Thorsten Sadowski

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.

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (ł50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

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.

Proteomic Profiling of Cardiomyocyte-Specific Cathepsin A Overexpression Links Cathepsin A to the Oxidative Stress Response

Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to β-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography-tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated p-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response.

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.