Resolving the Ligand-Binding Specificity in c‑MYC G‑Quadruplex DNA: Absolute Binding Free Energy Calculations and SPR Experiment

We report the absolute binding free energy calculation and surface plasmon resonance (SPR) experiment for ligand binding with the c-MYC G-quadruplex DNA. The unimolecular parallel DNA G-quadruplex formed in nuclease hypersensitivity element III₁ of the c-MYC gene promoter regulates the c-MYC transcription and is recognized as an emerging drug target for cancer therapy. Quindoline derivatives have been shown to stabilize the G-quadruplex and inhibit the c-MYC expression in cancer cells. NMR revealed two binding sites located at the 5′ and 3′ termini of the G-quadruplex. Questions about which site is more favored and the basis for the ligand-induced binding site formation remain unresolved. Here, we employ two absolute binding free energy methods, the double decoupling and the potential of mean force methods, to dissect the ligand-binding specificity in the c-MYC G-quadruplex. The calculated absolute binding free energies are in general agreement with the SPR result and suggest that quindoline has a slight preference for the 5′ site. The flanking residues around the two sites undergo significant reorganization as the ligand unbinds, which provides evidence for ligand-induced binding pocket formation. The results help interpret experimental data and inform rational design of small molecules targeting the c-MYC G-quadruplex

The Major G‑Quadruplex Formed in the Human BCL‑2 Proximal Promoter Adopts a Parallel Structure with a 13-nt Loop in K⁺ Solution

The human BCL-2 gene contains a 39-bp GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of BCL-2 gene expression. Inhibition of BCL-2 expression can decrease cellular proliferation and enhance the efficacy of chemotherapy. Here we report the major G-quadruplex formed in the Pu39 G-rich strand in this BCL-2 promoter region. The 1245G4 quadruplex adopts a parallel structure with one 13-nt and two 1-nt chain-reversal loops. The 1245G4 quadruplex involves four nonsuccessive G-runs, I, II, IV, V, unlike the previously reported bcl2 MidG4 quadruplex formed on the central four G-runs. The parallel 1245G4 quadruplex with the 13-nt loop, unexpectedly, appears to be more stable than the mixed parallel/antiparallel MidG4. Parallel-stranded structures with two 1-nt loops and one variable-length middle loop are found to be prevalent in the promoter G-quadruplexes; the variable middle loop is suggested to determine the specific overall structure and potential ligand recognition site. A limit of 7 nt in loop length is used in all quadruplex-predicting software. Thus, the formation and high stability of the 1245G4 quadruplex with a 13-nt loop is significant. The presence of two distinct interchangeable G-quadruplexes in the overlapping region of the BCL-2 promoter is intriguing, suggesting a novel mechanism for gene transcriptional regulation and ligand modulation

Selective Lighting Up of Epiberberine Alkaloid Fluorescence by Fluorophore-Switching Aptamer and Stoichiometric Targeting of Human Telomeric DNA G‑Quadruplex Multimer

Aptamers, that exist naturally in living cells as functional elements and can switch nonfluorescent natural targets to fluorophores, are very useful in developing highly sensitive and selective biosensors and screening functional agents. This work demonstrates that human telomeric G-quadruplex (HTG) can serve as a potential fluorophore-switching aptamer (FSA) to target a natural isoquinoline alkaloid. We found that, among the G-quadruplexes studied here and the various structurally similar alkaloids including epiberberine (EPI), berberine (BER), palmatine (PAL), jatrorrhizine (JAT), coptisine (COP), worenine (WOR), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT), only the HTG DNA, especially with a 5′-TA-3′ residue at the 5′ end of the G-quadruplex tetrad (5′-TAG₃(TTAG₃)₃-3′, TA­[Q]) as the minimal sequence, is the most efficient FSA to selectively light up the EPI fluorescence. Compared to the 5′ end flanking sequences, the 3′ end flanking sequences of the tetrad contribute significantly less to the recognition of EPI. The binding affinity of EPI to TA­[Q] (<i>K</i>_d = 37 nM) is at least 20 times tighter than those of the other alkaloids. The steady-state absorption, steady-state/time-resolved fluorescence, and NMR studies demonstrate that EPI most likely interact with the 5′ end flanking sequence substructure beyond the core [Q] and the G-quadruplex tetrad in a much more specific manner than the other alkaloids. The highly selective and tight binding of EPI with the FSA and significantly enhanced fluorescence suggest the potential development of a selective EPI sensor (detection limit of 10 nM). More importantly, EPI, as the brightest FSA emitter among the alkaloids, can also serve as an efficient conformation probe for HTG DNA and discriminate the DNA G-quadruplex from the RNA counterpart. Furthermore, EPI can bind stoichiometrically to each G-quadruplex unit of long HTG DNA multimer with the most significant fluorescence enhancement, which has not been achieved by the previously reported probes. Our work suggests the potential use of EPI as a bioimaging probe and a therapeutic DNA binder

A Series of β‑Carboline Alkaloids from the Seeds of <i>Peganum harmala</i> Show G‑Quadruplex Interactions

In this study, we screened 17 medicinal plants for binding activity to G-quadruplex d­(TTGGGTT)₄ by ¹H NMR spectroscopy and found that the crude extract of <i>Peganum harmala</i> L. seeds showed the most potential binding activity. Subsequently, ¹H NMR- and bioassay-guided isolation of the extract of <i>P. harmala</i> L. was performed to obtain four pairs of partially racemized β-carboline alkaloids, pegaharmines A–D (<b>1</b>–<b>4</b>). Their structures and absolute configurations were determined by extensive NMR analyses, X-ray crystallography, ECD calculations, and CD exciton chirality approaches. Interestingly, pegaharmine D (<b>4</b>), which showed the strongest G-quadruplex interaction, exhibited significant cytotoxic activity against three cancer cell lines. This work contributed a practical strategy for the discovery of novel G-quadruplex ligands from natural products and provided potential insights for using β-carboline alkaloids as anticancer lead compounds specifically targeting G-quadruplexes

Structurally Diverse Alkaloids from the Seeds of Peganum harmala

Investigation of the alkaloids from Peganum harmala seeds yielded two pairs of unique racemic pyrroloindole alkaloids, (±)-peganines A–B (<b>1</b>–<b>2</b>); two rare thiazole derivatives, peganumals A–B (<b>3</b>–<b>4</b>); six new β-carboline alkaloids, pegaharmines F–K (<b>5</b>–<b>10</b>); and 12 known analogues. Their structures, including stereochemistry, were elucidated through spectroscopic analyses, quantum chemistry calculations, and single-crystal X-ray diffraction. Notably, the incorporation of pyrrole and indole moieties in peganines A–B, thiazole fragments in peganumals A–B, and a C-1 α,β-unsaturated ester motif in pegaharmine F (<b>5</b>) are all rare, and their presence in the genus <i>Peganum</i> were demonstrated for the first time. All isolates were tested for antiproliferative activities against the HL-60, PC-3, and SGC-7901 cancer cell lines, and compounds <b>9</b>, <b>11</b>, <b>12</b>, and <b>13</b> exhibited moderate cytotoxicity against HL-60 cancer cell lines with IC₅₀ values in the range of 4.36–9.25 μM