Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

In-vitro modeling of intravenous drug precipitation by the optical spatial precipitation analyzer (OSPREY).

Intravenous (IV) administration of poorly water-soluble small molecule therapeutics can lead to precipitation during mixing with blood. This can limit characterization of pharmacological and safety endpoints in preclinical models. Most often, tests of kinetic and thermodynamic solubility are used to optimize the formulation for solubility prior to infusion in animals, but these do not capture the dynamic precipitation processes that take place during in-vivo administration. To better capture the fluid dynamic processes that occur during IV administration, we developed the Optical Spatial PREcipitation analYzer (OSPREY) as a method to quantify the amount and size of compound precipitates in whole blood using a flow-through system that mimics IV administration. Here, we describe the OSPREY device and its underlying imaging processing methods. We then validate the ability to accurately segment particles according to their size using monodisperse suspensions of microspheres (diameter 50 to 425 µm). Next, we use a tool compound, ABT-737, to study the effects of compound concentration, vessel flow rate, compound infusion rate and vessel diameter on precipitation. Finally, we use the physiological diameter and flow rate of rat femoral vein and dog saphenous vein to demonstrate the potential of OSPREY to model in-vivo precipitation in a controlled, dynamic in-vitro assay.

Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients.

BACKGROUND: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). METHODS: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. RESULTS: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. CONCLUSIONS: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs.

Comparative analysis of inactivated-state block of N-type (Ca(v)2.2) calcium channels.

OBJECTIVE: The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays. METHODS: The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology. RESULTS: N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 µM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable. CONCLUSIONS: The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.

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.

Drug-induced QT prolongation: Concordance of preclinical anesthetized canine model in relation to published clinical observations for ten CiPA drugs.

INTRODUCTION: The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative differentiates torsadogenic risk of 28 drugs affecting ventricular repolarization based on multiple in vitro human derived ionic currents. However, a standardized prospective assessment of the electrophysiologic effects of these drugs in an integrated in vivo preclinical cardiovascular model is lacking. This study questioned whether QTc interval prolongation in a preclinical in vivo model could detect clinically reported QTc prolongation and assign torsadogenic risk for ten CiPA drugs. METHODS: An acute intravenous administered ascending dose anesthetized dog cardiovascular model was used to assess QTc prolongation along with other electrocardiographic (PR, QRS intervals) and hemodynamic (heart rate, blood pressures, left ventricular contractility) parameters at plasma concentrations spanning and exceeding clinical exposures. hERG current block potency was characterized using IC50 values from automated patch clamp. RESULTS: All eight drugs eliciting clinical QTc prolongation also delayed repolarization in anesthetized dogs at plasma concentrations within four-fold clinical exposures. In vitro QTc safety margins (defined based on clinical Cmax values/plasma concentrations eliciting statistically significant QTc prolongation in dogs) were lower for high vs intermediate torsadogenic risk drugs. In comparison, hERG IC10 values represented as total drug concentrations were better predictors of preclinical QTc prolongation than hERG IC50 values. CONCLUSION: There was good concordance for QTc prolongation in the anesthetized dog model and clinical torsadogenic risk assignment. QTc assessment in the anesthetized dog remains a valuable part of a more comprehensive preclinical integrated risk assessment for delayed repolarization and torsadogenic risk as part of a global cardiovascular evaluation.

Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

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.

Discovery of N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N'-(2-fluoro-5-methylphenyl)urea (ABT-869), a 3-aminoindazole-based orally active multitargeted receptor tyrosine kinase inhibitor.

In our continued efforts to search for potent and novel receptor tyrosine kinase (RTK) inhibitors as potential anticancer agents, we discovered, through a structure-based design, that 3-aminoindazole could serve as an efficient hinge-binding template for kinase inhibitors. By incorporating an N,N'-diaryl urea moiety at the C4-position of 3-aminodazole, a series of RTK inhibitors were generated, which potently inhibited the tyrosine kinase activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor receptor families. A number of compounds with potent oral activity were identified by utilizing an estradiol-induced mouse uterine edema model and an HT1080 human fibrosarcoma xenograft tumor model. In particular, compound 17p (ABT-869) was found to possess favorable pharmacokinetic profiles across different species and display significant tumor growth inhibition in multiple preclinical animal models.

The effects of plasma proteins on delayed repolarization in vitro with cisapride, risperidone, and D, L-sotalol.

