The identification of small molecules that positively modulate the mitochondrial respiratory function has broad applications in fundamental research, therapeutic target validation, and drug ...discovery. We present an approach in which primary screens for mitochondrial function in yeast are used to efficiently identify a subset of high-value compounds that can in turn be rapidly tested against a broad range of mammalian cell lines. The ability of the yeast assay to successfully identify in a high-throughput format hit compounds that increase the mitochondrial membrane potential and adenosine triphosphate (ATP) levels by as little as 15% was demonstrated. In this study, 14 hits were identified from a collection of 13,680 compounds. Secondary testing with myotubes, fibroblasts, and PC-12 and HepG2 cells identified two compounds increasing ATP levels in hepatocytes and two other compounds increasing ATP in fibroblasts. The effect on hepatocytes was further studied using genomic and mitochondrial proteomic tools to characterize the changes induced by the two compounds. Changes in the accumulation of a series of factors involved in early gene response or apoptosis or linked to metabolic functions (i.e., β-Klotho, RORα, PGC-1α, G6PC, IGFBP1, FTL) were discovered.
The β‐subunit of voltage‐gated Ca2+ channels is essential for trafficking the channels to the plasma membrane and regulating their gating. It contains a Src homology 3 (SH3) domain and a guanylate ...kinase (GK) domain, which interact intramolecularly. We investigated the structural underpinnings of this intramolecular coupling and found that in addition to a previously described SH3 domain β strand, two structural elements are crucial for maintaining a strong and yet potentially modifiable SH3–GK intramolecular coupling: an intrinsically weak SH3–GK interface and a direct connection of the SH3 and GK domains. Alterations of these elements uncouple the two functions of the β‐subunit, degrading its ability to regulate gating while leaving its chaperone effect intact.
A series of conformationally restricted acetanilides were synthesized and evaluated as β3-adrenergic receptor agonists (β3-AR) for the treatment of overactive bladder (OAB). Optimization studies ...identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine β3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent β3-AR mediated responses in a rat bladder hyperactivity model.
The β-subunit of voltage-gated Ca
2+ channels is essential for trafficking the channels to the plasma membrane and regulating their gating. It contains a Src homology 3 (SH3) domain and a guanylate ...kinase (GK) domain, which interact intramolecularly. We investigated the structural underpinnings of this intramolecular coupling and found that in addition to a previously described SH3 domain β strand, two structural elements are crucial for maintaining a strong and yet potentially modifiable SH3–GK intramolecular coupling: an intrinsically weak SH3–GK interface and a direct connection of the SH3 and GK domains. Alterations of these elements uncouple the two functions of the β-subunit, degrading its ability to regulate gating while leaving its chaperone effect intact.
The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the ...S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.
The inner pore of voltage-gated Ca super(2+) channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K super(+) channels, this part of the pore is ...formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca super(2+) channel pore, however, is formed by four asymmetric domains of the same ( alpha sub(1)) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca super(2+) channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha sub(1) subunit (Ca sub(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K super(+) channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K super(+) channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca super(2+) channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 Aa x 10 Aa x 6 Aa suggests that the inner pore can open to >10 Aa. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.
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