Acquiring a divergent response from homologous protein domains is essential for selective ligand-protein interactions. Stereospecific fluorination of (-)-balanol, an ATP mimic, uncovers a new source of selectivity from integrated chemical and conformational perturbation that differentiates homologous sites by the level of congruency in their response to local and remote fluorine effects.

Epithelial sodium channel (ENaC) is a transmembrane protein that has an essential role in maintaining the levels of sodium in blood plasma. A person with a family history of hypertension has a high enough amount of ENaC protein in the kidneys or other organs, so that the ENaC protein acts as a marker that a person is susceptible to hypertension. An aptasensor involves aptamers, which are oligonucleotides that function similar to antibodies, as sensing elements. An electrochemical aptasensor for the detection of ENaC was developed using a screen-printed carbon electrode (SPCE) which was modified by electrodeposition of cerium oxide (CeO 2 ). The aptamer immobilization was via the streptavidin–biotin system. The measurement of changes in current of the active redox [Fe(CN) 6 ] 3−/4− was carried out by differential pulse voltammetry. The surfaces of SPCE and SPCE/CeO 2 were characterized using scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The Box–Behnken experimental optimization design revealed the streptavidin incubation time, aptamer incubation time and streptavidin concentrations were 30 min, 30 min and 10.8 µg ml −1 , respectively. Various concentrations of ENaC were used to obtain the linearity range of 0.05–3.0 ng ml −1 , and the limits of detection and quantification were 0.012 ng ml −1 and 0.038 ng ml −1 , respectively. This aptasensor method has the potential to measure the ENaC protein levels in urine samples as well as to be a point-of-care device.

Nocardiotide A has been successfully synthesized using a combination of solid‐ and solution‐phase synthesis. Peptides 1 and 2 of nocardiotide A, with different C and N termini, were synthesized on 2‐chlorotrityl chloride resin. A combination of N,N,N′,N′‐tetramethyl‐O‐(1H‐benzotriazol‐1‐yl)uronium hexafluorophosphate and hydroxybenzotriazole (HBTU/HOBt) was employed as coupling reagent and the release of the linear peptides from the resin was undertaken using AcOH and trifluoroethanol (TFE) reagents resulting in linear hexapeptides with protected side chains in good yields (83.4 % and 76 %, respectively). Peptide 1, which has Ala as C terminus and Trp as N terminus, was the only peptide that was successfully cyclized without epimerization under a dilute condition (1.25×10−3 M) using HBTU (3 equiv.) and 1 % N,N‐diisopropylethylamine (DIPEA) in dichloromethane. A combination of a small residue at the C terminus and a larger residue at the N terminus was found to be effective for the cyclization. The protecting group of the crude cyclic product was then deprotected by 95 % TFA in water and the crude was then purified to give white solid product with 16 % overall yield. The peptide was then characterized by HR‐ToFMS, 1H‐ and 13C‐NMR spectroscopy. In order to ensure the absence of epimerization during cyclization, the D‐analogue of nocardiotide A was synthesized using similar synthetic protocol. The NMR data of D‐analogue was found to have a larger difference compared to the synthetic and natural product, showing there is no epimerization at C‐terminus during cyclization.

Background(-)-Balanol is an ATP mimic that inhibits protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA) with limited selectivity. While PKA is a tumour promoter, PKC isozymes act as tumour promoters or suppressors, depending on the cancer type. In particular, PKCε is frequently implicated in cancer promotion, making it a potential target for anticancer drugs. To improve isozyme selectivity of balanol, exhaustive structural and activity relationship (SAR) studies have been performed in the last two decades, but with limited success. More recently, fluorination on balanol has shown improved selectivity for PKCε, although the fluorine effect is not yet clearly understood. Understanding the origin to this fluorine-based selectivity will be valuable for designing better balanol-based ATP mimicking inhibitors. Computational approaches such as molecular dynamics (MD) simulations can decipher the fluorine effect, provided that correct charges have been assigned to a ligand. Balanol analogues have multiple ionisable functional groups and the effect of fluorine substitutions on the exact charge state of each analogue bound to PKA and to PKCε needs to be thoroughly investigated in order to design highly selective inhibitors for therapeutic applications.ResultsWe explored the charge states of novel fluorinated balanol analogues using MD simulations. For different potential charge states of these analogues, Molecular Mechanics Generalized Born Surface Area (MMGBSA) binding energy values were computed. This study suggests that balanol and the most potent fluorinated analogue (5S fluorine substitution on the azepane ring), have charges on the azepane ring (N1), and the phenolic (C6′′OH) and the carboxylate (C15′′O2H) groups on the benzophenone moiety, when bound to PKCε as well as PKA.ConclusionsTo the best our knowledge, this is the first study showing that the phenolate group is charged in balanol and its analogues binding to the ATP site of PKCε. Correct charge assignments of ligands are important to obtain predicted binding energy values from MD simulations that reflect experimental values. Both fluorination and the local enzymatic environment of the ATP site can influence the exact charge states of balanol analogues. Overall, this study is highly valuable for further rational design of potent balanol analogues selective to PKCε.Electronic supplementary materialThe online version of this article (10.1186/s12859-017-1955-7) contains supplementary material, which is available to authorized users.

(-)-Balanol is an adenosine triphosphate mimic that inhibits protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA) with limited selectivity. While PKA is known as a tumor promoter, PKC isozymes can be tumor promoters or suppressors. In particular, PKCε is frequently involved in tumorigenesis and a potential target for anticancer drugs. We recently reported that stereospecific fluorination of balanol yielded a balanoid with enhanced selectivity for PKCε over other PKC isozymes and PKA, although the global fluorine effect behind the selectivity enhancement is not fully understood. Interestingly, in contrast to PKA, PKCε is more sensitive to this fluorine effect. Here we investigate the global fluorine effect on the different binding responses of PKCε and PKA to balanoids using molecular dynamics (MD) simulations. For the first time to the best of our knowledge, we found that a structurally equivalent residue in each kinase, Thr184 in PKA and Ala549 in PKCε, is essential for the different binding responses. Furthermore, the study revealed that the invariant Lys, Lys73 in PKA and Lys437 in PKCε, already known to have a crucial role in the catalytic activity of kinases, serves as the main anchor for balanol binding. Overall, while Thr184 in PKA attenuates the effect of fluorination, Ala549 permits remote response of PKCε to fluorine substitution, with implications for rational design of future balanol-based PKCε inhibitors.