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Find video protocols related to scientific articles indexed in Pubmed.
Autophagy gene atg16l1 prevents lethal T cell alloreactivity mediated by dendritic cells.
Immunity
PUBLISHED: 09-13-2014
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Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.
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Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.
J. Clin. Invest.
PUBLISHED: 02-24-2014
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Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.
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Design, synthesis, and biological evaluation of estrone-derived hedgehog signaling inhibitors.
Tetrahedron
PUBLISHED: 12-27-2011
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The design, synthesis and biological evaluation of new analogs of the naturally occurring compound cyclopamine, a Hedgehog signaling inhibitor, are described. Stucture-activity relationship studies lead to an evolving model for the pharmacophore of this medically promising compound class of anti-cancer chemotherapeutic agents.
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STUDIES DIRECTED TOWARD THE SYNTHESIS OF NAKADOMARIN A.
Tetrahedron Lett.
PUBLISHED: 10-21-2011
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The application of a Pummerer-initiated tandem reaction cascade leads to the highly stereoselective formation of the tetracyclic core of Nakadomarin A.
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Studies directed toward the elucidation of the pharmacophore of steroid-based Sonic Hedgehog signaling inhibitors.
Org. Lett.
PUBLISHED: 09-09-2011
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Previous work from our laboratory has established that the readily available steroid-based analog 2 of cyclopamine 1 is, like 1, a highly potent inhibitor of Hedgehog signaling. The first structure-activity relationship studies on 2, i.e., the synthesis and biological evaluation of both the C-17 epi analog 4 and the C-3 deoxy analog 11, both of which are more potent than cyclopamine 1, are described. The implications of these results for the emerging pharmacophore of these Sonic Hedgehog signaling inhibitors are discussed.
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Stereoselective synthesis of F-ring saturated estrone-derived inhibitors of Hedgehog signaling based on cyclopamine.
Org. Lett.
PUBLISHED: 08-15-2011
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Previous work in this laboratory established that the readily available F-ring aromatic analog of cyclopamine is a highly potent inhibitor of Hedgehog signaling. The synthesis and biological evaluation of two F-ring saturated analogs that are more potent than the F-ring aromatic structure are reported.
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Discovery of substituted 3-(phenylamino)benzoic acids as potent and selective inhibitors of type 5 17?-hydroxysteroid dehydrogenase (AKR1C3).
Bioorg. Med. Chem. Lett.
PUBLISHED: 01-03-2011
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Aldo-keto reductase 1C3 (AKR1C3) also known as type 5 17?-hydroxysteroid dehydrogenase has been implicated as one of the key enzymes driving the elevated intratumoral androgen levels observed in castrate resistant prostate cancer (CRPC). AKR1C3 inhibition therefore presents a rational approach to managing CRPC. Inhibitors should be selective for AKR1C3 over other AKR1C enzymes involved in androgen metabolism. We have synthesized 2-, 3-, and 4-(phenylamino)benzoic acids and identified 3-(phenylamino)benzoic acids that have nanomolar affinity and exhibit over 200-fold selectivity for AKR1C3 versus other AKR1C isoforms. The AKR1C3 inhibitory potency of the 4-substituted 3-(phenylamino)benzoic acids shows a linear correlation with both electronic effects of substituents and the pK(a) of the carboxylic acid and secondary amine groups, which are interdependent. These compounds may be useful in treatment and/or prevention of CRPC as well as understanding the role of AKR1C3 in endocrinology.
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Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species.
J. Biol. Chem.
PUBLISHED: 12-30-2010
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The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O(2) yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species.
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Design and synthesis of inhibitors of Hedgehog signaling based on the alkaloid cyclopamine.
Org. Lett.
PUBLISHED: 06-26-2009
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The synthesis and biological evaluation of structurally simplified, metabolically stable cyclopamine-like Sonic Hedgehog (SHH) signaling inhibitors, i.e., 5, is described in four chemical steps from commercially available steroidal precursors. Biological evaluation of this cyclopamine analogue in two different systems establishes the high potency of 5 as a SHH signaling inhibitor. This approach provides important new lead structures for the development of new cancer chemotherapeutic agents based on the inhibition on SHH signaling.
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De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-09-2009
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The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.
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An unusual pathway to cyclobutane formation via desulfurative intramolecular photocycloaddition of an enone benzothiazoline pair.
Org. Lett.
PUBLISHED: 03-24-2009
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Irradiation of the enone benzothiazoline 3 leads to the formation of cyclobutane 5. Preliminary mechanistic studies establish the intermediacy of an enecarbamate 14 in this photochemical transformation, which could be the result of sulfur extrusion from an episulfide intermediate. Photocycloaddition of the enecarbamate intermediate 14 leads to the formation of "crossed" photoadducts, i.e., 5, in excellent yield, with high levels of regio- and stereochemical control.
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Lys05: a new lysosomal autophagy inhibitor.
