Second-order Rate Constants For Inactivation Research Articles

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Effect of cetyltrimethylammonium chloride on various Escherichia coli strains and their inactivation kinetics by ozone and monochloramine

Cethyltrimethylammonium chloride (CTMA) is one of the most used quaternary ammonium compounds (QACs) in consumer products. CTMA and other QACs are only partially eliminated in municipal wastewater treatment and they can interact with bacteria in biological processes. Currently, there is only limited information on the antimicrobial efficiency of CTMA in matrices other than standard growth media and if and how CTMA influences conventional chemical disinfection. The results obtained in this study showed that the susceptibility of E. coli to CTMA was significantly enhanced in phosphate-buffered saline, lake water and wastewater compared to broth. In broth, a minimum inhibitory concentration (MIC) of CTMA of 20 mgL−1 was observed for E. coli, whereas a 4-log inactivation occurred for CTMA concentrations of about 4 mgL−1 in buffered ultra-purified water, a lake water and wastewater effluent. The impacts of the pre-exposure and the presence of CTMA on inactivation by ozone and monochloramine were tested with three different E. coli strains: AG100 with the efflux pump acrAB intact, AG100A with it deleted and AG100tet with it overexpressed. Pre-exposure of E. coli AG100 to CTMA led to an increased susceptibility for ozone with second-order inactivation rate constants (∼ 106 M−1s−1) increasing by a factor of about 1.5. An opposite trend was observed for monochloramine with second-order inactivation rate constants (∼ 103 M−1s−1) decreasing by a factor of about 2. For E. coli AG100tet, the second-order inactivation rate constant decreased by a factor of almost 2 and increased by a factor of about 1.5 for ozone and monochloramine, respectively, relative to the strain AG100. The simultaneous presence of CTMA and ozone enhanced the second-order inactivation rate constants for CTMA concentrations of 2.5 mgL−1 by a factor of about 3. For monochloramine also an enhancement of the inactivation was observed, which was at least additive but might also be synergistic. Enhancement by factors from about 2 to 4.5 were observed for CTMA concentrations > 2.5 mgL−1.

Inactivation and Site-specific Oxidation of Aquatic Extracellular Bacterial Leucine Aminopeptidase by Singlet Oxygen.

Extracellular enzymes are master recyclers of organic matter, and to predict their functional lifetime, we need to understand their environmental transformation processes. In surface waters, direct and indirect photochemical transformation is a known driver of inactivation. We investigated molecular changes that occur along with inactivation in aminopeptidase, an abundant class of extracellular enzymes. We studied the inactivation kinetics and localized oxidation caused by singlet oxygen, 1O2, a major photochemically derived oxidant toward amino acids. Aminopeptidase showed second-order inactivation rate constants with 1O2 comparable to those of free amino acids. We then visualized site-specific oxidation kinetics within the three-dimensional protein and demonstrated that fastest oxidation occurred around the active site and at other reactive amino acids. However, second-order oxidation rate constants did not correlate strictly with the 1O2-accessible surface areas of those amino acids. We inspected site-specific processes by a comprehensive suspect screening for 723,288 possible transformation products. We concluded that histidine involved in zinc coordination at the active site reacted slower than what was expected by its accessibility, and we differentiated between two competing reaction pathways of 1O2 with tryptophan residues. This systematic analysis can be directly applied to other proteins and transformation reactions.

Inactivation of fungal spores in water using ozone: Kinetics, influencing factors and mechanisms

Fungal contamination of drinking water sources is increasingly threatening the environment and human health. In this study, the inactivation of three genera of dominant fungi in drinking water sources using ozone was first reported. The inactivation of the fungal spores by ozone could be divided into two distinct stages: first a rapid reduction in survival, and then the inactivation at a slower rate. The secondary stage inactivation fitted the Chick–Watson model well, and there was no significant difference in the second-order inactivation rate constants of the three fungal spores (0.199–0.209 L mg−1 min−1). The inactivation rate constants of fungal spores by molecular ozone were much lower than those of viruses, which were equivalent to that of Cryptosporidium. The increase in pH and temperature showed a positive effect on the inactivation rate. Damage to cell membranes, leakage of intracellular compounds, and changes of reactive oxygen species and esterase activity in the spores were detected after inactivation. The results indicated that ozone inactivated fungal spores by firstly destroying cell walls and membranes and then causing the release of intracellular compounds. The fungicidal efficiency of ozone was superior to those of chlorine and chlorine dioxide. In addition, the inactivation efficiency of ozone on fungal spores in real water matrices was reduced to 50.7–91.2% of the efficiency in phosphate buffer. In conclusion, ozone showed high efficiency in the inactivation of fungal spores and could be used as an alternative disinfectant for fungal contamination in drinking water sources.

