ABSTRACT
Maple syrup urine disease (MSUD) is an inborn error of branched-chain amino acid metabolism affecting several thousand individuals worldwide. MSUD patients have elevated levels of plasma leucine and its metabolic product α-ketoisocaproate (KIC), which can lead to severe neurotoxicity, coma, and death. Patients must maintain a strict diet of protein restriction and medical formula, and periods of noncompliance or illness can lead to acute metabolic decompensation or cumulative neurological impairment. Given the lack of therapeutic options for MSUD patients, we sought to develop an oral enzyme therapy that can degrade leucine within the gastrointestinal tract prior to its systemic absorption and thus enable patients to maintain acceptable plasma leucine levels while broadening their access to natural protein. We identified a highly active leucine decarboxylase enzyme from Planctomycetaceae bacterium and used directed evolution to engineer the enzyme for stability to gastric and intestinal conditions. Following high-throughput screening of over 12 000 enzyme variants over 9 iterative rounds of evolution, we identified a lead variant, LDCv10, which retains activity following simulated gastric or intestinal conditions in vitro. In intermediate MSUD mice or healthy nonhuman primates given a whey protein meal, oral treatment with LDCv10 suppressed the spike in plasma leucine and KIC and reduced the leucine area under the curve in a dose-dependent manner. Reduction in plasma leucine correlated with decreased brain leucine levels following oral LDCv10 treatment. Collectively, these data support further development of LDCv10 as a potential new therapy for MSUD patients.
Subject(s)
Maple Syrup Urine Disease , Humans , Mice , Animals , Leucine , Amino Acids, Branched-Chain , Proteins , Enzyme Therapy , Primates/metabolismABSTRACT
K2P (KCNK) potassium channels form "background" or "leak" currents that have critical roles in cell excitability control in the brain, cardiovascular system, and somatosensory neurons. Similar to many ion channel families, studies of K2Ps have been limited by poor pharmacology. Of six K2P subfamilies, the thermo- and mechanosensitive TREK subfamily comprising K2P2.1 (TREK-1), K2P4.1 (TRAAK), and K2P10.1 (TREK-2) are the first to have structures determined for each subfamily member. These structural studies have revealed key architectural features that underlie K2P function and have uncovered sites residing at every level of the channel structure with respect to the membrane where small molecules or lipids can control channel function. This polysite pharmacology within a relatively small (~70 kDa) ion channel comprises four structurally defined modulator binding sites that occur above (Keystone inhibitor site), at the level of (K2P modulator pocket), and below (Fenestration and Modulatory lipid sites) the C-type selectivity filter gate that is at the heart of K2P function. Uncovering this rich structural landscape provides the framework for understanding and developing subtype-selective modulators to probe K2P function that may provide leads for drugs for anesthesia, pain, arrhythmia, ischemia, and migraine.
Subject(s)
Neurons , Binding Sites , HumansABSTRACT
The trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the "Keystone inhibitor site" under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR "super-responders" having nanomolar sensitivity. Together, the data define a "finger in the dam" inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.
Subject(s)
Coloring Agents/pharmacology , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Ruthenium Compounds/pharmacology , Ruthenium Red/pharmacology , Amines/chemistry , Amines/pharmacology , Animals , Coloring Agents/chemistry , Crystallography, X-Ray , Mice , Molecular Docking Simulation , Potassium Channels, Tandem Pore Domain/chemistry , Potassium Channels, Tandem Pore Domain/metabolism , Ruthenium/chemistry , Ruthenium/pharmacology , Ruthenium Compounds/chemistry , Ruthenium Red/chemistryABSTRACT
K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) â¼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.
Subject(s)
Potassium Channels, Tandem Pore Domain/drug effects , Tetrahydronaphthalenes/pharmacology , Tetrazoles/pharmacology , Animals , HEK293 Cells , Humans , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/metabolism , Oocytes , Patch-Clamp Techniques , Potassium Channels/drug effects , Potassium Channels/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Xenopus laevisABSTRACT
For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic ß-subunit (CaVß). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVß interactions and reduce the entropic penalty associated with AID binding to CaVß. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVß interaction that modulate CaV function in an CaVß isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design.