Toward improved characterization of biologically relevant isomeric and isobaric ions on mass spectrometry-based platforms

Mass spectrometry has frequently been employed in the analysis of biologically relevant molecules; however, mass spectrometry alone may not always be sufficient for the differentiation and characterization of isomeric and isobaric ions. In this work, infrared multiple photon dissociation (IRMPD) spectroscopy and ion mobility spectrometry (IMS) were evaluated as complementary techniques for the characterization and separation of isomeric and isobaric ions of biological relevance. In the first project, analysis of experimental IRMPD spectroscopy data shows that this technique is useful in the differentiation of hydroxyproline isomers. Absorption bands allow for the differentiation of three isomeric species: 1640 cm-1 (trans-4-hydroxyproline), 1718 cm-1 (cis-4-hydroxyproline), and 1734 cm-1 (cis-3-hydroxyproline). In the second project, theoretical CCS and IR spectroscopy predictions of isobaric modified amino acids and isomeric drugs have been carried out as predictions of IMS and IRMPD spectroscopy suitability. Preliminary IMS measurements suggest that the CCS predictions are at least qualitatively useful

A Combined Infrared Ion Spectroscopy and Computational Chemistry Study of Hydroxyproline Isomers

Contains fulltext : 221339.pdf (publisher's version ) (Closed access

Targeting a Novel G-Quadruplex in the <i>CARD11</i> Oncogene Promoter with Naptho(2,1-b)furan-1-ethanol,2-nitro- Requires the Nitro Group

The aggressive nature of the activated B cell such as (ABC) subtype of diffuse large B cell (DLBCL) is frequently associated with altered B cell Receptor (BCR) signaling through the activation of key components including the scaffolding protein, CARD11. Most inhibitors, such as ibrutinib, target downstream BCR kinases with often modest and temporary responses for DLBCL patients. Here, we pursue an alternative strategy to target the BCR pathway by leveraging a novel DNA secondary structure to repress transcription. We discovered that a highly guanine (G)-rich element within the CARD11 promoter forms a stable G-quadruplex (G4) using circular dichroism and polymerase stop biophysical techniques. We then identified a small molecule, naptho(2,1-b)furan-1-ethanol,2-nitro- (NSC373981), from a fluorescence-resonance energy transfer-based screen that stabilized CARD11 G4 and inhibited CARD11 transcription in DLBCL cells. In generating and testing analogs of NSC373981, we determined that the nitro group is likely essential for the downregulation of CARD11 and interaction with CARD11 G4, and the removal of the ethanol side chain enhanced this activity. Of note, the expression of BCL2 and MYC, two other key oncogenes in DLBCL pathology with known promoter G4 structures, were often concurrently repressed with NSC373981 and the highly potent R158 analog. Our findings highlight a novel approach to treat aggressive DLBCL by silencing CARD11 gene expression that warrants further investigation

Supplementary Figures and Tables from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Supplemental Methods, Supplementary Figures S1-11, Supplementary tables S2, 3, and 5.</p

Figure S3 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Original blot images from Figures 2E and 2F in the main text. (A) Uncropped blot of Figure 2E immunoblotted with a mixture of anti-NEK2 and anti-TBP primary antibodies and imaged as a composite of both Dylight 650 and 800 channels. (B) Uncropped blot of Figure 2F immunoblotted with a mixture of anti-NEK2 and anti-ACTIN primary antibodies. The blot is shown as imaged in each individual channel. Both blots were resolved with a mixture of Dylight 650 and Dylight 800 secondary antibodies and imaged using the BioRad ChemiDoc MP as described in the Methods.</p

Table S5 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Growth inhibitory concentrations (GI50) of NBI-961, doxorubicin and vincristine.</p

Figure S2 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

1H NMR spectra of NBI-961. Purity of NBI-961 was determined by 1H NMR after dissolved in methanol. Spectra was recorded using an Agilent HPLC with a run time of 15.01 mins and a flow rate of 0.8 mL/min.</p

Table S8 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Differentially expressed proteins identified from the total proteomic analysis in RIVA treated with NBI-961 relative to DMSO vehicle control.</p

Figure S5 from Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma

Original blot images from Supplementary Figure S9. Uncropped blot images of (A) Figure S9A and (B) Figure S9B. The blot in (B) also contained samples from a replicate of the SUDHL5 untreated cell lysate that was incubated without and with phosphatase with an additional molecular weight ladder to separate the two sample sets. Each blot was imaged in both the Dylight 650 and 800 channels and shown as the respective image (NEK2 from 650 in top panels and ACTIN from 800 in lower panels). All blots were immunoblotted with a mixture of anti-NEK2 and anti-ACTIN primary antibodies, resolved with a mixture of secondary antibodies Dylight 650 and Dylight 800 and imaged using the BioRad ChemiDoc MP as described in the methods.</p