Photoinduced disinfection in sunlit natural waters: Measurement of the second order inactivation rate constants between E. coli and photogenerated transient species

This work uncovers the implications of the estimation of exogenous inactivation rates for E. coli after the initial lag phase, and presents a strategy for the determination of the second-order inactivation rate constants (k2nd) of these bacteria with relevant transient species promoted by solar light in natural waters. For this purpose, specific precursors were considered (nitrate ion, rose bengal, anthraquinone-2-sulfonate) as well as the respective photo-generated transient species (i.e., hydroxyl radical (•OH), singlet oxygen (1O2) and triplet excited states). Under these conditions and by using suitable reference compounds (acesulfame K and 2,4,6-trimethylphenol in different series of experiments), the k2nd values were obtained after developing a proper competition kinetics methodology. The k2nd values were (2.5 ± 0.9) × 1011, (3.8 ± 1.6) × 107 and (1.8 ± 0.7) × 1010 M−1 s−1 for the inactivation of E. coli by •OH, 1O2 and the triplet state of anthraquinone-2-sulfonate (3AQ2S*), respectively. The measurement of a reaction rate constant that is higher than the diffusion-control limit for small molecules in aqueous solution implies that bacteria behave differently from molecules, e.g., because of the large size difference between bacteria and the transients. The obtained k2nd values were used for the modeling of the bacteria inactivation kinetics in outdoor systems (both water bodies and SODIS bottles), limited to the exponential decay phase that follows the initial lag time. Afterwards, the role of dissolved organic matter (DOM) as precursor of transient species for bacterial elimination was systematically studied. The interaction of different sunlight wavelength regions (UVB, UV-A, blue, green and yellow light) with Suwannee river (SW) and Nordic Lake organic matter (ND) was tested, and the photoinduced disinfection exerted by DOM isolates (SW DOM, Suwannee River Humic Acid, Suwannee River Fulvic Acid or Pony Lake Fulvic Acid) was compared. It was not possible to achieve a complete differentiation of the individual contributions of DOM triplet states (3DOM*) and 1O2 to bacterial inactivation. However, the application of competition kinetics to E. coli under solar irradiation in the presence of SW led to a k2nd value of (2.17 ± 0.40) × 1010 M−1 s−1, which is very near the value for inactivation by 3AQ2S* and suggests that the latter behaved very similar to SW-3DOM* and was a good 3DOM* proxy in the present case. The determination of the second-order inactivation rate constants of E. coli with •OH, 3DOM* and 1O2 represents a significant progress in the understanding of the external inactivation pathways of bacteria. It also allows predicting that, after the lag phase, 1O2 would contribute to photoinactivation to a far lesser extent than •OH and 3DOM*.

A Short Peptide Inhibitor as an Activity-Based Probe for Matriptase-2.

Matriptase-2 is a type II transmembrane serine protease and a key regulator of systemic iron homeostasis. Since the activation mechanism and several features of the physiological role of matriptase-2 are not fully understood, there is strong need for analytical tools to perform tasks such as distinguishing active and inactive matriptase-2. For this purpose we present a short biotinylated peptide derivative with a chloromethyl ketone group, biotin-RQRR-CMK, as an activity-based probe for matriptase-2. Biotin-RQRR-CMK was kinetically characterized and exhibited a second-order rate constant of inactivation (kinac/Ki) of 10,800 M−1 s−1 towards the matriptase-2 activity in the supernatant of transfected human embryonic kidney (HEK) cells. Biotin-RQRR-CMK was able to label active matriptase-2, as visualized in western blot experiments. Pretreatment with aprotinin, an active-site directed inhibitor of serine proteases, protected matriptase-2 from the reaction with biotin-RQRR-CMK.

