Aromatic residues affecting permeation and gating in dSlo BK channels

Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site

Comparative study for the IMI2-NeuroDeRisk project on microelectrode arrays to derisk drug-induced seizure liability.

In the framework of the IMI2-NeuroDeRisk consortium, three in vitro electrophysiology assays were compared to improve preclinical prediction of seizure-inducing liabilities.Two cell models, primary rat cortical neurons and human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with hiPSC-derived astrocytes were tested on two different microelectrode array (MEA) platforms, Maestro Pro (Axion Biosystems) and Multiwell-MEA-System (Multi Channel Systems), in three separate laboratories. Pentylenetetrazole (PTZ) and/or picrotoxin (PTX) were included in each plate as positive (n = 3-6 wells) and ≤0.2% DMSO was used as negative controls (n = 3-12 wells). In general, concentrations in a range of 0.1-30 μM were tested, anchored, when possible, on clinically relevant exposures (unbound Cmax) were tested. Activity thresholds for drug-induced changes were set at 20%. To evaluate sensitivity, specificity and predictivity of the cell models, seizurogenic responses were defined as changes in 4 or more endpoints. Concentration dependence trends were also considered.Neuronal activity of 33 compounds categorized as positive tool drugs, seizure-positive or seizure-negative compounds was evaluated. Acute drug effects (<60 min) were compared to baseline recordings. Time points < 15 min exhibited stronger, less variable responses to many of the test agents. For many compounds a reduction and cessation of neuronal activity was detected at higher test concentrations. There was not a single pattern of seizurogenic activity detected, even among tool compounds, likely due to different mechanisms of actions and/or off-target profiles. A post-hoc analysis focusing on changes indicative of neuronal excitation is presented.All cell models showed good sensitivity, ranging from 70 to 86%. Specificity ranged from 40 to 70%. Compared to more conventional measurements of evoked activity in hippocampal slices, these plate-based models provide higher throughput and the potential to study subacute responses. Yet, they may be limited by the random, spontaneous nature of their network activity

FRESH™ 3D bioprinted cardiac tissue, a bioengineered platform for in vitro pharmacology

There is critical need for a predictive model of human cardiac physiology in drug development to assess compound effects on human tissues. In vitro two-dimensional monolayer cultures of cardiomyocytes provide biochemical and cellular readouts, and in vivo animal models provide information on systemic cardiovascular response. However, there remains a significant gap in these models due to their incomplete recapitulation of adult human cardiovascular physiology. Recent efforts in developing in vitro models from engineered heart tissues have demonstrated potential for bridging this gap using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in three-dimensional tissue structure. Here, we advance this paradigm by implementing FRESH™ 3D bioprinting to build human cardiac tissues in a medium throughput, well-plate format with controlled tissue architecture, tailored cellular composition, and native-like physiological function, specifically in its drug response. We combined hiPSC-CMs, endothelial cells, and fibroblasts in a cellular bioink and FRESH™ 3D bioprinted this mixture in the format of a thin tissue strip stabilized on a tissue fixture. We show that cardiac tissues could be fabricated directly in a 24-well plate format were composed of dense and highly aligned hiPSC-CMs at >600 million cells/mL and, within 14 days, demonstrated reproducible calcium transients and a fast conduction velocity of ∼16 cm/s. Interrogation of these cardiac tissues with the β-adrenergic receptor agonist isoproterenol showed responses consistent with positive chronotropy and inotropy. Treatment with calcium channel blocker verapamil demonstrated responses expected of hiPSC-CM derived cardiac tissues. These results confirm that FRESH™ 3D bioprinted cardiac tissues represent an in vitro platform that provides data on human physiological response

Oxabicyclooctane-Linked Novel Bacterial Topoisomerase Inhibitors as Broad Spectrum Antibacterial Agents

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC₅₀ > 18 μM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and <i>Staphylococcus aureus</i> gyrase (IC₅₀ = 1.02 μM) and topo IV (IC₅₀ = 10.4 μM). AM8191 showed parenteral and oral efficacy (ED₅₀) at less than 2.5 mg/kg doses in a <i>S. aureus</i> murine infection model. A cocrystal structure of AM8191 bound to <i>S. aureus</i> DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker

Time for a Fully Integrated Nonclinical-Clinical Risk Assessment to Streamline QTc Prolongation Liability Determinations: A Pharma Perspective

The appropriate and efficient assessment of drug-induced clinical QTc prolongation (a surrogate marker of Torsades de Pointes arrhythmia) remains a concern of drug developers and regulators worldwide. Refined and validated over 15+ years, the nonclinical ICH S7B guidance describes two core assays (in vitro hERG/IKr current & in vivo QT studies) to assess the potential for delayed ventricular repolarization. Incorporating these assays early in candidate selection has lead to: i) a low prevalence of QTc prolonging drugs in clinical trials, and ii) no drugs have been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical findings still minimally influence ICH E14-based strategies for interpreting clinical QTc prolongation, and defining proarrhythmic risk. In particular, the value of ICH S7B-based findings for candidates demonstrating minimal hERG/IKr block and minimal in vivo QTc prolongation at comparable clinical exposures (i.e., “double-negative nonclinical findings” are underestimated. Such reassuring nonclinical data has clear value assessing the risk of clinical QTc prolongation when clinical evaluation may be challenged by heart rate changes, exposure limitations, or safety considerations. The time has come to meaningfully merge nonclinical and clinical approaches to provide a more comprehensive and integrated proarrhythmia risk assessment strategy that complements advances described in ICH E14 amendments to enable more flexible clinical QTc risk assessments. Adoption of a truly integrative nonclinical/clinical risk assessment in the context of the low prevalence of QTc prolonging drugs would positively impact regulatory science, relieve the burden of extensive QTc monitoring, and streamline drug development

Rationale, design, and baseline characteristics in Evaluation of LIXisenatide in Acute Coronary Syndrome, a long-term cardiovascular end point trial of lixisenatide versus placebo

BACKGROUND: Cardiovascular (CV) disease is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). Furthermore, patients with T2DM and acute coronary syndrome (ACS) have a particularly high risk of CV events. The glucagon-like peptide 1 receptor agonist, lixisenatide, improves glycemia, but its effects on CV events have not been thoroughly evaluated. METHODS: ELIXA (www.clinicaltrials.gov no. NCT01147250) is a randomized, double-blind, placebo-controlled, parallel-group, multicenter study of lixisenatide in patients with T2DM and a recent ACS event. The primary aim is to evaluate the effects of lixisenatide on CV morbidity and mortality in a population at high CV risk. The primary efficacy end point is a composite of time to CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. Data are systematically collected for safety outcomes, including hypoglycemia, pancreatitis, and malignancy. RESULTS: Enrollment began in July 2010 and ended in August 2013; 6,068 patients from 49 countries were randomized. Of these, 69% are men and 75% are white; at baseline, the mean ± SD age was 60.3 ± 9.7 years, body mass index was 30.2 ± 5.7 kg/m(2), and duration of T2DM was 9.3 ± 8.2 years. The qualifying ACS was a myocardial infarction in 83% and unstable angina in 17%. The study will continue until the positive adjudication of the protocol-specified number of primary CV events. CONCLUSION: ELIXA will be the first trial to report the safety and efficacy of a glucagon-like peptide 1 receptor agonist in people with T2DM and high CV event risk