GC-rich sequences can fold into G-quadruplexes and i-motifs and are known to control gene expression in many organisms. The potent G-quadruplex experimental anticancer drug QN-302 down-regulates a number of cancer-related genes, in particular S100P. Here we show this ligand has strong opposing effects with i-motif DNA structures and is one of the most potent i-motif destabilising agents reported to date. QN-302 down-regulates the expression of numerous cancer-related genes by pan-quadruplex targeting. QN-302 exhibits exceptional combined synergistic effects compared to many other G-quadruplex and i-motif interacting compounds. This work further emphasises the importance of considering G-quadruplex and i-motif DNA structures as one dynamic system

Alexandrou, Effrosyni

Guneri, Dilek

Neidle, Stephen

Waller, Zoë AE

English

UCL Discovery

Organic &Biomolecular ChemistryCOMMUNICATIONCite this: DOI: 10.1039/d3ob01464aReceived 12th September 2023,Accepted 24th October 2023DOI: 10.1039/d3ob01464arsc.li/obcQN-302 demonstrates opposing effects betweeni-motif and G-quadruplex DNA structures in thepromoter of the S100P gene†Effrosyni Alexandrou, Dilek Guneri, Stephen Neidle * andZoë A. E. Waller *GC-rich sequences can fold into G-quadruplexes and i-motifs andare known to control gene expression in many organisms. Thepotent G-quadruplex experimental anticancer drug QN-302down-regulates a number of cancer-related genes, in particularS100P. Here we show this ligand has strong opposing effects withi-motif DNA structures and is one of the most potent i-motif desta-bilising agents reported to date. QN-302 down-regulates theexpression of numerous cancer-related genes by pan-quadruplextargeting. QN-302 exhibits exceptional combined synergisticeffects compared to many other G-quadruplex and i-motif inter-acting compounds. This work further emphasises the importanceof considering G-quadruplex and i-motif DNA structures as onedynamic system.Promoter sequences within cancer-related genes frequentlycontain repeats of short G-tracts that can fold into higher-order quadruplex structures under appropriate conditions.1–5Their complementary C-tract strands can also fold into i-motifarrangements.6,7 Stabilization of these structures by appropri-ate small-molecule compounds can result in transcriptionalinhibition, and ultimately to cancer cell death.1,5 Several thou-sand such compounds have been described, and some showpromise as potential drug candidates.8–12 We have developedseveral series of substituted naphthalene diimidederivatives,13–15 and the most recent, QN-302 (Fig. 1), showshigh potency in cell growth inhibition assays, favourablepharmacological properties and antitumour activity in severalin vivo cancer models.16 The transcriptional profile in cancercells of genes down-regulated by QN-302 is in accordance withthe hypothesis that it is a pan-quadruplex stabilising agent,affecting genes in several important cancer-related pathways.16However, to date it cannot be excluded that QN-302 also stabil-ises i-motif structures formed on the complementary C-richstrand of G-quadruplex sequences. The present studyaddresses this issue with a major gene target as an exemplar.QN-302 is currently in clinical development with QualigenTherapeutics Inc. It has been granted Orphan DrugDesignation status for the treatment of pancreatic cancer,clearance has been granted by the FDA in the USA to proceedto clinical trials for human cancers, which are now underway.QN-302 down-regulates the expression of a number of sig-nificant cancer-related genes,16 including the S100P gene incancer cells and in a xenograft model of pancreatic cancer.17This gene codes for a small (10.4 kDa) calcium-bindingprotein and is highly upregulated in 70% of human pancreaticcancer patients, correlating with disease status.18,19 The S100Pprotein has been proposed as a plausible biomarker for diag-nostic purposes and as a therapeutic target in pancreaticcancer.20,21 The S100P promoter22 contains a C-rich sequencecontaining four C-tracts on the coding strand, 48 nucleotidesupstream from the transcription start site. The complementaryfour G-tract sequence on the template strand forms a stableG-quadruplex, which is further stabilised by QN-302.17 Here wereport on the biophysical characterisation and comparison ofboth the G-rich and C-rich sequences from the promoterregion of S100P.We initially characterised both the C-rich [5′-TCCCAACC-CCACTGTCCCACCCT-3′] and G-rich [5′- AGGGTGGGACAGT-GGGGTTGGGA-3′] sequences from the promoter region of theFig. 1 Structure of QN-302.