A BODIPY-Tagged Phosphono Peptide as Activity-Based Probe for Human Leukocyte Elastase.

Human leukocyte elastase plays a crucial role in a variety of inflammatory disorders and represents an important subject of biomedical studies. The chemotype of peptidic phosphonates was employed for the design of a new activity-based probe for human leukocyte elastase. Its structure combines the phosphonate warhead with an adequate peptide portion and a BODIPY fluorophore with a clickable ethinylphenyl moiety at meso position. The probe 6 was assembled by copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition. It was characterized as an active site-directed elastase inhibitor exhibiting a second-order rate constant of inactivation of 88400 M-1s-1. The suitability of 6 as a fluorescent probe for human leukocyte elastase was demonstrated by in-gel fluorescence analysis. Labeling experiments and inhibition data toward a panel of related proteases underlined the selectivity of the probe for the targeted leukocyte elastase.

Kinetics of the Interaction between BAL29880 and LK157 and the Class C β-Lactamase CHE-1.

The chromosome-encoded class C β-lactamase CHE-1 produced by Enterobacter cloacae exhibits a lower sensitivity to avibactam than the P99 enzyme from which it is derived by a 6-residue deletion in the H-10 helix. In the present study, we investigated the sensitivity of CHE-1 to two other β-lactamase inhibitors: LK-157 (or Lek 157), a tricyclic β-lactam, and BAL29880, a bridged monobactam. With both compounds, the second-order rate constants for inactivation were significantly lower for CHE-1, which can thus be considered an inactivator-resistant mutant of P99. However, the second-order rate constant for the inactivation by BAL29880 probably remains adequate for a rather rapid reaction with CHE-1 in the absence of protection by the substrate.

Synthesis and evaluation of vinyl sulfones as caspase-3 inhibitors. A structure–activity study

The first structure–activity relationship study of vinyl sulfones as caspase-3 inhibitors is reported. A series of 12 vinyl sulfones was synthesized and evaluated for two downstream caspases (caspases-3 and -7). Dipeptidyl derivatives were significantly superior to their counterparts containing only Asp at P 1, as caspase-3 inhibitors. Fmoc-Val-Asp- trans-CH CH–SO 2Me was the most potent inhibitor of caspase-3 in the series, with a IC 50 of 29 μM and a second-order rate constant of inactivation, k inact/ K i, of 1.5 M −1 s −1. Computational studies suggest that the second amino acid occupies position S 3 of the enzyme. In addition, Fmoc-Val-Asp- trans-CH CH–SO 2Ph was inactive for caspase-7 for the tested concentrations.

Isoform-Selective Inactivation of Human Arylamine N-Acetyltransferases by Reactive Metabolites of Carcinogenic Arylamines

Human arylamine N-acetyltransferases (NATs) are expressed as two polymorphic isoforms, NAT1 and NAT2, that have toxicologically significant functions in the detoxification of xenobiotic arylamines by N-acetylation and in the bioactivation of N-arylhydroxylamines by O-acetylation. NAT1 also catalyzes the N-acetylation of 4-aminobenzoylglutamic acid, a product of folic acid degradation, and is associated with endogenous functions in embryonic development. On the basis of earlier studies with hamster NAT1, hamster NAT2, and human NAT1, we proposed that human NAT2 would be more susceptible than NAT1 to inactivation by N-arylhydroxamic acid metabolites of arylamines. Kinetic analyses of the inactivation of recombinant NAT1 and NAT2 by the N-arylhydroxamic acid, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), as well as the inactivation of NAT2 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), resulted in second-order inactivation rate constants (k(inact)/K(I)) that were several fold greater for NAT2 than for NAT1. Mass spectrometric analysis showed that inactivation of NAT2 in the presence of the N-arylhydroxamic acids was due to formation of a sulfinamide adduct with Cys68. Treatment of HeLa cells with N-OH-4-AABP and N-OH-AAF revealed that the compounds were less potent inactivators of intracellular NAT activity than the corresponding nitrosoarenes, but unexpectedly, the hydroxamic acids caused a significantly greater loss of NAT1 activity than of NAT2 activity. Nitrosoarenes are the electrophilic products responsible for NAT inactivation upon interaction of the enzymes with N-arylhydroxamic acids, as well as being metabolic products of arylamine oxidation. Treatment of recombinant NAT2 with the nitrosoarenes, 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), caused rapid and irreversible inactivation of the enzyme by sulfinamide adduct formation with Cys68, but the k(inact)/K(I) values for inactivation of recombinant NAT2 and NAT1 did not indicate significant selectivity for either isoform. Also, the IC(50) values for inactivation of HeLa cell cytosolic NAT1 and NAT2 by 4-NO-BP were similar, as were the IC(50) values obtained with 2-NO-F. Treatment of HeLa cells with low concentrations (1-10 microM) of either 4-NO-BP or 2-NO-F resulted in preferential and more rapid loss of NAT1 activity than NAT2 activity. Because of its wide distribution in human tissues and its early expression in developing tissues, the apparent high sensitivity of intracellular NAT1 to inactivation by reactive metabolites of environmental arylamines may have important toxicological consequences.

