Calpain inhibitors based on the quiescent affinity label concept: high rates of calpain inactivation with leaving groups derived from N-hydroxy peptide coupling reagents.

A series of irreversible inhibitors of recombinant calpain has been synthesized and their rates of inactivation have been evaluated against calpain and cathepsin B, respectively. The design of the inhibitors was based on the quiescent affinity label concept. By choosing the appropriate affinity group and by employing leaving groups derived from N-hydroxy coupling reagents, good inhibitors of calpain with high rates of inactivation have been identified. However, poor aqueous stability limits their therapeutic utility.

P2-achiral, P'-extended alpha-ketoamide inhibitors of calpain I.

A series of potent P2-achiral, P'-extended alpha-ketoamide inhibitors of calpain I is described.

P'-extended alpha-ketoamide inhibitors of proteasome.

A series of potent P'-extended alpha-ketoamide inhibitors of chymotrypsin-like activity of proteasome is described.

A sensitive, continuously recording fluorogenic assay for calpain.

We have developed a sensitive and continuously recording fluorogenic assay for the thiol protease calpain. This assay uses the dipeptide substrate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA) in Tris buffer (pH 7.5) in the presence of 0.1% CHAPS. The assay is linear over a wide range of enzyme concentration and is capable of detecting 10 picomolar calpain making it more sensitive than any previously published method. Moreover, this assay gives a rate that is linear for over ten minutes making it useful for mechanistic studies of inhibitors. This assay can be easily adapted to a 96-well plate format facilitating the large scale screening of inhibitors.

P2-proline-derived inhibitors of calpain I.

The syntheses and biological activities of a series of calpain I inhibitors, derived from D- and L-Pro, are described.

Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Lead development: validation and application of high throughput screening for determination of pharmacokinetic parameters for enzyme inhibitors.

An approach utilizing robotics (automation) for the rapid and reliable determination of protease inhibitor concentration in rat plasma samples is described. The bioassay protocol using an immobilized peptide substrate allows high sample throughput, compatible with parallel synthesis/SAR development strategy.

Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

Inhibition of mature IL-1 beta production in murine macrophages and a murine model of inflammation by WIN 67694, an inhibitor of IL-1 beta converting enzyme.

The proinflammatory cytokine IL-1 beta is synthesized by activated monocytes and macrophages as a 31-kDa, biologically inactive precursor that is proteolytically processed to the biologically active 17-kDa mature molecule by the IL-1 beta converting enzyme (ICE). WIN 67694, Z-Val-Ala-Asp-CH2O(CO)[2,6-(CI2)]Ph, is a potent, selective inhibitor of human ICE. In activated murine peritoneal macrophages, WIN 67694 inhibited the release of mature IL-1 beta with an IC50 of 1.8 microM without any effect on the release of IL-1 alpha, IL-6, or TNF-alpha. The effect was specific to mature IL-1 beta release; the ICE inhibitor did not effect IL-1 beta RNA levels or precursor protein synthesis. In vivo, WIN 67694 was also able to inhibit selectively the release of IL-1 beta in a dose-dependent manner in a subcutaneous tissue chamber implant model of inflammation. IL-1 beta levels in tissue chamber fluid were inhibited 35 and 55% at 10 and 100 mg/kg, respectively. IL-1 alpha, IL-6, and TNF-alpha levels were not affected. The ability to selectively inhibit mature IL-1 beta release in vivo with ICE inhibitors will allow for detailed studies of the role of IL-1 beta and ICE in inflammatory diseases.

Preparation and evaluation of peptidic aspartyl hemiacetals as reversible inhibitors of interleukin-1 beta converting enzyme (ICE).

Aspartyl aldehyde, Ac-Tyr-Val-Ala-Asp-H 1 (L-709,049), has been reported to be a potent, reversible inhibitor of interleukin-1 beta converting enzyme (ICE) [Thornberry, N.A. et al. (1992) Nature (London) 356, 768-774]. In the context of our own work, we have developed a general synthetic approach to peptidic aspartyl aldehydes. Semicarbazone derivative, H-Asp(Ot-Bu)-Sc 4, was identified as a stable, masked aspartyl aldehyde equivalent. We have used 4 to synthesize a series of mono-, di- and tripeptide aldehydes, and multigram quantities of Ac-Tyr-Val-Ala-Asp-H 1, Ac-Tyr-Val-Lys-Asp-Sc 21 and Ac-Tyr-Val-Lys-Asp-H 2. Biological evaluation of these aspartyl aldehydes and derivatives suggests that the tripeptide scaffold, Z-Val-Ala-Asp, is a peptide scaffold that retains good potency and selectivity for ICE.

sigma recognition sites in brain and peripheral tissues. Characterization and effects of cytochrome P450 inhibitors.

