In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non‐covalent interaction inventories are essential to design follow‐up synthesis candidates. Two protocols are commonly applied to produce protein–ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment‐sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino‐acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein–ligand complexes.

Medicinal-chemistry optimization follows strategies replacing functional groups and attaching larger substituents at a promising lead scaffold. Well-established bioisosterism rules are considered, however, it is difficult to estimate whether the introduced modifications really match the required properties at a binding site. The electron density distribution and pK a values are modulated influencing protonation states and bioavailability. Considering the adjacent H-bond donor/ acceptor pattern of the hinge binding motif in a kinase, we studied by crystallography a set of fragments to map the required interaction pattern. Unexpectedly, benzoic acid and benzamidine, decorated with the correct substituents, are totally bioisosteric just as carboxamide and phenolic OH. A mono-dentate pyridine nitrogen out-performs bi-dentate functionalities. The importance of correctly designing pK a values of attached functional groups by additional substituents at the parent scaffold is rendered prominent.

Die medizinisch-chemische Optimierung folgt einer Strategie, funktionelle Gruppen zu ersetzen und grçßere Substituenten an ein vielversprechendes Leitstrukturgerüst anzufügen. Wohl etablierte Bioisosterie-Regeln werden berücksichtigt, dennoch ist es schwierig abzuschätzen, ob die durchgeführten Modifikationen auch wirklich den Anforderungen einer Bindestelle gerecht werden. Die Elektronendichteverteilung und die pK a-Werte der Liganden werden moduliert und beeinflussen so Protonierungszustände und Bioverfügbarkeiten. Unter Berücksichtigung des benachbarten H-Brücken-Donor/Akzeptor-Musters des Scharnierbindungsmotivs ("Hinge-Region") einer Kinase untersuchten wir kristallographisch eine Reihe von Fragmenten, um zu sehen, ob sie das erforderliche Interaktionsmuster erwidern kçnnen. Unerwarteterweise sind Benzoesäure und Benzamidin, mit den richtigen Substituenten dekoriert, ebenso wie ein Carboxamid oder eine phenolische OH-Gruppe vçllig bioisoster. Ein einzähniger Pyridinstickstoff übertrifft sogar zweizähnige Funktionalitäten am Liganden. Die Bedeutung der korrekten Einstellung von pK a-Werten der angefügten funktionellen Gruppen am Liganden durch zusätzliche Substituenten am Molekülgerüst wird damit offensichtlich.

Aims A key event in the regulation of cardiac contraction and relaxation is the phosphorylation of phospholamban (PLN) that relieves the inhibition of the sarco/endoplasmic Ca2+-ATPase SERCA2a. PLN exists in an equilibrium between monomers and pentamers. While only monomers can inhibit SERCA2a by direct interaction, the functional role of pentamers is still unclear. This study investigates the functional consequences of PLN pentamerization. Methods and Results We generated transgenic mouse models expressing either a PLN mutant that cannot form pentamers (TgAFA-PLN) or wildtype PLN (TgPLN) in a PLN-deficient background. TgAFA-PLN hearts demonstrated 3-fold stronger phosphorylation of monomeric PLN, accelerated Ca2+ cycling of cardiomyocytes and enhanced contraction and relaxation of sarcomeres and whole hearts in vivo. All of these effects were observed under baseline conditions and abrogated upon inhibition of protein kinase A (PKA). Mechanistically, far western kinase assays revealed that PLN pentamers are phosphorylated by PKA directly and independent of any subunit exchange for free monomers. In vitro-phosphorylation of synthetic PLN demonstrated that pentamers even provide a preferred PKA substrate and compete with monomers for the kinase, thereby reducing monomer phosphorylation and maximizing SERCA2a inhibition. However, β-adrenergic stimulation induced strong PLN monomer phosphorylation in TgPLN hearts and sharp acceleration of cardiomyocyte Ca2+ cycling and hemodynamic values that now were indistinguishable from TgAFA-PLN and PLN-KO hearts. The pathophysiological relevance of PLN pentamerization was evaluated using transverse aortic constriction (TAC) to induce left ventricular pressure overload. Compared to TgPLN, TgAFA-PLN mice demonstrated reduced survival after TAC, impaired cardiac hemodynamics, failure to respond to adrenergic stimulation, higher heart weight, and increased myocardial fibrosis. Conclusions The findings show that PLN pentamerization greatly impacts on SERCA2a activity as it mediates the full range of PLN effects from maximum inhibition to full release of SERCA2a. function. This regulation is important for myocardial adaptation to sustained pressure overload. Translational Perspective Pentamerization of PLN adds to the regulation of cardiac contractile function and facilitates myocardial transition to an energy saving mode during resting phases. Thus, PLN pentamers would protect cardiomyocytes from energetic deficits, and they improve stress adaptation of the heart as shown for sustained pressure overload in this study. Strategies that target PLN pentamerization promise therapeutic potential in the treatment of myocardial maladaptation to stress as well as cardiac pathologies associated with altered monomer-to-pentamer ratios, e.g., cardiomyopathies due to PLN mutations, certain types of heart failure, and aged hearts.

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