Maxim Frizler

Structural Optimization of Azadipeptide Nitriles Strongly Increases Association Rates and Allows the Development of Selective Cathepsin Inhibitors

Using the example of cathepsin K, we demonstrate the design of highly potent and selective azadipeptide nitrile inhibitors. A systematic scan with respect to P2 and P3 substituents was carried out. Structural modifications strongly affected the enzyme-inhibitor association (but not dissociation) rate. A combination of optimized P2 and P3 substituents with a methylation of the P3-P2 amide linker resulted in the picomolar cathepsin K inhibitor 19 with remarkable selectivity over cathepsins L, B, and S.

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.

Facing the gem-dialkyl effect in enzyme inhibitor design: preparation of homocycloleucine-based azadipeptide nitriles.

Cathepsin K belongs to the subfamily of papain-like cysteine proteases and plays an important role in the (patho)physiological processes of bone remodeling. Therefore, cathepsin K represents a promising target for the development of new therapeutic strategies for the treatment of osteoporosis and related diseases. The known low-molecular-weight inhibitors of cathepsin K are mainly peptidomimetic structures containing an electrophilic group to allow covalent interaction with the active-site cysteine. Among such inhibitors, nitrile-derived compounds attracted the most attention in various drug-development programs. The fact that the structural combination of a large P3 substituent with 1amino-1-cyclohexanecarboxylic acid (homocycloleucine) at the P2 position is preferable for the selective inhibition of cathepsin K has also been explored. Recently, azadipeptide nitriles have been introduced as a new inhibitor class for papain-like cysteine proteases, which exhibited Ki values toward human cysteine cathepsins L, S and K in the picomolar range. The nitrogen atoms of the aza-amino nitrile moiety of such inhibitors are essentially alkylated for reasons of synthetic access. Using the example of cathepsin K, it was further demonstrated that the structural optimization of azadipeptide nitriles increases the enzyme–inhibitor association rates and allows the development of selective cathepsin inhibitors. Moreover, falcipaininhibiting azadipeptide nitriles with modified P1 residues displayed antimalarial activity against Plasmodium falciparum. Recently, organelle-specific drug-delivery systems for azadipeptide nitriles were reported. Although azadipeptide nitriles containing a leucine residue at the P2 position could be easily obtained, the synthesis of homocycloleucine-derived counterparts is considerably more difficult. In this study, we discuss the gem-dialkyl effect as the reason for an enhanced tendency to undergo cyclization reactions, which, along with the decreased reactivity of homocycloleucine, complicate the synthesis of the target compounds. However, herein we present a successful synthetic route to the first azadipeptide nitriles with homocycloleucine at the P2 position. The preparation of a first pair of dipeptide and azadipeptide inhibitors with homocycloleucine at the P2 position, that is, compounds 2 and 4, is shown in Scheme 1. The dipeptide nitrile 2 was synthesized by the reaction of the carbobenzyloxy (Cbz)-protected homocycloleucine with aminoacetonitrile by a mixed anhydride. Compound 2 was obtained after chromatographic separation in a moderate yield of 53 %. The carbamate 2.1 was isolated as the main byproduct, as a result of nucleophilic attack of the aminoacetonitrile at the carbonate carbon of the mixed anhydride. When compound 1 was treated, by the same procedure, with 1,2dimethylhydrazine, the desired dimethylhydrazide 3 was formed in a yield of only 18 %. The carbazate 3.1 was identi-

α,β → β,γ double bond migration in corallopyronin A biosynthesis

In polyketide biosynthesis the reduction of β-carbonyl groups to an alkene usually results in a α,β double bond. However, in a few antibiotics the rare case of such a carbon–carbon double bond in β,γ position is observed. The in vivo active antibiotic corallopyronin A represents such a molecule, whereby a α,β → β,γ double bond migration takes place during the assembly of the molecule. Here we report the in vitro analysis of the enzyme domain responsible for this double bond isomerization. This “shift domain” was heterologously expressed and assayed with its acyl carrier protein bound substrate. To facilitate this analysis the biosynthetic corallopyronin A intermediate was chemically synthesized as a SNAC-derivative. Enzyme activity was analyzed by NMR and high-resolution MS measurements, the latter enabled by performing the assay in deuterated buffer. Mutated enzyme variants gave first experimental evidence for the essential amino acids involved in double bond migration. These results further support the proposed corallopyronin A biosynthesis.

Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore

An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.

Synthesis and Radiopharmacological Characterisation of a Fluorine-18-Labelled Azadipeptide Nitrile as a Potential PET Tracer for in vivo Imaging of Cysteine Cathepsins

A fluorinated cathepsin inhibitor based on the azadipeptide nitrile chemotype was prepared and selected for positron emission tomography (PET) tracer development owing to its high affinity for the oncologically relevant cathepsins L, S, K and B. Labelling with fluorine‐18 was accomplished in an efficient and reliable two‐step, one‐pot radiosynthesis by using 2‐[18F]fluoroethylnosylate as a prosthetic agent. The pharmacokinetic properties of the resulting radiotracer compound were studied in vitro, ex vivo and in vivo in normal rats by radiometabolite analysis and small‐animal positron emission tomography. These investigations revealed rapid conjugate formation of the tracer with glutathione in the blood, which is associated with slow blood clearance. The potential of the developed 18F‐labelled probe to image tumour‐associated cathepsin activity was investigated by dynamic small‐animal PET imaging in nude mice bearing tumours derived from the human NCI‐H292 lung carcinoma cell line. Computational analysis of the obtained image data indicated the time‐dependent accumulation of the radiotracer in the tumours. The expression of the target enzymes in the tumours was confirmed by immunohistochemistry with specific antibodies. This indicates that azadipeptide nitriles have the potential to target thiol‐dependent cathepsins in vivo despite their disadvantageous pharmacokinetics.

Fluorescent nitrile-based inhibitors of cysteine cathepsins.

Cysteine cathepsins play an important role in many (patho)physiological conditions. Among them, cathepsins L, S, K and B are subjects of several drug discovery programs. Besides their role as drug targets, cysteine cathepsins are additionally considered to be possible biomarkers for inflammation and cancer. Herein, we describe the design, synthesis, biological evaluation and spectral properties of fluorescently labeled dipeptide- and azadipeptide nitriles.

Selective Nitrile Inhibitors To Modulate the Proteolytic Synergism of Cathepsins S and F

A series of dipeptide nitriles with different P3 substituents was designed to explore the S3 binding pocket of cathepsin S. Racemic 7-16 and the enantiopure derivative (R)-22 proved to be potent inhibitors of human cathepsin S and exhibited notable selectivity over human cathepsins L, K, and B. Inhibition of cathepsin F, the functional synergist of cathepsin S, was not observed. The azadipeptide analogue of 22, compound 26, was highly potent but nonselective.

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.

Synthesis and elastase-inhibiting activity of 2-pyridinyl-isothiazol-3(2H)-one 1,1-dioxides.

The synthesis of a series of new isothiazol-3(2H)-one 1,1-dioxides with halogenated (mostly fluorinated) pyridinyl and pentafluorophenyl substituents at 2-position is reported. These compounds (18-24) became easily accessible from 2-thiocyanato-1-carboxaldehydes and aminopyridines, pentafluoroaniline, respectively, by an isothiazolium cyclization-oxidation route. Compound 21 exhibited an IC(50) value of 3.1 microM toward human leukocyte elastase. The proteases cathepsin G, trypsin, cathepsin L, and angiotensin-converting enzyme, and the serine esterases acetylcholinesterase and cholesterol esterase were not inhibited by 21.