11-Phenyl-substituted indoloquinolines have been found to exhibit significant antiproliferative potency in cancer cells but to show only moderate affinity toward genomic double-helical DNA. In this study, parallel as well as antiparallel triple-helical DNA targets are employed to evaluate the triplex binding of these ligands. UV melting experiments with parallel triplexes indicate considerable interactions with the drug and a strong preference for TAT-rich triplexes in line with an increasing number of potential intercalation sites of similar binding strength between two TAT base triads. Via substitution of a singly charged aminoethylamine side chain by a longer and doubly charged bis(aminopropyl)amine substituent at the ligand, binding affinities increase and also start to exhibit long-range effects as indicated by a strong correlation between the binding affinity and the overall length of the TAT tract within the triplex stem. Compared to parallel triplexes, an antiparallel triplex with a GT-containing third strand constitutes a preferred target for the indoloquinoline drug. On the basis of pH-dependent titration experiments and corroborated by a Job analysis of continuous variation, binding of the drug to the GT triplex not only is strongly enhanced when the solution pH is lowered from 7 to 5 but also reveals a pH-dependent stoichiometry upon formation of the complex. Calorimetric data demonstrate that stronger binding of a protonated drug at acidic pH is associated with a more exothermic binding process. However, at pH 7 and 5, binding is enthalpically driven with additional favorable entropic contributions.

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions.

A-Kinase Anchoring Proteins (AKAPs) act as spatial and temporal regulators of Protein Kinase A (PKA) by localizing PKA along with multiple proteins into discrete signaling complexes. AKAPs interact with the PKA holoenzyme through an α-helix that docks into a groove formed on the dimerization/docking domain of PKA-R in an isoform-dependent fashion. In an effort to understand isoform selectivity at the molecular level, a library of protein-protein interaction (PPI) disruptors was designed to systematically probe the significance of an aromatic residue on the AKAP docking sequence for RI selectivity. The stapled peptide library was designed based on a high affinity, RI-selective disruptor of AKAP binding, RI-STAD-2. Phe, Trp and Leu were all found to maintain RI selectivity, while multiple intermediate-sized hydrophobic substitutions at this position either resulted in loss of isoform selectivity (Ile) or a reversal of selectivity (Val). Since a limited number of RI-selective sequences are currently known, this study aids in our understanding of isoform selectivity and establishing parameters for discovering additional RI-selective AKAPs.

Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIβ or RIIα versus RIIβ. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIβ while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors.

A number of unsymmetrically substituted N-methylaminoboranes, each with a phenyl ring at nitrogen and boron, were synthesized. The ratio of the cis-h.ans isomers has been investigated by means of 'HNMR spectroscopy and its dependence on the size of the aromatic moieties and the second substituent on boron is discussed. The structures of the compounds were established from the position of the N-methyl signal and were based on X-ray structure determinations of (4-bromophenylmethylamino) chlorophenylborane and (4-bromo-2-methylphenylmethylamino)chloro(2-methylphenyl)borane. In the case of (methylphenylamino)chlorophenylborane, the isomer with the phenyl group in cis position is highly favoured (90%) in the thermal equilibrium. Substitution of one of the phenyl groups by a 2-methyl-or 2,6-dimethylphenyl group decreases the fraction of the cis isomer. The same occurs when the chlorine substituent at boron is replaced by bromine or the methyl group. In absolute terms, the trans isomers are energetically more stable than the cis isomers only if one of the substituents at boron is a methyl or a 2,6-dimethylphenyl group or if there is a 2-methylphenyl substituent both at the nitrogen and the boron atom. Steric hindrance and electronic repulsion are probably responsible for the observed substitution effects. In addition, these experiments show that the isomer favoured in the crystal also predominates in solution.Eine Reihe unsymmetrisch substituierter N-Methylaminoborane rnit je einem Phenylring am Stickstoff und am Bor wurde synthetisiert und das in verdunnten Lijsungen 'H NMR-spektroskopisch bestimmte cistrans-Isomerenverhaltnis in Abhangigkeit von der GroRe der aromatischen Reste und vom Zweitsubstituenten am Bor diskutiert. Die Strukturbestimmung aller untersuchter Verbindungen folgte aus der Lage des N-Methylsignals rnit Hilfe eines Zuordnungskriteriums, das durch die Rontgenstrukturanalysen von Methyl(4-Br-pheny1)amino-chlorphenylboran und Methyl(2-methyl-4-Br-phenyl)amino-chlor(~-methylpheny1)boran erstellt wurde. Von Methylphenylamino-chlorphenylboran ist im thermischen Gleichgewicht das Isomere rnit cis-standigen Phenylringen stark begiinstigt (goo/,). Beim Ersatz eines Phenylringes durch den 2-Methyl-oder 2,6-Dimethylphenylrest nimmt stets der Anteil des cis-Isomeren ah. Substitution des Chloratoms am Bor durch Brom oder die Methylgruppe fuhrt ebenfalls zu einer Verschiebung des Isomerenverhaltnisses zum trans-Isomeren hin. Absolut betrachtet sind jedoch nur bei den Verbindungen rnit einer Methylgruppe oder rnit einem 2,6-Dimethylphenylrest am Bor, sowie bei denen mit je einem 2-Methylphenylrest am Stickstoff und am Bor die trans-Isomeren energetisch gunstiger als die cis-komeren. Fur die gefundenen Substituenteneffekte werden sterische Hinderung und elektronische AbstoBung verantwortlich gemacht. Die Experimente zeigen auBerdem, dal3 in allen Fallen das im Kristall bevorzugte Isomere auch im Losungsgleichgewicht ubenviegt.