Characterization of A-935142, a hERG enhancer, in the presence and absence of standard hERG blockers.

AIMS: In a previous study we found that A-935142 enhanced hERG current in a concentration-dependent manner by facilitating activation, reducing inactivation, and slowing deactivation (Su et al., 2009). A-935142 also shortened action potential duration (APD90) in canine Purkinje fibers and guinea pig atrial tissue. This study focused on the combined effects of the prototypical hERG enhancer, A-935142 and two hERG current blockers (sotalol and terfenadine). MAIN METHODS: The whole-cell voltage clamp method with HEK 293 cells heterologously expressing the hERG channel (Kv 11.1) was used. KEY FINDINGS: A-935142 did not compete with 3H-dofetilide binding, suggesting that A-935142 does not overlap the binding site of typical hERG blockers. In whole-cell voltage clamp studies we found: 1) 60 µM A-935142 enhanced hERG current in the presence of 150 µM sotalol (57.5±5.8%) to a similar extent as seen with A-935142 alone (55.6±5.1%); 2) 150 µM sotalol blocked hERG current in the presence of 60 µM A-935142 (43.5±1.5%) to a similar extent as that seen with sotalol alone (42.0±3.2%) and 3) during co-application, hERG current enhancement was followed by current blockade. Similar results were obtained with 60 nM terfenadine combined with A-935142. SIGNIFICANCE: These results suggest that the hERG enhancer, A-935142 does not compete with these two known hERG blockers at their binding site within the hERG channel. This selective hERG current enhancement may be useful as a treatment for inherited or acquired LQTS (Casis et al., 2006).

Ventricular rate adaptation: a novel, rapid, cellular-based in-vitro assay to identify proarrhythmic and torsadogenic compounds.

INTRODUCTION: Delayed cardiac repolarization is an established risk factor for proarrhythmia and Torsades-de-Pointes (TdeP) that is typically measured in vitro during slow, regular stimulation. We have developed an alternative, novel, and rapid cellular-based approach for predicting drug-induced proarrhythmia that detects changes in electrical refractoriness based on mechanical responses (measured optically) during increasingly rapid trains of stimulation interspersed with pauses (mimicking the clinically observed short-long-short (SLS) stimulation sequence associated with the TdeP initiation). METHODS: Acutely isolated rabbit ventricular myocytes were superfused and electrically stimulated using an accelerating pacing protocol (APP) consisting of 12 consecutive pacing segments (10 beats per segment) with incrementally faster cycle lengths; trains were separated by pauses to identify loss of stimulus capture as well as to mimic clinically observed SLS sequences. Drug effects were evaluated based on a myocyte's ability to contract during progressively faster pacing segments (rate-adaptation); the earliest rate during which the myocyte fails to respond (longest cycle length with incomplete capture (CLIC)) was used to quantify electrophysiologic effects. RESULTS: Torsadogenic drugs known to delay repolarization during slow stimulation prolonged CLIC and dramatically limited the ability to respond to progressively rapid stimulation. The recognized proarrhythmic compounds E-4031, cisapride, grepafloxacin, and haloperidol rapidly prolonged CLIC at and above therapeutic concentrations in a concentration-dependent manner, while negative controls (captopril, indomethacin, and loratidine) do not affect rate-adaptation. DISCUSSION: Ventricular rate adaptation represents a novel approach for rapidly detecting drugs with torsadogenic risk using rapid rhythms that are typically not employed when evaluating proarrhythmic risk. This method is well suited for detecting and avoiding potential cardiac liabilities early in drug discovery ("frontloading") prior to final selection of candidate drugs.

In vitro studies on a class of quinoline containing histamine H3 antagonists.

A series of quinoline containing histamine H(3) antagonists is reported herein. These analogs were synthesized via the Friedlander quinoline synthesis between an aminoaldehyde intermediate and a methyl ketone allowing for a wide diversity of substituents at the 2-position of the quinoline ring.

Validation of diacyl glycerolacyltransferase I as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor.

A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1(-/-) mice.

1,4-Dihydroindeno[1,2-c]pyrazoles with acetylenic side chains as novel and potent multitargeted receptor tyrosine kinase inhibitors with low affinity for the hERG ion channel.

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

Novel heterocyclic-substituted benzofuran histamine H3 receptor antagonists: in vitro properties, drug-likeness, and behavioral activity.

Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.

Constrained 7-fluorocarboxychromone-4-aminopiperidine based Melanin-concentrating hormone receptor 1 antagonists: the effects of chirality on substituted indan-1-ylamines.

The incorporation of constrained tertiary amines into an existing class of N-benzyl-4-aminopiperidinyl chromone-based MCHr1 antagonists led to the identification of a series of chiral racemic compounds that displayed good to excellent functional potency, binding affinity, and selectivity over the hERG channel. Further separation of two distinct chiral racemic compounds into their corresponding pairs of enantiomers revealed a considerable selectivity for MCHr1 for one configuration, in addition to a striking difference in oral exposure between one pair of enantiomers in diet-induced obese mice. Oral administration of the most potent compound in this class in the same animal model led to significant reduction of fat mass in a semi-chronic model for weight loss.

The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K+]o.

INTRODUCTION: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (IKr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [3H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value. METHODS: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [3H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K+ channels. The object of these experiments was to (1) compare binding Ki values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K+ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing Ki values from binding studies at 5 and 60 mM [K+]o to functional IC50 values for hERG current block using 56 structurally diverse drugs. RESULTS: We found (a) comparable Ki values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K+]o lowered the Kd and increased the Bmax for [3H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding Ki values and functional IC50 values for hERG current block. DISCUSSION: In conclusion, increasing K+ concentrations results in an increase in both [3H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between Ki values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K+]o within assays. There is good correlation between binding Ki values and functional (whole-cell patch clamp) IC50 values at both 5 and 60 mM K+ concentrations (R2 values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [3H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K+ channel.

Oral bimoclomol elevates heat shock protein 70 and reduces myocardial infarct size in rats.

Bimoclomol has been shown to increase an inducible member of the heat shock protein 70 family (HSP70) and cytoprotect in vitro. Here, we addressed whether oral pretreatment of rats with bimoclomol could elevate myocardial HSP70 and reduce infarct size in a rat model of ischemia and reperfusion. Rats were pretreated with bimoclomol at 3, 6 or 18 h or with 42 degrees thermal stress 24 h before ischemia. Infarct size was significantly decreased 6 h after oral administration of bimoclomol and 24 h after thermal stress. Left ventricles from a separate group of rats were examined for HSP70 levels. Western blots showed a significant increase in HSP70 6 h after oral administration of bimoclomol and 24 h after thermal stress. There was a significant correlation (P<0.05) between HSP70 induction and infarct size reduction, whether produced by thermal stress or oral administration of bimoclomol. Thus, bimoclomol can increase HSP70 and reduce infarct size in a rat model of ischemia and reperfusion.