory function of SLPI.

Despite the homology with alpha 1-protease inhibitor (alpha 1PI), wild-type antichymotrypsin (ACT) is a substrate for HNE rather than an inhibitor of the enzyme. In order to investigate the nature of the specificity between serpins and serine proteases, the reactions of human neutrophil elastase (HNE) with wild-type recombinant ACT and recombinant variants of ACT were studied. ACT variants were generated where (1) the primary interaction site, the P1 position, was replaced with the P1 residue of alpha 1PI, (2) the residues corresponding to P3-P3' were replaced with those of alpha 1PI, and (3) the residues corresponding to the canonical recognition sequence as well as flanking residues encompassing the exposed reactive loop of the inhibitor were replaced with the corresponding residues of alpha 1PI. Each variant was analyzed to determine the effect of the replacements on reactions with human neutrophil elastase and chymotrypsin with regard to (1) the second-order rate constant for enzyme-serpin complex formation, (2) the number of moles of serpin required to completely inhibit 1 mol of enzyme (the stoichiometry of inhibition, SI), and (3) the stability of the enzyme-serpin complex. Replacing Leu with Met in the P1 position (rACT-L358M) was sufficient to convert rACT into an inhibitor of HNE with an apparent second-order rate constant (k'/[I]) of 4 x 10(4) M-1 s-1 and an SI of 5. The high SI was due to a concurrent hydrolytic reaction at sites in the reactive loop.(ABSTRACT TRUNCATED AT 250 WORDS)

In GC cells, a growth hormone-producing rat pituitary cell line, 3,5,3'-triiodo-L-thyronine (L-T3) rapidly stimulates the transcription rate of the growth hormone gene which parallels the level of chromatin-associated L-T3-receptor complexes (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). In this study we have functionally mapped the elements of the gene which are involved in mediating basal and hormone-regulated expression. Stable transformation studies indicate that transcriptional regulation of the gene by L-T3 is mediated by sequences in the 5'-flanking region. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the chloramphenicol acetyltransferase gene. Transient expression occurred only in cells which expressed the endogenous growth hormone gene. Sequences between -104 and +7 were found to be essential for basal expression. One of the most highly conserved regions (-105 to -145) contains elements which further enhance the level of basal expression but are not necessary for regulated expression by L-T3. DNA between -210 and -181 was found to be essential for stimulation by L-T3 and was shown to function most efficiently with the homologous rat growth hormone promoter (-104 to +7). Sequences from -206 to -198 show about 80% homology with a sequence in the 5'-flanking region of two other rat genes which are regulated by thyroid hormone. Glucocorticoid hormones, which also transcriptionally stimulate the rat growth hormone gene, elicited only minimal effects in both stable and transient expression studies. This suggests that the elements which mediate glucocorticoid regulation of the endogenous gene are found either upstream of the cloned 5'-flanking region (1800 base pairs) or 3' of the cap site.