Irreversible inhibition of human cathepsins B, L, S and K by hypervalent tellurium compounds

The inhibition of human cysteine cathepsins B, L, S and K was evaluated by a set of hypervalent tellurium compounds (telluranes) comprising both organic and inorganic derivatives. All telluranes studied showed a time- and concentration-dependent irreversible inhibition of the cathepsins, and their second-order inactivation rate constants were determined. The organic derivatives were potent inhibitors of the cathepsins and clear specificities were detected, which were parallel to their known substrate specificities. In all cases, the activity of the tellurane-inhibited cathepsins was recovered by treatment of the inactivated enzymes with reducing agents. The maximum stoichiometry of the reaction between cysteine residues and telluranes were also determined. The presented data indicate that it is possible to design organic compounds with a tellurium(IV) moiety as a novel warhead that covalently modifies the catalytic cysteine, and which also form strong interactions with subsites of cathepsins B, L, S and K, resulting in more specific inhibition.

Irreversible inhibition of dihydrodipicolinate synthase by 4-oxo-heptenedioic acid analogues

We report the synthesis of (2 E,5 E)-4-oxoheptadienedioic acid and (2 E)-4-oxoheptenedioic acid and evaluation of both diester and diacid analogues as inhibitors of bacterial dihydrodipicolinate synthase. Enzyme kinetic studies allowed the determination of second-order rate constants of inactivation; and substrate co-incubation studies have shown the inhibitors act at the active-site. Mass spectrometric analyses have further explored the enzyme–inhibitor interaction and determined the sites of enzyme alkylation.

Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPα)

Dipeptidyl peptidase IV (DPP-IV) and seprase belong to a small group of membrane-bound, proline-specific serine proteases, the serine integral membrane proteases (SIMPs). Whilst DPP-IV is the most exhaustively studied peptidase in this class, relatively less is known about the inhibitor/substrate specificity of its close homolog seprase. Additionally, whereas, DPP-IV expression is largely ubiquitous, seprase expression is restricted to tumour and tissue remodelling sites in vivo. Consequently, the highly restricted expression and distribution of seprase potentially make it an excellent therapeutic target for the modulation of neoplastic invasion and metastasis. Against this background, we now wish to report on the design, synthesis, and kinetic testing of a series of dipeptide proline diphenyl phosphonates, against DPP-IV and seprase. The most potent inhibitor of DPP-IV and seprase was found to be Gly-Pro P(OPh) 2, which exhibited overall second-order rate constants of inactivation of 5.24 × 10 5 M −1 min −1 and 1.06 × 10 4 M −1 min −1 against DPP-IV and seprase, respectively. Both proteases displayed differing profiles of susceptibility towards the other members of the series of inhibitors synthesised. In addition, Gly-Pro P(OPh) 2 and Tyr-Pro P(OPh) 2 were found to exert a considerable, dose-dependent anti-invasive effect on the LOX melanoma cell line, in vitro.

Inhibition of beta-lactamases by monocyclic acyl phosph(on)ates.

