Modulation of Ligand-Gated Glycine Receptors Via Functional Monoclonal Antibodies.

Ion channels are targets of considerable therapeutic interest to address a wide variety of neurologic indications, including pain perception. Current pharmacological strategies have focused mostly on small molecule approaches that can be limited by selectivity requirements within members of a channel family or superfamily. Therapeutic antibodies have been proposed, designed, and characterized to alleviate this selectivity limitation; however, there are no Food and Drug Administration-approved therapeutic antibody-based drugs targeting ion channels on the market to date. Here, in an effort to identify novel classes of engineered ion channel modulators for potential neurologic therapeutic applications, we report the generation and characterization of six (EC50 < 25nM) Cys-loop receptor family monoclonal antibodies with modulatory function against rat and human glycine receptor alpha 1 (GlyRα1) and/or GlyRα3. These antibodies have activating (i.e., positive modulator) or inhibiting (i.e., negative modulator) profiles. Moreover, GlyRα3 selectivity was successfully achieved for two of the three positive modulators identified. When dosed intravenously, the antibodies achieved sufficient brain exposure to cover their calculated in vitro EC50 values. When compared head-to-head at identical exposures, the GlyRα3-selective antibody showed a more desirable safety profile over the nonselective antibody, thus demonstrating, for the first time, an advantage for GlyRα3-selectivity. Our data show that ligand-gated ion channels of the glycine receptor family within the central nervous system can be functionally modulated by engineered biologics in a dose-dependent manner and that, despite high protein homology between the alpha subunits, selectivity can be achieved within this receptor family, resulting in future therapeutic candidates with more desirable drug safety profiles. SIGNIFICANCE STATEMENT: This study presents immunization and multiplatform screening approaches to generate a diverse library of functional antibodies (agonist, potentiator, or inhibitory) raised against human glycine receptors (GlyRs). This study also demonstrates the feasibility of acquiring alpha subunit selectivity, a desirable therapeutic profile. When tested in vivo, these tool molecules demonstrated an increased safety profile in favor of GlyRα3-selectivity. These are the first reported functional GlyR antibodies that may open new avenues to treating central nervous system diseases with subunit selective biologics.

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.

Activation of the epithelial sodium channel by the metalloprotease meprin ß subunit.

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin ß (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin ß or α/ß in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin ß, but not the α subunit. Meprin ß promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin ß regulates the activity of ENaC in a metalloprotease-dependent fashion.

Effect of macrolides on in vivo ion transport across cystic fibrosis nasal epithelium.

Fourteen- and 15-member macrolide antibiotics are under investigation as potential therapeutic agents for cystic fibrosis (CF). The nonantibiotic mechanisms of action of these compounds in CF are not understood. We used nasal potential difference (NPD) measurements to test the effect of macrolides on airway epithelial ion (chloride, sodium) transport of CF mice and humans. We tested clarithromycin and azithromycin in mice, and clarithromycin in patients with CF. Baseline and post-treatment NPD was measured in two strains (C57Bl6 and BalbC) of CF transmembrane regulator "knockout" and littermate control mice, and in DeltaF508/DeltaF508 mice. In addition, NPD was measured in 18 human subjects with CF (17 DeltaF-508/DeltaF-508 and 1 DeltaF-508/other) who were undergoing a 12-month, randomized, double-blind crossover study of the effects of clarithromycin on pulmonary outcome in CF. Neither clarithromycin nor azithromycin affected ion transport characteristics of normal or CF nasal epithelium in either mouse or humans. We conclude that the apparent beneficial effects of macrolides on pulmonary outcome in CF are not mediated by their modulation of ion transport.

Pulmonary oedema fluid induces non-alpha-ENaC-dependent Na(+) transport and fluid absorption in the distal lung.

To determine if pulmonary oedema fluid (EF) alters ion and fluid transport of distal lung epithelium (DLE), EF was collected from rats in acute heart failure. EF, but not plasma, increased amiloride-insensitive short circuit current (I(sc)) and Na(+)-K(+) ATPase protein content and pump activity of DLE grown in primary culture. Inhibitors of Cl(-) transport or cGMP-gated cation channels had a significant (P < 0.05), but limited ability to block the increased I(sc). EF increased amiloride-insensitive, but not amiloride-sensitive, DLE apical membrane Na(+) conductance. The level of mRNA encoding epithelial sodium channel (ENaC) subunits was unchanged (alpha, beta), or decreased (gamma, P < 0.05) in EF-exposed DLE. EF also induced an amiloride-insensitive increase in the potential difference across murine tracheal cysts. Distal lung explants from late gestation wild-type and alpha-ENaC-deficient fetal mice, which normally expand due to liquid secretion, decreased in size due to liquid absorption when exposed to EF. Trypsin digestion or heat treatment of EF abrogated the ability of EF to increase amiloride-insensitive I(sc) in DLE and liquid absorption by distal lung explants. Thus proteins or protein-dependent factors within cardiogenic EF induce an alpha-ENaC-independent and amiloride-insensitive apical membrane Na(+) conductance and liquid absorption in the distal lung.