12 research outputs found
Analogs of Cinnamic Acid Benzyl Amide As Nonclassical Inhibitors of Activated JAK2 Kinase.
Scaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design. To identify the potential mechanisms of action of this class of compounds, direct interactions of the selected CABA analogs with JAK2 kinase were examined. Inhibition of JAK2 enzymatic activity was assessed, and molecular modeling studies of selected compounds-(E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide (WP1065), (E)-2-cyano-N-[(S)-1-phenylbutyl]- 3-(3-bromopyridin-2-yl)acrylamide (WP1130), and (E)-2-cyano-N-[(S)-1,4-diphenylbutyl]-3-(3-bromopyridin-2-yl)acrylamide (WP1702)-in the JAK2 kinase domain were used to support interpretation of the experimental data. Our results indicated that the tested CABA analogs are nonclassical inhibitors of activated (phosphorylated) JAK2, although markedly weaker than clinically tested ATP-competitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstrate-competitive. These results demonstrated that direct inhibition of JAK2 enzymatic activity by the WP1065 (half-maximal inhibitory concentration [IC50] = 14.8 ”M), WP1130 (IC50 = 3.8 ”M), and WP1702 (IC50 = 2.9 ”M) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathways in cancer cells and that additional specific structural modifications may amplify JAK2-inhibitory effects
Clinical characterization of the CMT2A patient harboring the p.Arg274Trp mutation.
<p>A. Wasting of distal muscles (upper and lower limbs) in the proband. B. Severe wasting of the small hand muscles and forearms in the proband. C. <i>Pes cavus</i> deformity in the proband.</p
The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient
<div><p>Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (<i>MFN2</i>). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a <i>de novo</i> Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions.</p></div
Mitochondrial morphology in control and proband-derived fibroblasts cultured for 72 hours in glucose and glucose-free medium.
<p>Fibroblasts were stained with 50 nM MitoTracker for 30 min in 37°C and visualized using confocal microscopy. Representative images of MitoTracker stained mitochondria are shown. Bar = 10 Όm.</p
GTP-hydrolysis activity of mitofusin 2 obtained from control and proband-derived fibroblasts.
<p>GTPase activity of control and mutated MFN2 presented as HPLC traces (A) and GDP/GTP ratio (B). Endogenous MFN2 was immunoprecipitated from 300 ÎŒg PNS lysates obtained from control (IP MFN2 C) and proband-derived fibroblasts (IP MFN2 Arg274Trp) and incubated with GTP at 37°C for 60 minutes, before HPLC analysis as described in Methods. In parallel, immunoprecipitation with nonspecific rabbit antibody was performed with PNS lysates obtained from control (IP IgG C) and proband-derived fibroblasts (IP IgG Arg274Trp). RBâreaction buffer containing 100 ÎŒM GTP.</p
The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient - Fig 2
<p>Morphology, lactate formation and oxygen consumption in proband-derived fibroblasts A. Transmitted light images of control and proband-derived fibroblasts cultured for 24 and 72 hours in glucose and glucose-free medium. Bar = 200 ÎŒm. B. Lactate synthesis was measured enzymatically in control and proband-derived fibroblasts cultured in glucose and glucose-free medium for 72 h. Data are expressed as mean amount of lactate (nanomoles per mg of protein) ± S.D.; n = 3, *p < 0.05. C. Oxygen consumption of cells grown in glucose or glucose-free media for 72 h was measured polarographically at 37°C. Data are expressed as pmoles O<sub>2</sub> x s<sup>-1</sup>/mg protein and show mean values ± S.D.; n = 2â3.</p
Mitochondrial DNA content was determined in control and proband-derived fibroblasts cultured for 72 hours in glucose and glucose-free medium by real time PCR.
<p>Quantification was based on mtDNA to nuclear Kir4.1 (nDNA) gene encoding ratio. Mean values and standard deviations were calculated from at least X independent experiments. *p<0.05</p
MFN2 GTP binding site and interactions.
<p>A. 3D structure of GTPase pocket with GTP ligand present. Oxygen atoms depicted in red, nitrogen in blue, phosphorus in purple. Carbon colors represent specific regions: G1 (brown), G2 (yellow), G3 (orange), G4 (mauve), G5 (green) and additional interacting residues (grey). Ligand carbons are cyan-colored. B. 2D scheme of GTP interactions with the MFN2 pocket.</p
Overall MFN2 model structure.
<p>Colors demonstrate domain division: GTPase (blue), HR-1 (green), HR-2 (yellow), and paddle region (orange). A. MFN2 monomer structure with visible GTP showed as Van der Waals spheres. B. Sequence-oriented MFN2 domain composition, with boundary residues. C. MFN2 homodimer embedded in lipid bilayer. Paddle residues 620â662 deeply embedded, residues 438â479 localized above hydrophobic layer.</p