Design, synthesis and biological evaluation of 1,5-disubstituted α-amino tetrazole derivatives as non-covalent inflammasome-caspase-1 complex inhibitors with potential application against immune and inflammatory disorders.

Compounds targeting the inflammasome-caspase-1 pathway could be of use for the treatment of inflammation and inflammatory diseases. Previous caspase-1 inhibitors were in great majority covalent inhibitors and failed in clinical trials. Using a mixed modelling, computational screening, synthesis and in vitro testing approach, we identified a novel class of non-covalent caspase-1 non cytotoxic inhibitors which are able to inhibit IL-1ß release in activated macrophages in the low µM range, in line with the best activities observed for the known covalent inhibitors. Our compounds could form the basis of further optimization towards potent drugs for the treatment of inflammation and inflammatory disorders including also dysregulated inflammation in Covid 19.

Modeling caspase-1 inhibition: Implications for catalytic mechanism and drug design.

The metabolic product of caspase-1, IL-1ß, is an important mediator in inflammation and pyroptosis cell death process. Alzheimer's disease, septic shock and rheumatoid arthritis are IL-1ß mediated diseases, making the caspase-1 an interesting target of pharmacological value. Many inhibitors have been developed until now, most of them are peptidomimetic with improved potency. In the present study, all-atom molecular dynamics simulations and the MM/GBSA method were employed to reproduce and interpret the results obtained by in vitro experiments for a series of inhibitors. The analysis shows that the tautomeric state of the catalytic His237 impact significantly the performance of the prediction protocol, providing evidence for a His237 tautomeric state different to the proposed in the putative mechanism. Additionally, analysis of inhibitor-enzyme interactions indicates that the differences in the inhibitory potency of the tested ligands can be explained mainly by the interaction of the inhibitors with the S2-S4 protein region. These results provide guidelines for subsequent studies of caspase-1 catalytic reaction mechanism and for the design of novel inhibitors.

Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide.

The synthetic tellurium compound, AS101, is a novel inhibitor of IL-1beta converting enzyme.

The organotellurium compound, trichloro(dioxoethylene-O,O') tellurate (AS101) has been shown previously to exert diverse biologic activities both in vitro and in vivo. This compound was recently found to react with thiols and to catalyze their oxidation. This property of AS101 raises the possibility that it may serve as a cysteine protease inhibitor. In the present study, using a substrate-specific enzymatic assay, we show that treatment of caspase-1 (interleukin-1beta [IL-1beta] converting enzyme [ICE]) with AS101 inhibits its enzymatic activity in a dose-dependent manner. Moreover, the results show that AS101 treatment causes a significant reduction in the active form of IL-18 and IL-1beta in peripheral blood mononuclear cells (PBMC) and in human HaCat keratinocytes. We further demonstrate that the inhibitory effect of AS101 does not involve nitric oxide (NO) or interferon-gamma (IFN-gamma), two possible regulators of IL-18 production, and does not occur at the mRNA level, suggesting a posttranscriptional mechanism of action. More importantly, AS101 downregulates IL-18 and IL-1beta serum levels in a mouse model of lipopolysaccharide (LPS)-induced sepsis, resulting in increased survival. Recent studies emphasize the pathophysiologic role of IL-18 and IL-1beta in a variety of inflammatory diseases. Thus, their blockage by the nontoxic compound, AS101, currently used in clinical studies, may provide clinical advantage in the treatment of these diseases.

Synthesis and evaluation of unsaturated caprolactams as interleukin-1beta converting enzyme (ICE) inhibitors.

Peptidomimetic compounds possessing a caprolactam ring constraint were prepared and evaluated as interleukin-1beta converting enzyme (ICE) inhibitors. The caprolactam ring was used to constrain the P3 region of our inhibitors. This strategy proved to be effective for the synthesis of ICE inhibitors, maintaining key hydrogen bond interactions with the enzyme and invoking a preferred conformation for binding. Several compounds exhibited IC(50) values less than 10nM in a caspase-1 enzyme assay and less than 100nM in a THP-1 whole cell assay measuring IL-1beta production. Two compounds, 13c and 13j, were found to have good oral bioavailability (>50%) in rats when administered as prodrugs.

Total synthesis of the interleukin-1beta converting enzyme inhibitor EI-1941-2 using tandem oxa-electrocyclization/oxidation1.

[reaction: see text] The total synthesis of the interleukin-1beta converting enzyme inhibitor EI-1941-2 was achieved utilizing tandem oxidation/oxa-electrocyclization/oxidation to access a key alpha-pyrone intermediate. Support for the tandem reaction mechanism was obtained by evaluation of a stepwise oxidation protocol.

The design and synthesis of sulfonamides as caspase-1 inhibitors.

A series of sulfonamides (1) has been prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase 1. These compounds were designed to improve potency by rigidifying the enzyme bound molecule through an intramolecular hydrogen bond. An X-ray crystal structure of a representative member of this series bound to the active site of ICE, confirms the presence of the hydrogen bonding interaction.

Synthesis and biological properties of the seven alanine-modified analogues of the marine cyclopeptide hymenamide C.

The synthesis and biological activity of the marine cyclopeptide hymenamide C(1), showing an inhibitory effect on human neutrophil elastase degranulation release, were recently described. Based on this result, it was decided to undertake a systematic structure-activity relationship study of this cyclopeptide, based on the Ala-scan technique, in order to obtain useful information for the rational design of additional analogues. The synthesis and characterization of the seven Ala modified analogues are reported and their biological and pharmacological properties are described.

Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity.