The cyclic acyl phosph(on)ates, 1-hydroxy-5-phenyl-2,6-dioxaphosphorinone(3)-1-oxide, its 4-phenyl isomer, and the phosphonate (2-oxo) analogue of the latter inhibited typical class A (TEM-2) and class C (Enterobacter cloacae P99) beta-lactamases in a time-dependent fashion. No enzyme-catalyzed turnover was detected in any case. The interactions occurring were interpreted in terms of the reaction scheme E + I left arrow over right arrow EI left arrow over right arrow EI', where EI is a reversibly formed noncovalent complex, and EI' is a covalent complex. Reactions of the cyclic phosphates with the P99 beta-lactamase were effectively irreversible, while that of the 4-phenyl cyclic phosphate with the TEM beta-lactamase was slowly reversible. The 4-phenyl cyclic phosphate was generally the most effective inhibitor, both kinetically and thermodynamically, with second-order rate constants of inactivation of both enzymes around 10(4) s(-1) M(-1). This compound also bound noncovalently to both enzymes, with dissociation constants of 25 microM from the P99 enzyme and 100 microM from the TEM. It is unusual to find an inhibitor equally effective against the TEM and P99 enzymes; the beta-lactamase inhibitors currently employed in medical practice (e.g., clavulanic acid) are significantly more effective against class A enzymes. The results of lysinoalanine analysis after hydroxide treatment of the inhibited enzymes and of a (31)P nuclear magnetic resonance spectrum of one such complex were interpreted as favoring a mechanism of inactivation by enzyme acylation rather than phosphylation. Molecular modeling of the enzyme complexes of the 4-phenyl phosphate revealed bound conformations where recyclization and thus reactivation of the enzyme would be difficult. The compounds studied were turned over slowly or not at all by acetylcholinesterase and phosphodiesterase I.

Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves by disulfiram

Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents. As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P. aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs. DSF totally inactivated both enzymes in a time- and dose-dependent manner. In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9±0.4 M −1 s −1, whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8±0.6 and 0.33±0.04 M −1 s −1. At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was 1×10 3 M −1 s −1, which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism. Both BADHs were inactivated faster in the presence of NAD(P) + than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme. The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione. The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P) +, together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria.

Studies on the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA: elucidation of the inactivation mechanism and identification of cysteine-114 as the entrapped nucleophile.

The inhibitory properties of (methylenecyclopropyl)formyl-CoA (MCPF-CoA), a metabolite derived from a natural amino acid, (methylenecyclopropyl)glycine, against bovine liver enoyl-CoA hydratase (ECH) were characterized. We have previously demonstrated that MCPF-CoA specifically targets ECHs, which catalyze the reversible hydration of alpha,beta-unsaturated enoyl-CoA substrates to the corresponding beta-hydroxyacyl-CoA products. Here, we synthesized (R)- and (S)-diastereomers of MCPF-CoA to examine the stereoselectivity of this inactivation. Both compounds were shown to be competent inhibitors for bovine liver ECH with nearly identical second-order inactivation rate constants (k(inact)/K(I)) and partition ratios (k(cat)/k(inact)), indicating that the inactivation is nonstereospecific with respect to ring cleavage. The inhibitor, upon incubation with bovine liver ECH, labels a tryptic peptide, ALGGGXEL, near the active site of the protein, where X is the amino acid that is covalently modified. Cloning and sequence analysis of bovine liver ECH gene revealed the identity of the amino acid residue entrapped by MCPF-CoA as Cys-114 (mature sequence numbering). On the basis of gHMQC (gradient heteronuclear multiple quantum coherence) analysis with [3-(13)C]-labeled MCPF-CoA, the ring cleavage is most likely induced by the nucleophilic attack at the terminal carbon of the exomethylene group (C(2)'). We propose a plausible inactivation mechanism that involves relief of ring strain and is consistent with examples found in the literature. In addition, these studies provide important clues for future design of more efficient and selective inhibitors to control and/or regulate fatty acid metabolism.

Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi.

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.

