Structure-guided design of antibacterials that allosterically inhibit DNA gyrase.

A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.

Tetrasubstituted pyridines as potent and selective AKT inhibitors: Reduced CYP450 and hERG inhibition of aminopyridines.

The synthesis and evaluation of tetrasubstituted aminopyridines, bearing novel azaindazole hinge binders, as potent AKT inhibitors are described. Compound 14c was identified as a potent AKT inhibitor that demonstrated reduced CYP450 inhibition and an improved developability profile compared to those of previously described trisubstituted pyridines. It also displayed dose-dependent inhibition of both phosphorylation of GSK3beta and tumor growth in a BT474 tumor xenograft model in mice.

Correction to Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].

Discovery and electrophysiological characterization of SKF-32802: A novel hERG agonist found through a large-scale structural similarity search.

Despite the importance of the hERG channel in drug discovery and the sizable number of antagonist molecules discovered, only a few hERG agonists have been discovered. Here we report a novel hERG agonist; SKF-32802 and a structural analog of the agonist NS3623, SB-335573. These were discovered through a similarity search of published hERG agonists. SKF-32802 incorporates an amide linker rather than NS3623's urea, resulting in a compound with a different mechanism of action. We find that both compounds decrease the time constant of open channel kinetics, increase the amplitude of the envelope of tails assay, mildly increased the amplitude of the IV curve, bind the hERG channel in either open or closed states, increase the plateau of the voltage dependence of activation and modulate the effects of the hERG antagonist, quinidine. Neither compound affects inactivation nor deactivation kinetics, a property unique among hERG agonists. Additionally, SKF-32802 induces a leftward shift in the voltage dependence of activation. Our structural models show that both compounds make strong bridging interactions with multiple channel subunits and are stabilized by internal hydrogen bonding similar to NS3623, PD-307243 and RPR26024. While SB-335573 binds in a nearly identical fashion as NS3623, SKF-32802 makes an additional hydrogen bond with neighboring threonine 623. In summary, SB-335573 is a type 4 agonist which increases open channel probability while SKF-32802 is a type 3 agonist which induces a leftward shift in the voltage dependence of activation.

Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 µM).

Development of a high-throughput electrophysiological assay for the human ether-à-go-go related potassium channel hERG.

INTRODUCTION: Drug-induced prolongation of the QT interval via block of the hERG potassium channel is a major cause of attrition in drug development. The advent of automated electrophysiology systems has enabled the detection of hERG block earlier in drug discovery. In this study, we have evaluated the suitability of a second generation automated patch clamp instrument, the IonWorks Barracuda, for the characterization of hERG biophysics and pharmacology. METHODS: All experiments were conducted with cells stably expressing hERG. Recordings were made in perforated patch mode either on a conventional patch clamp setup or on the IonWorks Barracuda. On the latter, all recordings were population recordings in 384-well patch plates. RESULTS: HERG channels activated with a V(1/2)=-3.2±1.6mV (n=178) on the IonWorks Barracuda versus -11.2±6.1mV (n=9) by manual patch clamp. On the IonWorks Barracuda, seal resistances and currents were stable (<30% change) with up to six cumulative drug additions and 1-min incubations per addition. Over 27 experiments, an average of 338 concentration-response curves were obtained per experiment (96% of the 352 test wells on each plate). HERG pharmacology was examined with a set of 353 compounds that included well-characterized hERG blockers. Astemizole, terfenadine and quinidine inhibited hERG currents with IC(50) values of 159nM, 224nM and 2µM, respectively (n=51, 10 and 18). This set of compounds was also tested on the PatchXpress automated electrophysiology system. We determined through statistical methods that the two automated systems provided equivalent results. DISCUSSION: Evaluating drug effects on hERG channels is best performed by electrophysiological methods. HERG activation and pharmacology on the IonWorks Barracuda automated electrophysiology platform were in good agreement with published electrophysiology results. Therefore, the IonWorks Barracuda provides an efficient way to study hERG biophysics and pharmacology.

Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and NaV1.5 blockade.

INTRODUCTION: The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa(V)1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture. METHODS: Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds. RESULTS: Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC(50)s (paired values denote constantly cultured and frozen, respectively) of 4.8±0.4 and 5.1±0.4, 1.4±0.1 and 1.1±0.1, 24.4±2.4 and 21.9±1.8, 2.1±0.4 and 2.1±0.1, 5.2±0.4 and 4.0±0.2µM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa(V)1.5 with IC(50)s of 46.6±4.3 and 28.0±2.3, 7.6±0.7 and 6.2±0.5, 153.5±13.0 and 106.0±4.7, 5.5±0.5 and 4.8±0.2µM. Voltage dependences of activation (V(1/2)) for hERG were statistically identical, 0.4±0.8mV and 2.5±0.5mV. In hNa(V)1.5, the V(1/2) of inactivation and activation were statistically identical, -82.7±0.1mV versus -84.9±0.3mV, -47.5±0.3mV versus -45.0±0.6mV. Current density in both conditions in hERG experiments was similar, 47.0±4.1pA versus 42.3±6.0pA/pF. DISCUSSION: hERG and hNa(V)1.5 screens run using frozen cells have statistically identical IC(50)s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.