The monohydrazone compound 15 identified by Amato et al. (J. Med. Chem. 2020) stabilizes G-quadruplexes (G4s) with a striking in-vitro preference: in a fluorescence-indicated displacement assay it displaces thiazole orange from the parallel telomeric G4 Tel23-p at DC50 = 0.48 μM but from the parallel c-Kit2 promoter G4 at DC50 = 7.80 μM, an approximately 16-fold difference between two G4s both assigned the parallel topology. In cancer cells the same compound stabilizes G4s and R loops and induces DNA damage that results mainly in cell killing together with micronucleus formation, yet the stabilized G4s are not enriched at telomeres. The source paper leaves open three questions: the molecular basis of the differential affinity (interactions with G4 loops and grooves versus π–π stacking on external G-tetrads); why the in-vitro telomeric preference does not translate into telomeric specificity in cells, including whether Tel23-p-like parallel G4s exist elsewhere in the genome as operative targets; and the biological role of G4 sequence selectivity in steering the outcome between direct cell killing and genome instability. The problem remains open.

Background

Guanine-rich nucleic acid sequences can fold into G-quadruplexes (G4s), four-stranded structures built from stacks of planar G-tetrads, each a hydrogen-bonded square of four guanines stabilized by a central cation. G4-forming motifs are enriched at telomeres — the repetitive DNA–protein caps that protect chromosome ends — and in oncogene promoters, and small molecules that bind and stabilize G4s are widely pursued as potential anticancer agents. A G4's topology — the relative orientation and connectivity of its four strands — determines whether its external G-tetrads are exposed for aromatic stacking: in parallel G4s all strands run in the same direction and the outer tetrads are fully exposed at the top and bottom, whereas hybrid topologies leave flanking segments partially covering the tetrads. The 23-nucleotide human telomeric repeat sequence forms the parallel G4 termed Tel23-p under the conditions used in the source study, while c-Kit2, a G4 formed in the promoter of the c-KIT oncogene, is also parallel; the two differ mainly in the length and base composition of the loops that connect their G-tract segments. The source study's CD data indicate, however, that Tel23-p is not a pure parallel G4 population under these conditions (a shoulder at about 290 nm in its CD spectrum). Ligand affinity is commonly quantified by fluorescence-indicated displacement (FID): a fluorescent probe (thiazole orange) pre-bound to the G4 is titrated with ligand, and DC50 is the ligand concentration displacing 50% of the probe. Building on an earlier bis-guanylhydrazone lead, Amato et al. synthesized a series of monohydrazone derivatives and identified compound 15, an indolinone–benzylidene monohydrazone, as the best of the series: selective for G4s over duplex DNA (duplex DC50 = 8.57 μM), preferring parallel over hybrid telomeric G4, and strongly preferring Tel23-p over c-Kit2. In cells, compound 15 killed both U2OS osteosarcoma and HeLa cells at low-micromolar concentrations; in U2OS cells it increased the number of nuclear foci detected by BG4, a structure-specific antibody that binds G4 structures, raised R loops (three-stranded RNA–DNA hybrid plus displaced single-stranded DNA structures linked to DNA damage), and tripled micronucleus frequency — micronuclei being membrane-bounded fragments of mis-segregated or broken chromosomes that serve as a readout of genome instability. Notably, immunofluorescence showed that most ligand-stabilized G4s were not at telomeres, echoing a known precedent in which even the classic ligand Braco-19, which inhibits telomerase (the enzyme that extends telomeres), stabilizes mostly non-telomeric G4s in cells. The authors proposed that loop and groove interactions, which differ between Tel23-p and c-Kit2, may explain the affinity difference — a hypothesis supported by the qualitatively different FID displacement curves for the two G4s — and speculated that Tel23-p-like parallel G4 structures elsewhere in the genome might be the compound's real cellular targets. They further proposed that the in-vivo target selectivity of G4 binders may modulate whether DNA damage manifests as cell death or as genome instability in surviving cells, and stated that establishing the biological role of G4-binder sequence selectivity is a question for future investigations. Since then, the same and adjacent groups have actively developed the biology of hydrazone G4 ligands — cytostatic immune-modulating activity at non-cytotoxic doses, interferon-β gene activation governed by the balance of G4 affinity and cytotoxic potency, and synthetic-lethal sensitization of cells deficient in FANCJ, a G4-resolving DNA helicase — but the three selectivity questions raised by the source paper have not been answered.

Problem Statement

For compound 15, the monohydrazone G4 ligand identified by Amato et al. (2020), establish the molecular and cellular determinants of G4 sequence selectivity and the biological role of that selectivity in the outcome of G4 stabilization, specifically: (i) determine the molecular basis of the approximately 16-fold difference in FID DC50 measured for compound 15 between the two nominally parallel G4s Tel23-p (DC50 = 0.48 μM) and c-Kit2 (DC50 = 7.80 μM) — that is, establish what underlies the differential response, whether different binding interactions of the ligand with the two G4s (contacts with G4 loops and grooves versus π–π stacking on the external G-tetrads, including their electrostatic and sequence-dependent components), the differing thiazole-orange displacement behavior reported for the two FID curves (complete displacement for c-Kit2 versus up to 80% for Tel23-p, where displacement might result from both direct and indirect competition), ligand-induced structural changes in the G4s, or a combination of these; (ii) explain why the in-vitro preference for the parallel telomeric G4 does not translate into telomeric specificity of G4 stabilization in cells, including whether Tel23-p-like parallel G4 structures elsewhere in the genome — whose existence the source paper noted cannot be excluded — are operative cellular targets of compound 15; and (iii) establish the biological role of G4 sequence selectivity in modulating the balance between direct cell killing and micronucleus-mediated genome instability, that is, whether, as the source paper proposed, the in-vivo target selectivity of G4 binders modulates the biological activity of the compound, either cell killing or the formation of micronuclei.

References

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