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Four loop-mutants of the blue copper protein amicyanin from Paracoccus versutus have been constructed and characterized. The mutations replaced the loop containing three (Cys93, His96, Met99) of the four copper ligands in amicyanin by the “ligand loops” of P. aeruginosa azurin (AmiAzu), A. faecalis pseudoazurin (AmiPaz), P. nigra plastocyanin (AmiPcy), and P. aureofaciens nitrite reductase (AmiNiR). The copper centers of all variants appear to be perfect type-1 Cu sites although the AmiNiR variant exhibits diminished stability. The optical spectra of the AmiAzu and AmiPaz variants display a significant dependence on temperature. Excitation at 457 nm as well as 647 nm results in similar resonance Raman spectra. The reduction potentials of the three stable variants are all higher than that of wt amicyanin. The reduced forms of the loop-mutants protonate at the C-terminal histidine, with pK a values of 5.6 (AmiAzu), 5.4 (AmiPaz), and 5.7 (AmiPcy) (6.8 for wt amicyanin). AmiAzu is the first known cupredoxin with more than two amino acids between the cysteine and histidine ligands that undergoes this protonation. The electron self-exchange rate constants at 25 °C are 5.7−7.5 times lower for the loop mutants than for wt amicyanin (1.2 × 105 M-1 s-1). The results are interpreted by taking into consideration that three metal ligands are intimately connected with the stable β-sandwich structure of the cupredoxin. This leaves considerable freedom in positioning the fourth one, the C-terminal His, on the “ligand loop”. This explains the ease by which the C-terminal histidine ligand can be excised and replaced by external ligands without losing the metal binding property of the protein. The results also help understand the remarkable evolutionary success of the combination of cupredoxin fold and Cu site for mediating biological electron transfer.

In this Perspective, recent advances and challenges in the development of GPR119 agonists as new oral antidiabetic drugs will be discussed. Such agonists are expected to exhibit a low risk to induce hypoglycemia as well as to have a beneficial impact on body weight. Many pharmaceutical companies have been active in the search for GPR119 agonists, making it a highly competitive area in the industrial environment. Several GPR119 agonists have been entered into clinical studies, but many have failed either in phase I or II and none has progressed beyond phase II. Herein we describe the strategies chosen by the different medicinal chemistry teams in academia and the pharmaceutical industry to improve potency, physicochemical properties, pharmacokinetics, and the safety profile of GPR119 agonists in the discovery phase in order to improve the odds for successful development.

FLEXX-PHARM, an extended version of the flexible docking tool FLEXX, allows the incorporation of information about important characteristics of protein-ligand binding modes into a docking calculation. This information is introduced as a simple set of constraints derived from receptor-based type pharmacophore features. The constraints are determined by selected FLEXX interactions and inclusion volumes in the receptor active site. They guide the docking process to produce a set of docking solutions with particular properties. By applying a series of look-ahead checks during the flexible construction of ligand fragments within the active site, FLEXX-PHARM determines which partially built docking solutions can potentially obey the constraints. Solutions that will not obey the constraints are deleted as early as possible, often decreasing the calculation time and enabling new docking solutions to emerge. FLEXX-PHARM was evaluated on various individual protein-ligand complexes where the top docking solutions generated by FLEXX had high root mean square deviations (RMSD) from the experimentally observed binding modes. FLEXX-PHARM showed an improvement in the RMSD of the top solutions in most cases, along with a reduction in run time. We also tested FLEXX-PHARM as a database screening tool on a small dataset of molecules for three target proteins. In two cases, FLEXX-PHARM missed one or two of the active molecules due to the constraints selected. However, in general FLEXX-PHARM maintained or improved the enrichment shown with FLEXX, while completing the screen in considerably less run time.

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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.

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.

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