Autophagy
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Lys05 is a previously undescribed dimeric chloroquine which more potently accumulates in the lysosome and blocks autophagy compared with HCQ. Lys05 produced more potent antitumor activity as a single agent both in vitro and in vivo in multiple human cancer cell lines and xenograft models compared with HCQ. Initial structure-activity relationship studies demonstrated that the increased activity associated with Lys05 was due to the bivalent aminoquinoline rings, C7-Chlorine, and a short triamine linker. While lower doses of Lys05 were well tolerated and associated with antitumor activity, at the highest dose tested, Lys05 produced Paneth cell dysfunction and intestinal toxicity, similar to what can be observed in mice and humans with genetic defects in the autophagy gene ATG16L1. Lys05 is therefore a new lysosomal autophagy inhibitor that has potential to be developed further into a drug for cancer and other medical applications.
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Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency.
Proc. Natl. Acad. Sci. U.S.A.
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Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer.
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Identification of a novel family of BRAF(V600E) inhibitors.
J. Med. Chem.
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The BRAF oncoprotein is mutated in about half of malignant melanomas and other cancers, and a kinase activating single valine to glutamate substitution at residue 600 (BRAF(V600E)) accounts for over 90% of BRAF-mediated cancers. Several BRAF(V600E) inhibitors have been developed, although they harbor some liabilities, thus motivating the development of other BRAF(V600E) inhibitor options. We report here the use of an ELISA based high-throughput screen to identify a family of related quinolol/naphthol compounds that preferentially inhibit BRAF(V600E) over BRAF(WT) and other kinases. We also report the X-ray crystal structure of a BRAF/quinolol complex revealing the mode of inhibition, employ structure-based medicinal chemistry efforts to prepare naphthol analogues that inhibit BRAF(V600E) in vitro with IC(50) values in the 80-200 nM range under saturating ATP concentrations, and demonstrate that these compounds inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed.
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Crystal structures of AKR1C3 containing an N-(aryl)amino-benzoate inhibitor and a bifunctional AKR1C3 inhibitor and androgen receptor antagonist. Therapeutic leads for castrate resistant prostate cancer.
Bioorg. Med. Chem. Lett.
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Castrate resistant prostate cancer (CRPC) is associated with increased androgen receptor (AR) signaling often brought about by elevated intratumoral androgen biosynthesis and AR amplification. Inhibition of androgen biosynthesis and/or AR antagonism should be efficacious in the treatment of CRPC. AKR1C3 catalyzes the formation of potent AR ligands from inactive precursors and is one of the most upregulated genes in CRPC. AKR1C3 inhibitors should not inhibit the related isoforms, AKR1C1 and AKR1C2 that are involved in 5?-dihydrotestosterone inactivation in the prostate. We have previously developed a series of flufenamic acid analogs as potent and selective AKR1C3 inhibitors [Adeniji, A. O. et al., J. Med. Chem.2012, 55, 2311]. Here we report the X-ray crystal structure of one lead compound 3-((4-(trifluoromethyl)phenyl) amino)benzoic acid (1) in complex with AKR1C3. Compound 1 adopts a similar binding orientation as flufenamic acid, however, its phenylamino ring projects deeper into a subpocket and confers selectivity over the other AKR1C isoforms. We exploited the observation that some flufenamic acid analogs also act as AR antagonists and synthesized a second generation inhibitor, 3-((4-nitronaphthalen-1-yl)amino)benzoic acid (2). Compound 2 retained nanomolar potency and selective inhibition of AKR1C3 but also acted as an AR antagonist. It inhibited 5?-dihydrotestosterone stimulated AR reporter gene activity with an IC(50)=4.7 ?M and produced a concentration dependent reduction in androgen receptor levels in prostate cancer cells. The in vitro and cell-based effects of compound 2 make it a promising lead for development of dual acting agent for CRPC. To illuminate the structural basis of AKR1C3 inhibition, we also report the crystal structure of the AKR1C3·NADP(+)·2 complex, which shows that compound 2 forms a unique double-decker structure with AKR1C3.
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Development of potent and selective inhibitors of aldo-keto reductase 1C3 (type 5 17?-hydroxysteroid dehydrogenase) based on N-phenyl-aminobenzoates and their structure-activity relationships.
J. Med. Chem.
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Aldo-keto reductase 1C3 (AKR1C3; type 5 17?-hydroxysteroid dehydrogenase) is overexpressed in castration resistant prostate cancer (CRPC) and is implicated in the intratumoral biosynthesis of testosterone and 5?-dihydrotestosterone. Selective AKR1C3 inhibitors are required because compounds should not inhibit the highly related AKR1C1 and AKR1C2 isoforms which are involved in the inactivation of 5?-dihydrotestosterone. NSAIDs, N-phenylanthranilates in particular, are potent but nonselective AKR1C3 inhibitors. Using flufenamic acid, 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid, as lead compound, five classes of structural analogues were synthesized and evaluated for AKR1C3 inhibitory potency and selectivity. Structure-activity relationship (SAR) studies revealed that a meta-carboxylic acid group relative to the amine conferred pronounced AKR1C3 selectivity without loss of potency, while electron withdrawing groups on the phenylamino B-ring were optimal for AKR1C3 inhibition. Lead compounds did not inhibit COX-1 or COX-2 but blocked the AKR1C3 mediated production of testosterone in LNCaP-AKR1C3 cells. These compounds offer promising leads toward new therapeutics for CRPC.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.