N. Boggetto

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

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

Inhibition of human leukocyte elastase by functionalized N-aryl azetidin-2-ones: substituent effects at C-3 and benzylic positions

Summary A series of functionalized N -aryl azetidin-2-ones with a latent alkylating group was prepared by a flexible four-step synthesis. They met criteria expected for a suicide-type inactivation of human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE), with no inactivation of trypsin- and chymotrypsin-like proteases. The inhibition potency was dependent on the halogen substituents at C-3 (F, F; Cl, Cl; Br, Br) and the nature and the position relative to nitrogen of the latent benzylic leaving group (F, Cl, Br). Better inactivations of HLE compared with PPE were observed with azetidinones gem -disubstituted by Cl and Br rather than by F. Their protio analogs, which are devoid of the latent quinoniminium methide electrophile, behave as simple substrates of elastases.

Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors

A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

The yeast metacaspase is implicated in oxidative stress response in frataxin-deficient cells.

Friedreich ataxia is the most common recessive neurodegenerative disease and is caused by reduced expression of mitochondrial frataxin. Frataxin depletion causes impairment in iron–sulfur cluster and heme biosynthesis, disruption of iron homeostasis and hypersensitivity to oxidants. Currently no pharmacological treatment blocks disease progression, although antioxidant therapies proved to benefit patients. We show that sensitivity of yeast frataxin‐deficient cells to hydrogen peroxide is partially mediated by the metacaspase. Metacaspase deletion in frataxin‐deficient cells results in recovery of antioxidant capacity and heme synthesis. In addition, our results suggest that metacaspase is associated with mitochondrial respiration, intracellular redox control and genomic stability.

Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells

Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15–30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H2O2. On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.

Synthesis and inhibition of human leucocyte elastase by functionalized N-aryl azetidin-2-ones: effect of different substituents on the aromatic ring.

N‐aryl‐3,3‐difluoroazetidin‐2‐ones featured by a latent electrophilic methylene quinoniminium function have been synthesized and evaluated as inhibitors of human leucocyte elastase.

Evaluation of the Inhibitory Activity on Serine and Aspartic Proteases of 4-Amino-4H-1,2,4-triazole and 5-Aminothiazole Derivatives Structurally Related to β-Lactam Antibiotics

Abstract— Twenty new derivatives of 4‐amino‐4H‐1,2,4‐triazole and 5‐aminothiazole have been examined for their inhibitory potential towards serine and aspartic proteases. Upon prolonged incubation with enzyme, the phenylacetylaminothiazolium salts exhibit progressive, time‐dependent inhibition of chymotrypsin according to a first‐order process. The formation of a tetrahedral transition state‐like complex by attack of the active‐site serine at the C2‐position of the pseudobase form of the thiazolium may be responsible for the observed effect. Triazolium salts appeared to be simple competitive inhibitors of this enzyme, effective in the Mm range concentration. Poor inhibitions of trypsin and pepsin were also obtained in the triazolium series. In spite of their structural analogy with β‐lactams, the selected derivatives failed to inhibit human leucocyte elastase.

AZT and AZT-monophosphate Prodrugs Incorporating HIV-protease Substrate Fragment: Synthesis and Evaluation as Specific Drug Delivery Systems

With the view to deliver anti-HIV nucleoside and nucleoside-monophosphate (MP) analogues specifically into HIV-infected cells, we synthesized a series of ester and phosphoramidate peptide conjugates of zidovudine (AZT) and of AZT-MP, respectively, wherein the peptide sequences derive from a HIV-protease (PR) hydrolysable substrate. Their in vitro stability with respect to hydrolysis, anti-HIV activity and cytotoxicity, and ability to inhibit the HIV-PR activity were investigated. Concerning the ester AZT-peptide conjugates, their antiviral activity level in thymidine kinase-expressing (TK+) CEM-SS and MT-4 cells was in most cases closely correlated to their hydrolysis rate: the faster the hydrolysis, the closer the anti-HIV activity to that of AZT. None of them was a HIV-PR substrate, indicating that their antiviral activity was not related to their intracellular hydrolysis by this enzyme. None of them inhibited HIV in TK-deficient (TK−) CEM cells, demonstrating that they probably act as prodrugs of AZT. Most of the phosphoramidate peptide conjugates of AZT-MP were rapidly degraded in a physiological buffer into several metabolites including AZT. Their anti-HIV activity in TK+ CEM-SS and MT-4 cells was much lower than that of AZT, indicating that only low amounts of AZT or AZT-MP were released into cells during incubation. Antiviral activities measured on TK− CEM cells for some phosphoramidates suggest that low amounts of AZT-MP could be released intracellularly. However, this AZT-MP release was not initiated by a HIV-PR hydrolysis, as no evidence for peptide cleavage was obtained by HPLC analysis of one representative compound after incubation with HIV-PR.

Progress towards new conformationally constrained HIV-1 protease inhibitors

Summary Two series of molecules containing a trisubstituted cyclopentyl group as the central unit were synthesized and evaluated as inhibitors of HIV-1 protease (HIV PR). In the first series of molecules ( 13–20 ), the central unit A, 3-( N -acyl)amino-2-hydroxy-1-cyclopentylacetyl, was designed so as to reproduce three of the central interactions found in the ‘classical’ complex HIV PR-JG365 inhibitor. Significant inhibitions (IC 50 ∼ 10 μM) were obtained with compound 20 in which the central unit was elongated by Z-Ile-Phe at the N-terminus and by Val-OMe at the C-terminus. In the second series of molecules ( 21–28 ), the central unit B, 3-hydroxy-2-( N -acyl)amino-1-cyclopentylacetyl, was obtained in the first steps of the synthesis. Unexpectedly better inhibitions were observed with these derivatives ( K i = 2 μM for compound 28 ). Docking and molecular dynamics simulations performed with compound 28 into HIV PR suggested that the HIV PR- 28 complex should have a structure analogous to that of the recently described HIV PR-urea complex.

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.