INTRODUCTION: Drug-induced long QT syndrome (LQTS) has been linked to arrhythmias (including Torsades de Pointes and sudden cardiac death), and has led to an increased awareness of the potential risk of delayed repolarization in vitro and in vivo. However, in vitro assessments of delayed repolarization have not been fully predictive of in vivo effects. METHODS: To define the extent to which plasma protein binding (ppb) contributes to such disparities in repolarization studies, we compared drug-induced prolongation of the canine Purkinje fiber action potential duration (APD(90)) in vitro during superfusion with 100% Tyrode's solution (Tyrodes), canine plasma [50% plasma/50% Tyrodes] and a 5% solution of recombinant human serum albumin in Tyrodes (HSA). Drugs evaluated included cisapride (>98% ppb), risperidone (90% ppb), and d, l-sotalol (negligible ppb). Effects on APD were monitored using standard microelectrode techniques under physiologic conditions and temperature ([K(+)]=4 mM, 37 degrees C) during slow stimulation (2 s basic cycle length). RESULTS: The effects of cisapride and risperidone on Purkinje fiber APD(90) were significantly attenuated in the presence of plasma proteins. However, with cisapride, the extent of reduction with plasma proteins was significantly less than predicted based on calculated free drug levels. DISCUSSION: We conclude that while plasma protein binding does reduce APD prolongation seen with bound drugs, this effect is not well correlated with the calculated plasma protein binding or expected clinical free fraction. Because of the complex drug interactions that occur in plasma, the electrophysiological effects seen with bound drugs are not well correlated with the calculated free fraction and thus caution should be exercised when assigning a predictive safety window. Thus, the canine Purkinje fiber assay is useful for defining the modulation of delayed repolarization due to plasma protein binding of novel therapeutic agents.

Block of hERG channel by ziprasidone: biophysical properties and molecular determinants.

Ziprasidone, an antipsychotic agent, delays cardiac repolarization and, thus, prolongs the QT interval of the cardiac ECG. In this study, we examined the biophysical properties and the molecular determinants of the ziprasidone block of wild-type hERG potassium channels stably expressed in HEK-293 cells or wild-type and mutant hERG channels expressed in Xenopus oocytes. In stably transfected HEK-293 cells, ziprasidone blocked wild-type hERG current in a voltage- and concentration-dependent manner (IC(50)=120nM, 0mV, 37 degrees C). Ziprasidone showed minimal tonic block of hERG current estimated during a depolarizing voltage (-20 or +30mV) or evaluated by the envelope of tails test (+30mV). Rate of the block onset was rapid, but not significantly affected by test potentials ranging from -20 to +30mV (time constant (tau)=114+/-14ms at +30mV). The time constant of the slow component of hERG current deactivation (at -50mV) was significantly increased by ziprasidone (tau=1776+/-90 versus 1008+/-71ms, P<0.01). Time course of channel inactivation was slowed by ziprasidone in a voltage-dependent manner. The V(1/2) values for steady-state activation and inactivation of hERG channel in HEK-293 cells were not significantly altered by ziprasidone. In Xenopus oocytes, ziprasidone exhibited less potent block of wild-type hERG current (IC(50)=2.8microM, 0mV, 23 degrees C). Mutation of the aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of hERG dramatically reduced the potency of channel block by ziprasidone (IC(50)>0.4 and 1mM at 0mV for Y652A and F656A, respectively). In conclusion, ziprasidone preferentially binds to and blocks open hERG channels. Tyr-652 and Phe-656 are two critical residues in the ziprasidone-binding site.

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.

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.

Negative inotropic effect of a CB2 agonist A-955840 in isolated rabbit ventricular myocytes is independent of CB1 and CB2 receptors.

