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Long-term severe diabetes only leads to mild cardiac diastolic dysfunction in Zucker diabetic fatty rats.

Type 2 diabetes mellitus (DM) leads to cardiac dysfunction irrespective of hypertension and coronary artery disease; this is called diabetic cardiomyopathy. Here, we investigated the severity of diabetic cardiomyopathy and myocardial remodelling in aged Zucker diabetic fatty (ZDF) rats.

Novel β-amino acid derivatives as inhibitors of cathepsin A.

Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel β-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling.

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μm, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μm RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.

Identification and synthesis of novel inhibitors of acetyl-CoA carboxylase with in vitro and in vivo efficacy on fat oxidation.

Acetyl CoA carboxylase isoforms 1 and 2 (ACC1/2) are key enzymes of fat utilization and their inhibition is considered to improve aspects of the metabolic syndrome. To identify pharmacological inhibitors of ACC1/2, a high throughput screen was performed which resulted in the identification of the lead compound 3 ( Gargazanli , G. ; Lardenois , P. ; Frost , J. ; George , P. Patent WO9855474 A1, 1998 ) as a moderate selective ACC2 inhibitor. Optimization of 3 led to 4m ( Zoller , G. ; Schmoll , D. ; Mueller , M. ; Haschke , G. ; Focken , I. Patent WO2010003624 A2, 2010 ) as a submicromolar dual ACC1/2 inhibitor of the rat and human isoforms. 4m possessed favorable pharmacokinetic parameters. This compound stimulated fat oxidation in vivo and reduced plasma triglyceride levels in a rodent model after subchronic administration. 4m is a suitable tool compound for the elucidation of the pharmacological potential of ACC1/2 inhibition.

Identification of High-Affinity P2Y12 Antagonists Based on a Phenylpyrazole Glutamic Acid Piperazine Backbone

A series of novel, highly potent P2Y₁₂ antagonists as inhibitors of platelet aggregation based on a phenylpyrazole glutamic acid piperazine backbone is described. Exploration of the structural requirements of the substituents by probing the structure-activity relationship along this backbone led to the discovery of the N-acetyl-(S)-proline cyclobutyl amide moiety as a highly privileged motif. Combining the most favorable substituents led to remarkably potent P2Y₁₂ antagonists displaying not only low nanomolar binding affinity to the P2Y₁₂ receptor but also a low nanomolar inhibition of platelet aggregation in the human platelet rich plasma assay with IC₅₀ values below 50 nM. Using a homology and a three-dimensional quantitative structure-activity relationship model, a binding hypothesis elucidating the impact of several structural features was developed.

Inhibition of CatA: an emerging strategy for the treatment of heart failure

The lysosomal serine carboxypeptidase CatA has a very important and well-known structural function as well as a, so far, less explored catalytic function. A complete loss of the CatA protein results in the lysosomal storage disease galactosialidosis caused by intralysosomal degradation of β-galactosidase and neuraminidase 1. However, mice with a catalytically inactive CatA enzyme show no signs of this disease. This observation establishes a clear distinction between structural and catalytic functions of the CatA enzyme. Recently, several classes of orally bioavailable synthetic inhibitors of CatA have been identified. Pharmacological studies in rodents indicate a remarkable influence of CatA inhibition on cardiovascular disease progression and identify CatA as a promising novel target for the treatment of heart failure.

Cathepsin A mediates susceptibility to atrial tachyarrhythmia and impairment of atrial emptying function in Zucker diabetic fatty rats

AIMS Type 2 diabetes (T2D) is an independent risk factor for atrial fibrillation (AF) and stroke. The serine protease cathepsin A (CatA) is up-regulated in diabetes and plays an important role in the degradation of extracellular peptides. This study sought to delineate the role of CatA for the development of atrial remodelling under diabetic conditions. METHODS AND RESULTS Zucker Diabetic Fatty rats (ZDF) were treated with vehicle (n = 20) or CatA-inhibitor (SAR; 50 mg/kg; n = 20), and compared with age-matched non-diabetic littermates (Ctr, n = 20). Left-atrial (LA) emptying function [magnetic resonance imaging (MRI)] and atrial electrophysiological parameters were measured before sacrifice for histological and biochemical analysis. The impact of enhanced cardiac CatA expression on atrial remodelling was determined using CatA-transgenic mice. At the age of 9.5 months, atrial tissues of ZDF rats showed increased CatA gene expression and CatA-activity, along with increased AF-susceptibility and impaired LA-emptying function. CatA-inhibition reduced CatA-activity in ZDF comparable to Ctr values and decreased LA-fibrosis formation and connexin 43 lateralization. This was associated with shorter median duration of LA-tachyarrhythmia (12.0 ± 1.7 vs. 1.2 ± 0.47 s, P < 0.01) induced by burst pacing and diminished regions of slow conduction. Cardiac MRI revealed better LA-emptying function parameters (active per cent emptying: 29 ± 1 vs. 23 ± 2%, P < 0.01) after CatA-inhibition. CatA-inhibition reduced LA bradykinin-degrading activity in ZDF. Transgenic mice overexpressing CatA demonstrated enhanced atrial fibrosis formation and increased AF-susceptibility. CONCLUSION T2D leads to arrhythmogenic atrial remodelling in ZDF rats. CatA-inhibition reduces LA bradykinin-degrading activity in ZDF and suppresses the development of atrial structural changes and AF-promotion, implicating CatA as an important mediator for AF-substrate in T2D.

Tolerability, safety and pharmacokinetics of the novel Cathepsin A inhibitor SAR164653 in healthy subjects

Cathepsin A (CathA) is a lysosomal protein where it forms a stable complex with neuraminidase and ß‐galactosidase. CathA also has enzymatic activity and is involved in the degradation of many peptides. CathA was recently discovered as a target for heart failure, fostering the development of CathA inhibitors with SAR164653 as a frontrunner. The first‐in‐man study investigated single oral doses from 20 to 800 mg of SAR164653 followed by repeat dose studies at doses up to 800 mg in healthy young and elderly subjects. SAR164653 was safe and well tolerated at doses up to 800 mg in healthy subjects, and a maximum tolerated dose could not be determined from the study. Activity of ß‐galactosidase measured in leukocytes did not show any abnormalities. The tmax was 1.0 to 2.5 hours, and the t1/2 was ∼5–11 after single dosing; exposure increased less than dose proportional. Following multiple dosing, accumulation was not observed, Cmax and AUC0–24 increased in a dose‐proportional manner, and t1/2 was around 14–20 hours. The novel CathA inhibitor SAR164653 was found to have a favorable safety profile in these early phase 1 studies, but further studies are required to confirm if SAR164653 is equally safe in patients undergoing long‐term treatment.

Crystal Structure of Cathepsin A, a Novel Target for the Treatment of Cardiovascular Diseases.

The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order-disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253-Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains. This network can only form if at least half of the carboxylate groups involved are protonated, which explains the acidic pH optimum of the enzyme.