Reik Löser

Azadipeptide nitriles: highly potent and proteolytically stable inhibitors of papain-like cysteine proteases.

(Chemical Presented) Nitrogen instead of carbon: Azadipeptide nitriles resulting from CH/N exchange in the P position (see picture) are hitherto unknown. To access these compounds by conversion of amino acid-derived hydrazides with cyanogen bromide both nitrogen atoms of the hydrazide must be substituted. Despite a methylated P-P peptide bond, the azadipeptide nitriles show a strong inhibitory activity against cysteine proteases, and a high stability towards chymotryptic hydrolysis.

Noncovalent Tripeptidyl Benzyl- and Cyclohexyl-Amine Inhibitors of the Cysteine Protease Caspase-1

Potent and noncovalent inhibitors of caspase-1 were produced by incorporating a secondary amine (reduced amide) isostere in place of the conventional electrophile (e.g., aldehyde) that normally confers high potency to cysteine protease inhibitors. Benzyl- or cyclohexylamines produced potent, reversible, and competitive inhibitors that were selective for caspase-1 (e.g., K(i) = 47 nM) over caspases 3 and 8 with minimal cytotoxicity. Unlike most cysteine protease inhibitors, these compounds do not react covalently and indiscriminately with thiols.

Antimalarial activity of azadipeptide nitriles

Azadipeptide nitriles-novel cysteine protease inhibitors-display structure-dependent antimalarial activity against both chloroquine-sensitive and chloroquine-resistant lines of cultured Plasmodium falciparum malaria parasites. Inhibition of parasites hemoglobin-degrading cysteine proteases was also investigated, revealing the azadipeptide nitriles as potent inhibitors of falcipain-2 and -3. A correlation between the cysteine protease-inhibiting activity and the antimalarial potential of the compounds was observed. These first generation azadipeptide nitriles represent a promising new class of compounds for antimalarial drug development.

Microplate assay for quantitative determination of cathepsin activities in viable cells using derivatives of 4-methoxy-β-naphthylamide

A method is described allowing the selective determination of four cathepsins (B, H, K, and L) in live cells. Adherently growing cells are incubated with partially selective substrates for each cathepsin (peptidic derivatives of 4-methoxy-beta-naphthylamine) in microtiter plates together with nitrosalicylaldehyde. Using an appropriate reader accumulating fluorescent products may be detected continously or by end point measurement. Selectivity is achieved by running parallel assays containing inhibitors that are partially selective for each of the cathepsins (in case of cathepsin H, the nonlysosomal aminopeptidases are inhibited by bestatin). Individual cathepsin activities can then be calculated by the difference between the uninhibited and the inhibited assay. The method was validated by measurements in cells isolated from cathepsin B(-/-)-, K(-/-)-, and L(-/-)- mice. This strategy suggests that the combination of two partially selective reaction partners, substrate and inhibitor can yield selective cathepsin assays.

Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors

Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low‐molecular‐weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent–reversible inhibitors, was performed to draw structure–activity relationships for the non‐primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N‐(4‐phenylbenzoyl)‐leucylglycine nitrile) and 12 (N‐(4‐phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values <10 nM. With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure–activity relationships for this series of cathepsin F inhibitors.

Dipeptide-derived nitriles containing additional electrophilic sites: Potentially irreversible inhibitors of cysteine proteases

Heterocyclic and open-chain dipeptide-derived nitriles have been synthesized, containing an additional electrophilic center enabling the subsequent covalent modification of the thioimidate nitrogen formed in situ at the active site of the enzyme. The inhibitory potential of these nitriles against the cysteine proteases papain and cathepsins L, S, and K was determined. The open-chain dipeptide nitriles 8 and 10 acted as moderate reversible inhibitors, but no evidence for an irreversible inhibition of these enzymes was discernable.

Cathepsin B: Active Site Mapping with Peptidic Substrates and Inhibitors

The potential of papain-like cysteine proteases, such as cathepsin B, as drug discovery targets for systemic human diseases has prevailed over the past years. The development of potent and selective low-molecular cathepsin B inhibitors relies on the detailed expertise on preferred amino acid and inhibitor residues interacting with the corresponding specificity pockets of cathepsin B. Such knowledge might be obtained by mapping the active site of the protease with combinatorial libraries of peptidic substrates and peptidomimetic inhibitors. This review, for the first time, summarizes a wide spectrum of active site mapping approaches. It considers relevant X-ray crystallographic data and discloses propensities towards favorable protein-ligand interactions in case of the therapeutically relevant protease cathepsin B.

Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290–319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290–319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290–319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

Corrigendum: Synthesis and Kinetic Characterisation of Water-Soluble Fluorogenic Acyl Donors for Transglutaminase 2.

Scheme 5. Kinetic model for substrate hydrolysis by TGase 2 and the corresponding differential rate laws derived from it. The kinetic model was prepared according to ref. [32]. For all calculations, rate constants kacyl and kdeacyl were combined to rate constant kcat accordkcat = kacyl V kdeacyl/(kacyl + kdeacyl). Abbreviations used in the scheme. E: enzyme. SL: substrate (fluorogenic acyl donor). ESL: initial enzyme·substrate complex. L: coumarin derivative. E–S: acylated enzyme intermediate. P: product.