A-955840, a selective CB2 agonist, has been shown to elicit concentration-dependent decreases in cardiac contractility in the anesthetized dog (decreased maximal velocity of left ventricular pressure development [LV dP/dt max]). However, it is unknown whether this represents a direct effect or a response dependent on other factors (such as autonomic tone and neurohumoral factors) present in vivo. This study examined if A-955840 had a direct effect on contractility of isolated cardiac myocytes, and if so to determine the potential mechanisms. Contractility was assessed in vitro using percent changes in maximal shortening velocity of sarcomeres (dL/dt max) and fractional shortening of sarcomere length (FS) in rabbit left ventricular myocytes. L-type calcium current in myocytes was recorded using wholecell voltage-clamp techniques. A-955840 reduced dL/dt max and FS in a reversible and concentration-dependent manner with an IC50 of 11.4 µg/mL (based on dL/dt max) which is similar to the estimated IC50 value of 9.8 µg/mL based on the effects of A-955840 on LV dP/dt max in anesthetized dogs. A-955840 (4.1 µg/mL) reduced myocyte contractility (%FS) to a similar extent in the absence and presence of a CB2 antagonist, SR-2 (24.0 ± 3.4 vs 23.1 ± 3.0 %, n=5) or a CB1 antagonist, Rimonabant (18.8 ± 2.3 vs 19.8 ± 2.7 %, n=5). A-955840 (4.1 µg/mL) also reduced L-type calcium current of rabbit ventricular myocytes (1.05 ± 0.11 vs 0.70 ± 0.12 nA, n=5, P < 0.01). These results suggest that A-955840 exerts direct negative inotropic effects on isolated rabbit ventricular myocytes, which is mediated by neither CB2 nor CB1 receptors, and consistent with off-target negative inotropy mediated by inhibition of the cardiac L-type calcium current.

A novel secretagogue increases cardiac contractility by enhancement of L-type Ca2+ current.

N'1-(3,3,6,8-tetramethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yliden)-2-cyanoethanohydrazide (TTYC) increases secretion of glucagon-like peptide-1 and intracellular Ca(2+) concentration in GLUTag cells. The purpose of the present study was to examine if TTYC exerts positive inotropic effects on isolated rabbit ventricular myocytes and in vivo heart in anesthetized rats, and if so to further define the potential mechanism of action. Contractility was assessed in vitro using changes in fractional shortening (FS) of myocyte sarcomere length and in vivo using changes in the velocity of left ventricular pressure. Changes in L-type Ca(2+) current of ventricular myocytes were evaluated using whole-cell voltage-clamp techniques. TTYC increased FS of myocyte sarcomere length in a concentration-dependent manner. The positive inotropic effect was not abrogated by beta-adrenergic blockade (propranolol) or protein kinase A inhibition. TTYC enhanced peak L-type Ca(2+) current in a voltage-dependent manner (current amplitudes increased by 4.0-fold at -10 mV and 1.5-fold at +10 mV). Voltage-dependence of steady-state activation of L-type Ca(2+) current was shifted by 15 mV in the negative direction. Inactivation time course of the L-type Ca(2+) currents at voltages of -10 to 20 mV was significantly slowed by 0.3 microM TTYC. In vivo studies demonstrated that TTYC increased cardiac contractility in a dose-dependent manner. In conclusion, TTYC is a novel L-type Ca(2+) current activator with positive cardiac inotropic effects. Negative shifting of the voltage-dependence of L-type Ca(2+) current activation and reduced inactivation are two mechanisms responsible for the enhanced L-type Ca(2+) current that contribute to the positive inotropic effects.

Electrophysiologic characterization of a novel hERG channel activator.

Activators of the human ether-a-go-go-related gene (hERG) K(+) channel have been reported recently to enhance hERG current amplitude (five synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound A-935142 ({4-[4-(5-trifluoromethyl-1H-pyrazol-3-yl)-phenyl]-cyclohexyl}-acetic acid) on guinea-pig atrial and canine ventricular action potentials (microelectrode techniques) and hERG channels expressed in HEK-293 cells (whole-cell patch clamp techniques). A-935142 shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current-voltage relationship revealed that this compound (60 microM) increased both outward and inward K(+) current as well as the slope conductance of the linear portion of the fully activated I-V relation. A-935142 significantly reduced the time constants (tau) of hERG channel activation at two example voltages (-10 mV: tau=100+/-17 ms vs. 164+/-24 ms, n=6, P<0.01; +30 mV: tau=16.7+/-1.8 ms vs. 18.9+/-1.8 ms, n=5, P<0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9 mV. The time course of hERG channel deactivation was slowed at multiple potentials (-120 to -70 mV). A-935142 also reduced the rate of inactivation and shifted the voltage-dependence of inactivation in the depolarizing direction by 15 mV. Recovery of hERG channel from inactivation was not affected by A-935142. In conclusion, A-935142 enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.

Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties.

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Functional consequences of methionine oxidation of hERG potassium channels.

Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in human ether à go-go related gene 1 (hERG1), which encodes cardiac I(Kr) channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 microM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to -60 or -100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or -10mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel.