Influence of Solvent Polarity and DNA-Binding on Spectral Properties of Quaternary Benzo[<i>c</i>]phenanthridine Alkaloids

<div><p>Quaternary benzo[<i>c</i>]phenanthridine alkaloids are secondary metabolites of the plant families <i>Papaveraceae</i>, <i>Rutaceae</i>, and <i>Ranunculaceae</i> with anti-inflammatory, antifungal, antimicrobial and anticancer activities. Their spectral changes induced by the environment could be used to understand their interaction with biomolecules as well as for analytical purposes. Spectral shifts, quantum yield and changes in lifetime are presented for the free form of alkaloids in solvents of different polarity and for alkaloids bound to DNA. Quantum yields range from 0.098 to 0.345 for the alkanolamine form and are below 0.033 for the iminium form. Rise of fluorescence lifetimes (from 2–5 ns to 3–10 ns) and fluorescence intensity are observed after binding of the iminium form to the DNA for most studied alkaloids. The alkanolamine form does not bind to DNA. Acid-base equilibrium constant of macarpine is determined to be 8.2–8.3. Macarpine is found to have the highest increase of fluorescence upon DNA binding, even under unfavourable pH conditions. This is probably a result of its unique methoxy substitution at C₁₂ a characteristic not shared with other studied alkaloids. Association constant for macarpine-DNA interaction is 700000 M^-1.</p></div

Structure, numbering and acid-base equilibrium of studied QBAs.

<p>Structure, numbering and acid-base equilibrium of studied QBAs.</p

Lippert–Mataga plot of QBAs.

<p>Black–macarpine, red–sanguilutine, blue–chelirubine, green–sanguinarine, gold–sanguirubine, violet–chelerythrine; 1 –benzene, 2 –diethyl ether, 3 –octanol, 4 –ethanol, 5 –methanol, 6–0.01M borate buffer, pH 9.45. Samples in borate buffer (dashed box) excluded from fitting.</p

Representative fit of macarpine-DNA interaction.

<p>Macarpine (10 μM) binding to salmon testes DNA (0–116 μM bp) in 0.05 M citrate buffer, pH 6.15, [Na⁺] = 0.122 M was measured as a fluorescence change at 625 nm. Data were fitted to 1:1 binding model using DynaFit software.</p

Acid-base properties of 3 μM macarpine.

<p>Dependence of absorbance at 495 nm (black) and fluorescence at 450 nm (red) on pH, n = 3. Mean values ± SD and fits to Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.e004" target="_blank">4</a>) are shown. Inset: Absorption (black) and emission (red) spectra of iminium (—) and alkanolamine (…) form. Ordinates are the same as for bigger figure.</p

Absorbance and fluorescence spectra of QBAs in absence and presence of ctDNA.

<p>QBAs (3 μM in 20mM acetate buffer, 200 mM NaCl, 2mM EDTA, pH 5) in absence (—) and presence (…) of ctDNA (DNA base pair-to-drug ratio 15.9:1). a-f–absorption spectra, g-l–emission spectra; a, g–sanguinarine, b, h–chelerythrine, c, i–chelirubine, d, j–sanguilutine, e, k–sanguirubine, f, l–macarpine. Note that intensities are in arbitrary units due to conversion to wavenumber scale using Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.e001" target="_blank">1</a>).</p

Spectroscopic properties of alkanolamine (QOH) and iminium (Q⁺) forms of QBAs.

<p>Where two results were obtained (pK_ROH by two methods, two lifetimes), second value is indicated in brackets.</p><p>a) all QBAs except macarpine ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.ref030" target="_blank">30</a>].</p><p>b) this work, spectrophotometry, n = 3, SE = 0.02</p><p>c) this work, spectrofluorometry, n = 3, SE = 0.06</p><p>d) n = 4, estimated RSD ca. 15%</p><p>e) n = 3, SE ≤ 0.1 ns</p><p>f) 3.3 ns (ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.ref022" target="_blank">22</a>])</p><p>g) 3.5 ns (610 nm)</p><p>h) 5.8 ns (610 nm)</p><p>i) 2.4 ns (ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.ref022" target="_blank">22</a>])</p><p>Spectroscopic properties of alkanolamine (QOH) and iminium (Q⁺) forms of QBAs.</p

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1–5. Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein6—is phosphorylated during spliceosome activation7–10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing