Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel

Koster, Anna K.; Reese, Austin L; Kuryshev, Yuri A.; Wen, Xianlan; McKiernan, Keri A.; Gray, Erin E.; Wu, Caiyun; Huguenard, John R.; Maduke, Merritt; Bois, J. Du

doi:10.1073/pnas.2009977117

Cited by 15 publications

(45 citation statements)

References 81 publications

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“…These polar interactions are consistent with mutagenesis data. At 30 nM AK-42, inhibition of S392A is reduced ∼50% compared to WT, consistent with disruption of the observed hydrogen bond, while inhibition of K394A is not significantly different from WT, consistent with a backbone rather than a side-chain interaction (Koster et al, 2020; Ma et al, 2023). A third hydrogen bond – between K204 and the pyridine nitrogen of AK-42 – also contributes to AK-42’s stability in the binding pocket.…”

Section: Resultssupporting

confidence: 66%

“…This observation is surprising because WT CLC-2 has been characterized as passing little to no current at positive voltages (Park et al, 1998; Arreola et al, 2002). We initially considered that the increase in current at +80 mV could be due to the patch-clamp seal becoming leaky over time or that a background channel is being activated; however, such ‘leak’ currents would not be expected to be blocked by the small molecule inhibitor AK-42, which is highly selective for CLC-2 over all other CLCs and anion channels (Koster et al, 2020). AK-42 block of the augmented +80 mV currents seen at the end of each WT CLC-2 recording supports the idea that the hairpin peptide may contribute to the inward rectification of CLC-2 ( Figure 6F, Figure 6 – figure supplement 2 ).…”

Section: Resultsmentioning

confidence: 99%

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CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a ball-and-chain gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

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Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

Self Cite

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“…AK-42 is a small molecule that inhibits ClC-2 with nanomolar potency (IC 50 = 17 ± 1 nM) and rapidly and reversibly blocks ClC-2 currents. It displays unprecedented selectivity over ClC-1 and exhibits no off-target engagement against a panel of other common channels, receptors, and transporters expressed in brain tissue (Koster et al, 2020). This development provides a precise tool for future investigation on chloride-involved neurophysiology and the discovery of ClC-2-related therapeutics.…”

Section: Clc-2mentioning

confidence: 99%

Pharmacological modulation of chloride channels as a therapeutic strategy for neurological disorders

Wang

Choi²

2023

Front. Physiol.

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Chloride homeostasis is critical in the physiological functions of the central nervous system (CNS). Its concentration is precisely regulated by multiple ion-transporting proteins such as chloride channels and transporters that are widely distributed in the brain cells, including neurons and glia. Unlike ion transporters, chloride channels provide rapid responses to efficiently regulate ion flux. Some of chloride channels are also permeable to selected organic anions such as glutamate and γ-aminobutyric acid, suggesting neuroexcitatory and neuroinhibitory functions while gating. Dysregulated chloride channels are implicated in neurological disorders, e.g., ischemia and neuroinflammation. Modulation of chloride homeostasis through chloride channels has been suggested as a potential therapeutic approach for neurological disorders. The drug design for CNS diseases is challenging because it requires the therapeutics to traverse the blood-brain-barrier. Small molecules are a well-established modality with better cell permeability due to their lower molecular weight and flexibility for structure optimization compared to biologics. In this article, we describe the important roles of chloride homeostasis in each type of brain cells and introduce selected chloride channels identified in the CNS. We then discuss the contribution of their dysregulations towards the pathogenesis of neurological disorders, emphasizing the potential of targeting chloride channels as a therapeutic strategy for CNS disease treatment. Along with this literature survey, we summarize the small molecules that modulate chloride channels and propose the potential strategy of optimizing existing drugs to brain-penetrants to support future CNS drug discovery.

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“…4 For example, lead optimization generally involves adding one or two fragments at a time to a starting molecule, followed by iterative rounds of testing and further modifications. 5,6 In other cases, it is useful to add multiple fragments; in fragment-based drug discovery, multiple fragments are often added to a small starting molecule obtained from structural screening methods. 7−9 Expanding these starting molecules can result in more desirable drug properties, such as higher affinity and specificity for the protein target.…”

Section: ■ Introductionmentioning

confidence: 99%

“…A key part of this design process is adding chemical groups (termed “fragments” in this work) to a starting molecule known to bind to the target in order to tune its properties such as affinity, selectivity, and solubility . For example, lead optimization generally involves adding one or two fragments at a time to a starting molecule, followed by iterative rounds of testing and further modifications. , In other cases, it is useful to add multiple fragments; in fragment-based drug discovery, multiple fragments are often added to a small starting molecule obtained from structural screening methods. − Expanding these starting molecules can result in more desirable drug properties, such as higher affinity and specificity for the protein target. − However, in practice it is still exceedingly difficult to propose the optimal expansions as the desired properties are hard to predict a priori and the space of possible chemical modifications is vast. − …”

Section: Introductionmentioning

confidence: 99%

Geometric Deep Learning for Structure-Based Ligand Design

Powers,

Yu,

Suriana

et al. 2023

ACS Cent. Sci.

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A pervasive challenge in drug design is determining how to expand a ligand�a small molecule that binds to a target biomolecule�in order to improve various properties of the ligand. Adding single chemical groups, known as fragments, is important for lead optimization tasks, and adding multiple fragments is critical for fragmentbased drug design. We have developed a comprehensive framework that uses machine learning and three-dimensional protein−ligand structures to address this challenge. Our method, FRAME, iteratively determines where on a ligand to add fragments, selects fragments to add, and predicts the geometry of the added fragments. On a comprehensive benchmark, FRAME consistently improves predicted affinity and selectivity relative to the initial ligand, while generating molecules with more drug-like chemical properties than docking-based methods currently in widespread use. FRAME learns to accurately describe molecular interactions despite being given no prior information on such interactions. The resulting framework for quality molecular hypothesis generation can be easily incorporated into the workflows of medicinal chemists for diverse tasks, including lead optimization, fragment-based drug discovery, and de novo drug design.

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Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel

Cited by 15 publications

References 81 publications

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Pharmacological modulation of chloride channels as a therapeutic strategy for neurological disorders

Geometric Deep Learning for Structure-Based Ligand Design

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