3 research outputs found
Simultaneous Targeting of NPC1 and VDAC1 by Itraconazole Leads to Synergistic Inhibition of mTOR Signaling and Angiogenesis
The
antifungal drug itraconazole was recently found to exhibit
potent antiangiogenic activity and has since been repurposed as an
investigational anticancer agent. Itraconazole has been shown to exert
its antiangiogenic activity through inhibition of the mTOR signaling
pathway, but the molecular mechanism of action was unknown. We recently
identified the mitochondrial protein VDAC1 as a target of itraconazole
and a mediator of its activation of AMPK, an upstream regulator of
mTOR. However, VDAC1 could not account for the previously reported
inhibition of cholesterol trafficking by itraconazole, which was also
demonstrated to lead to mTOR inhibition. In this study, we demonstrate
that cholesterol trafficking inhibition by itraconazole is due to
direct inhibition of the lysosomal protein NPC1. We further map the
binding site of itraconazole to the sterol-sensing domain of NPC1
using mutagenesis, competition with U18666A, and molecular docking.
Finally, we demonstrate that simultaneous AMPK activation and cholesterol
trafficking inhibition leads to synergistic inhibition of mTOR, endothelial
cell proliferation, and angiogenesis
Activation of the c-Jun N-terminal Kinase/Activating Transcription Factor 3 (ATF3) Pathway Characterizes Effective Arylated Diazeniumdiolate-Based Nitric Oxide-Releasing Anticancer Prodrugs
Improved therapies are needed for nonsmall cell lung
cancer. Diazeniumdiolate-based
nitric oxide (NO)-releasing prodrugs are a growing class of promising
NO-based therapeutics. Recently, we have shown that <i>O</i><sup>2</sup>-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate
(JS-K, <b>1</b>) is effective against nonsmall cell lung cancer
(NSCLC) cells in culture and in vivo. Here we report mechanistic studies
with compound <b>1</b> and its homopiperazine analogue and structural
modification of these into more stable prodrugs. Compound <b>1</b> and its homopiperazine analogue were potent cytotoxic agents against
NSCLC cells in vitro and in vivo, concomitant with activation of the
SAPK/JNK stress pathway and upregulation of its downstream effector
ATF3. Apoptosis followed these events. An aryl-substituted analogue,
despite extended half-life in the presence of glutathione, did not
activate JNK or have antitumor activity. The data suggest that rate
of reactivity with glutathione and activation of JNK/ATF3 are determinants
of cancer cell killing by these prodrugs
Rapamycin-inspired macrocycles with new target specificity
Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.</p