Binding to sigma sites in subcellular fractions of brain and in crude homogenates from peripheral tissues of the guinea pig was characterized with the [3H]ligands (+)pentazocine and di(2-tolyl)guanidine (DTG). The inhibitory effects of representative sigma compounds and cytochrome P450 inhibitors were evaluated in guinea pig tissues, and the effects of cytochrome P450 induction on sigma binding in the rat were investigated. For both ligands, the majority of sites were localized to the microsomal fractions. The KD values for [3H](+)pentazocine- or [3H]DTG-labeled sigma sites in guinea pig liver and testes were 2-fold lower than those in brain and heart. The number of sites labeled by [3H](+)pentazocine varied, with an order of liver > testes > brain > heart. In contrast, the Bmax values for [3H]DTG-defined sigma sites were greatest in testes, followed by liver, brain and heart. The rank order of potency for representative sigma and P450 compounds was similar in brain, liver and testes for both [3H]ligands, and the potency of selective compounds to displace sigma binding in guinea pig liver failed to correlate with their abilities to inhibit cytochrome P450IID1 activity in human liver. Following induction of cytochrome P450IIB1 with phenobarbital or cytochrome P450IA1 with beta-naphthoflavone, neither the affinity nor the number of sigma sites was altered in rat brain or liver. These results suggest that sigma sites in the periphery are similar to those in the brain, and that the sigma binding site is not identical with cytochrome P450IIB1, P450IA1 or P450IID1.

Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, and catalyzes the conversion of nucleotides to deoxynucleotides. The mechanism of inactivation of RDPR by 2'-azido-2'-deoxynucleoside 5'-diphosphate (N3UDP) has been examined using a variety of isotopically labeled derivatives: (1'-, 2'-, 3'-, or 4'-[2H])-N3UDPs and 2'-[15N3, 13C]-N3UDP. Electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopy studies using these compounds indicate that the 2' carbon-nitrogen bond to the azide moiety is cleaved prior to or upon formation of the nitrogen-centered radical derived from the azide moiety of N3UDP. EPR studies reveal no hyperfine interactions of the nitrogen-centered radical with the 1', 2', 3', or 4' hydrogens of N3UDP. ESEEM studies however, reveal that the 1' and 4' deuterons are 3.3 +/- 0.2 and 2.6 +/- 0.3 A, respectively, from the nitrogen-centered radical. Further support for carbon-nitrogen bond cleavage is derived from studies of the interaction of oxidized R1, C225SR1, and C462SR1 with R2 and N3UDP. In all three cases, in contrast to the results with the wild type R1, azide is detected. Nitrogen-centered radical is not observed with either oxidized R1 or C225SR1 but is observed with C462SR1. These results suggest that C225 is required for the conversion of azide into N2 and a nitrogen-centered radical. The dynamics of the inactivation of RDPR by N3UDP have also been examined. Use of [3'-2H]N3UDP reveals an isotope effect of approximately 2 on the loss of the tyrosyl radical and the rate of inactivation of RDPR. In both cases the kinetics are complex, suggesting multiple modes of inactivation. In addition, several modes of inactivation are required to explain the observation that loss of the tyrosyl radical is slower than the rate of inactivation. Studies using [5'-3H]N3UDP reveal that the rapid inactivation is the result of the formation of a tight noncovalent complex between modified nucleotide, nitrogen-centered radical and RDPR. Destruction of the nitrogen-centered radical is a slow process which appears to be accompanied by decomposition of the modified nucleotide into PPi, uracil, and 2-methylene-3(2H)-furanone. The latter covalently modifies R1 and ultimately leads to loss of approximately 50% of the activity of R1.