BACKGROUND: Molecular diversity in nature is developed through a combination of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering tool. It combines the ability to introduce non-natural amino acids into a protein using native chemical ligation with exhaustive targeted mutagenesis of the protein via phage-display mutagenesis. RESULTS: A fully functional biosynthetic version of the protease inhibitor eglin c was constructed. The amino-terminal fragment (residues 8-40) was chemically synthesized with a non-natural amino acid at position 25. The remaining carboxy-terminal fragment was expressed as a 30-residue peptide extension of gIIIp or gVIIIp on filamentous phage in a phage-display mutagenesis format. Native chemical ligation was used to couple the two fragments and produced a protein that refolded to its active form. To facilitate the packing of the introduced non-natural amino acid, residues 52 and 54 in the carboxy-terminal fragment were fully randomized by phage-display mutagenesis. Although the majority of the observed solutions for residues 52 and 54 were hydrophobic - complementing the stereochemistry of the introduced non-natural amino acid - a significant number of residues (unexpected because of stereochemical and charge criteria) were observed in these positions. CONCLUSIONS: Peptide synthesis and phage-display mutagenesis can be combined to produce a very powerful protein engineering tool. The physical properties of the environment surrounding the introduced non-natural residue can be selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.

Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis.

Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stabilized primarily by disulfide bridges flanking the reactive-site peptide bond, eglin c possesses an enzyme-binding loop supported predominantly by extensive electrostatic/H-bonding interactions involving three Arg residues (Arg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As an adjacent residue, the C-terminal Gly70 participates in these interactions via its alpha-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H-bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the structural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH(2)CH(2)CONH(2)) or by alpha-hydroxylacetamide (HOCH(2)CONH(2)). NMR analysis indicated that the core structure of eglin c was maintained in the analogues, but that the binding loop was significantly perturbed. It was found that deletion or replacement of Gly70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in melting temperature. As a result, these inhibitors become substrates for their target enzymes. Binding assays on these analogues with a catalytically incompetent subtilisin BPN' mutant indicated that loss or weakening of the interactions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic inhibitors, the relative free energy changes (DeltaDeltaG) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0. 9996) with the DeltaDeltaG values derived from a decreased binding to the enzyme.

An efficient stereoselective synthesis of [3S(1S,9S)]-3-[[[9-(benzoylamino) octahydro-6,10-dioxo-6H-pyridazino-(1,2-a)(1,2)-diazepin-1-yl]-carbonyl ]amino]-4-oxobutanoic acid, an interleukin converting enzyme (ICE) inhibitor.

The title compound 1 is a potent interleukin-1beta-converting enzyme (ICE) inhibitor. Recently, an efficient chiral synthesis of compound 1 has been accomplished in our labs. The overall yield of this 18-step stereoselective synthesis was 9.8%.

Design of selective eglin inhibitors of HCV NS3 proteinase.

Hepatitis C virus (HCV) infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals, estimated to be 100-200 million worldwide. Unfortunately, immunotherapy or other effective treatments for HCV infection are not yet available, and interferon administration has limited efficacy. Different approaches to HCV therapy are being explored, and these include inhibition of the viral proteinase, helicase, and RNA-dependent RNA polymerase and development of a vaccine. Here we present the design of selective inhibitors with nanomolar potencies of HCV NS3 proteinase based on eglin c. These eglin c mutants were generated by reshaping the inhibitor active site-binding loop, and the results emphasize the role played by residues P5-P4' in enzyme recognition. In addition, alanine scanning experiments provide evidence that the N terminus of eglin c also contributes to NS3 binding. These eglin inhibitors offer a unique tool for accurately assessing the requirements for effective inhibition of the enzymatic activity of NS3 and at the same time can be considered lead compounds for the identification of other NS3 inhibitors in targeted design efforts.

Amino acids and peptides. XXXVII. Synthesis of stereoisomeric nonapeptides corresponding to sequence 41-49 of eglin c and examination of their inhibitory activity against human leukocyte cathepsin G and alpha-chymotrypsin.

A nonapeptide, H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OH, corresponding to sequence 41-49 of eglin c inhibited leukocyte cathepsin G and alpha-chymotrypsin with Ki values of 2.2 x 10(-5) and 7.2 x 10(-6) M, respectively, although eglin c itself inhibited leukocyte elastase, cathepsin G and alpha-chymotrypsin with Ki values of 6.0 x 10(-9), 5.5 x 10(-9) and 2.5 x 10(-9) M, respectively. The inhibitory activity of the nonapeptide decreased following incubation with cathepsin G due to the cleavage of the Leu45-Asp46 peptide bond. Therefore, Leu45 and/or Asp46 were replaced with D-amino acids and the inhibitory activities of the resultant nonapeptides were examined. Their inhibitory activities against cathepsin G and alpha-chymotrypsin were much weaker than those of the all-L-type nonapeptide, suggesting that the amino acids at the active site, Leu45 and Asp46 are required to be in the L-configuration for potent activity.

Synthetic peptide inhibitors of complement serine proteases--III. Significant increase in inhibitor potency provides further support for the functional equivalence hypothesis.

Synthetic peptides based on functionally equivalent (as defined by similar patterns of chemically equivalent amino acids) serine protease inhibitor (serpin) C-terminal sequences inhibit both classical and alternative pathways of complement activation. Inhibition was also found with hybrid peptides consisting of the cleavage site of one serpin (antithrombin III, alpha-1-antitrypsin, or antichymotrypsin) attached to the short and long functionally equivalent protease binding cores of the other two serpins. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to the long binding core of antithrombin III was selective in inhibiting the classical pathway with no effect on the alternative pathway at a concn of 10 microM. Extension of the functional equivalence hypothesis has produced inhibitors of complement activation named generic and generic +, whose sequences differ by 77% or 87%, respectively, from those of all three serpin sequences. A hybrid peptide composed of the antithrombin III cleavage site attached to the generic peptide is an inhibitor of complement activation at 500 nM, the most potent inhibitor found in this study.