Peptidyl inverse esters of p-methoxybenzoic acid: a novel class of potent inactivator of the serine proteases

A series of novel synthetic peptides, containing a C-terminal beta-amino alcohol linked to p-methoxybenzoic acid via an ester linkage, have been prepared and tested as inhibitors against typical members of the serine protease family. For example, the sequences Ac-Val-Pro-NH-CH-(CH2-C6H5)-CH2O-CO-C6H4-OCH3 (I) and Ac-Val-Pro-NH-CH-[CH-(CH3)2]-CH2O-CO-C6H4-OCH3 (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and elastase respectively, have been found to behave as exceptionally potent irreversible inactivators of their respective target protease. Thus I was found to inactivate chymotrypsin with an overall second-order rate constant (k2/Ki) of approx. 6.6x10(6) M-1. s-1, whereas II is an even more potent inactivator of human neutrophil elastase, exhibiting a second-order rate constant of inactivation of approx. 1.3x10(7) M-1.s-1. These values represent the largest rate constants ever reported for the inactivation of these proteases with synthetic peptide-based inactivators. On prolonged incubation in substrate-containing buffers, samples of the inactivated proteases were found to regain activity slowly. The first-order rate constants for the regeneration of enzymic activity from chymotrypsin and human neutrophil elastase inactivated by I and II respectively were determined to be approx. 5.8x10(-5) s-1 and approx. 4.3x10(-4) s-1. We believe that the most likely mechanism for the inactivation and regeneration of enzymic activity involves the formation and subsequent slow hydrolysis of long-lived acyl enzyme intermediates.

Highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized DNA-valine phosphonate library.

We incorporated a phosphonate irreversible inhibitor of neutrophil elastase into a randomized DNA library and, using the SELEX process, iteratively selected these assemblies for the most potent elastase inhibitors. The inhibitors were selected against purified elastase and against secreted elastase in the presence of activated neutrophils. Very active aptamer inhibitors were obtained by both methods, with second-order rate constants for inactivation of human neutrophil elastase ranging (1-3) x 10(8) M(-1) min(-1). These rates exceed those of any reported irreversible inhibitor of elastase and exceed the previous best phosphonate inhibitors by 80-fold. The selected inhibitors are also significantly more potent than alpha-1 proteinase inhibitor in blocking degradation of elastin by activated neutrophils. In contrast to a previous experiment [Smith et al. (1995) Chem. Biol. 2, 741-750], a single-enantiomer form of the valyl phosphonate was used rather than a racemic mixture. Our analysis shows that this use of a chirally resolved valyl phosphonate results in selection of much more potent inhibitors and that these inhibitors specifically potentiate a single enantiomeric form of the phosphonate.

Distinct Properties of Prohormone Thiol Protease (PTP) Compared to Cathepsins B, L, and H: Evidence for PTP as a Novel Cysteine Protease

The prohormone thiol protease (PTP) has been demonstrated as a major processing enzyme involved in converting the enkephalin precursor to active opiate enkephalin peptides. In this report, PTP was distinguished from other mammalian cysteine proteases, cathepsins B, L, and H, with regard to selectivity for monobasic and paired basic residue-containing peptide-MCA substrates, particular types of proteolytic activities, and sensitivity to active-site-directed peptide inhibitors. PTP cleaved at both COOH- and NH 2-terminal sides of basic residues within peptide-MCA substrates containing monobasic and dibasic sites. PTP showed greatest activity with Z-Phe-Arg-MCA and Bz-Val-Leu-Lys-MCA, and lower levels of activity with peptide substrates containing the paired basic residues Arg-Arg, Lys-Arg, and Lys-Lys. Cathepsins B and L also cleaved monobasic and paired basic residues at both COOH- and NH 2-terminal sides of basic residue(s), but differed from PTP in their preference for the same series of peptide-MCA substrates. Cathepsin H appeared to prefer cleaving at the NH 2-terminal side of basic residues. PTP showed no aminopeptidase activity which is known for cathepsin H. Also, PTP does not exhibit dipeptidylcarboxypeptidase activity, a property of cathepsin B; PTP demonstrates no elastinolytic activity, a characteristic of cathepsin L. Importantly, the sensitivity of PTP to active-site-directed peptide diazomethane inhibitors and E-64c differs from that of cathepsins B, L, and H. E-64c, a selective cysteine protease inhibitor, was the most effective inhibitor of PTP with a second-order rate constant of inactivation, k 2, of 6,710,000 M −1 s −1. These biochemical properties of PTP distinguish it from cathepsins B, L, and H, providing further support for PTP as a novel member of the family of cysteine proteases.