Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

The acid-sensitive, anesthetic-activated potassium leak channel, KCNK3, is regulated by 14-3-3ß-dependent, protein kinase C (PKC)-mediated endocytic trafficking.

The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3ß and a novel potassium channel endocytic motif, because depleting either 14-3-3ß protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3ß-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs.

Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease.

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel ß-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ∼20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.

O-glycosylation of the cardiac I(Ks) complex.

Post-translational modifications of the KCNQ1­KCNE1 (Kv7) K+ channel complex are vital for regulation of the cardiac IKs current and action potential duration. Here, we show the KCNE1 regulatory subunit is O-glycosylated with mucin-type glycans in vivo. As O-linked glycosylation sites are not recognizable by sequence gazing, we designed a novel set of glycosylation mutants and KCNE chimeras and analysed their glycan content using deglycosylation enzymes. Our results show that KCNE1 is exclusively O-glycosylated at Thr-7, which is also required for N-glycosylation at Asn-5. For wild type KCNE1, the overlapping N- and O-glycosylation sites are innocuous for subunit biogenesis; however, mutation of Thr-7 to a non-hydroxylated residue yielded mostly unglycosylated protein and a small fraction of mono-N-glycosylated protein. The compounded hypoglycosylation was equally deleterious for KCNQ1­KCNE1 cell surface expression, demonstrating that KCNE1 O-glycosylation is a post-translational modification that is integral for the proper biogenesis and anterograde trafficking of the cardiac IKs complex. The enzymatic assays and panel of glycosylation mutants used here will be valuable for identifying the different KCNE1 glycoforms in native cells and determining the roles N- and O-glycosylation play in KCNQ1­KCNE1 function and localization in cardiomyocytes,

Chemical control of metabolically-engineered voltage-gated K+ channels.

Metabolic oligosaccharide engineering is a powerful approach for installing unnatural glycans with unique functional groups into the glycocalyx of living cells and animals. Using this approach, we showed that K(+) channel complexes decorated with thiol-containing sialic acids were irreversibly inhibited with scorpion toxins bearing a pendant maleimide group. Irreversible inhibition required a glycosylated K(+) channel subunit and was completely reversible with mild reductant when the tether connecting the toxin to the maleimide contained a disulfide bond. Cleavage of the disulfide bond not only restored function, but delivered a biotin moiety to the modified K(+) channel subunit, providing a novel approach for preferentially labeling wild type K(+) channel complexes functioning in cells.

Identification of a protein-protein interaction between KCNE1 and the activation gate machinery of KCNQ1.

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K(+) channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein-protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4-S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1-KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K(+) channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1-KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1-KCNE1 cytoplasmic region where these protein-protein interactions are poised to slow activation gate opening.

Rearrangements in the relative orientation of cytoplasmic domains induced by a membrane-anchored protein mediate modulations in Kv channel gating.

Interdomain interactions between intracellular N and C termini have been described for various K(+) channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the "docking loop" at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.

Formation of the full SNARE complex eliminates interactions of its individual protein components with the Kv2.1 channel.

Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.

VAMP2 interacts directly with the N terminus of Kv2.1 to enhance channel inactivation.

Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.