†Electronic supplementary information (ESI) available: Experimental details,supporting UV melting, CD melting and CD titrations. See DOI: https://doi.org/10.1039/d3ob01464aSchool of Pharmacy, University College London, 29-39 Brunswick Square, London,WC1N 1AX, UK. E-mail: s.neidle@ucl.ac.uk, z.waller@ucl.ac.ukThis journal is © The Royal Society of Chemistry 2023 Org. Biomol. Chem.Open Access Article. Published on 06 November 2023. Downloaded on 11/21/2023 3:53:58 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView JournalS100P gene. All experiments were performed in 10 mM lithiumcacodylate and 100 mM KCl. The G-quadruplex formingsequence was examined at pH 7.0 and the i-motif at betweenpHs 4.0 and 8.0. UV melting and annealing experimentsshowed that the G-rich sequence had a Tm of 75.0 ± 0.2 °Cand a Ta of 73.3 ± 0.7 °C at pH 7.0 (Fig. S1†). This is con-sistent with our previous CD experiments indicating that theG-quadruplex structure formed would be highly stable underphysiological conditions.17 The complementary C-richsequence had a Tm of 45.4 ± 0.6 °C and a Ta of 43.0 ±0.0 °C at pH 5.5 (Fig. S2†). This Tm is similar to themelting temperature of other i-motifs of this length withthree-cytosine long tracks at the same pH.23 UV thermaldifference spectroscopy on the C-rich sequence showed posi-tive peaks at 240 and 265 nm and a negative peak at295 nm (Fig. 2, left), consistent with i-motif structure.24Circular dichroism studies at acidic pH gave a spectrumwith a positive peak at 288 nm and a negative peak at260 nm, which is also consistent with an i-motif structure25(Fig. 2, right). The i-motif forming sequence was found tohave a transitional pH (pHT) of 6.4, which indicates thatthis C-rich sequence can form an i-motif at near-neutralpH.23,26QN-302 is one of the most potent G-quadruplex bindingligands reported to date with a Kd of 4.9 nM for theG-quadruplex forming sequence from hTERT.15 It was pre-viously shown to stabilise the G-quadruplex from the promoterregion of S100P with ΔTm values of 7.4 ± 0.2 °C at 10 μM (1eq.), 17.0 ± 0.1 °C at 20 μM (2 eq.) and 20.0 ± 1.3 °C at 50 μM(5 eq.) (Table 1).17 These data indicates that QN-302 has astrong stabilising effect on the G-quadruplex structure formed.We then focused in detail on the effects of QN-302 on theC-rich sequence from the S100P promoter. ΔTm values in thepresence of QN-302 were determined in 10 mM lithium caco-dylate, 100 mM KCl at pH 5.5, where the S100P sequencewould be fully folded (Fig. 2). At 10 μM (1 eq.) of QN-302 theΔTm values were found to be −6.5 ± 1.7 °C, −14.3 ± 0.1 °C at20 μM (2 eq.) and −20.7 ± 1.1 °C at 50 μM (5 eq.), demonstrat-ing a dose-dependent destabilisation of i-motif structure byQN-302 (Fig. 3, Table 1 and Fig. S4†). Other knownG-quadruplex ligands such as berberine, BRACO-19, Phen-DC3, pyridostatin, RHPS4 and TmPyP4 have previously beenshown to destabilise i-motifs, but to a lesser extent.27–29 Forexample, BRACO-19 has a ΔTm values of −7.3 ± 0.7 °C for thei-motif forming sequence from the promoter region of the DAPgene28 and −13.4 ± 0.5 °C for the i-motif from the human telo-mere. These ΔTm values are significantly smaller compared toour observations with QN-302. Di Porzio, Galli et al. have syn-thesised bis-triazolyl-pyridine derivatives that appear to havehighly destabilising effects on the c-Myc and the hTelo i-motifswith ΔTm values of up to −29 ± 1 °C in one case. However, thisdestabilisation was achieved with double the number of ligandequivalents (10 molar equivalents) in phosphate buffer at pH5.0.29 These ligands did not have the same high stabilisingeffect on their respective G-quadruplexes as we observe withQN-302. To the best of our knowledge QN-302 is one of themost potent destabilising agents for i-motifs reported to date.Highly destabilising activity was also observed when QN-302was tested against the i-motif forming sequences from thehuman genome including the telomeric sequence (hTelo), theinsulin linked polymorphic region (ILPR) and the promoterregion of DAP (Fig. S5–S7 and Table S1†).To further investigate the destabilising effects of the S100Pi-motif by QN-302 CD titrations were performed (Fig. 4). Uponaddition of QN-302 at a concentration range from 0 to 110 μM,the CD signal intensity at 288 nm was found to decrease in adose-dependent fashion until a point at ∼50 μM beyond whichno further reduction in the ellipticity was observed. TheFig. 2 Left: thermal difference spectra of the C-rich i-motif formingS100P sequence: at 2.5 µM in 10 mM lithium cacodylate and 100 mMKCl buffer at pH 5.5. Right: CD spectra of the C-rich S100P sequence at10 µM DNA in 10 mM lithium cacodylate and 100 mM KCl buffer at pHas indicated. Inset: corresponding plot of ellipticity at 288 nm at thedifferent pHs to determine the transitional pH.Table 1 Change in melting temperature (ΔTm) of the S100PG-quadruplex and i-motif with QN-302 measured by CD meltingexperiments[QN-302] μMΔTm (°C) ΔTm (°C)S100P G – quadruplex17 S100P i – motif10 7.4 ± 0.2 −6.5 ± 1.720 17.0 ± 0.1 −14.3 ± 0.150 20.0 ± 1.3 −20.7 ± 1.1Fig. 3 Representative CD melting experiments with 10 µM S100Pi-motif in 10 mM lithium cacodylate 100 mM KCl buffer (pH 5.5), and 0,10, 20 or 50 µM QN-302, as indicated.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2023Open Access Article. Published on 06 November 2023. Downloaded on 11/21/2023 3:53:58 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinedecrease in the CD signal suggested ligand-dependent disrup-tion of the S100P i-motif, consistent with unfolding of thestructure to a single strand. This agrees with the CD meltingexperiments showing destabilisation. A plot of ellipticityagainst QN-302 concentration gave a sigmoidal-shaped curve(Fig. 4), indicative of a cooperative unfolding effect. By fittingthe sigmoidal-shaped curves to the Hill 1 equation usingOrigin software, we obtained Hill coefficients (n) of 2.3 ± 0.2.This reveals that the binding of QN-302 exhibits positive coop-erativity (n > 1) for the S100P i-motif. Additionally, the concen-tration of QN-302 that is required to reach 50% reduction ofthe molar ellipticity was determined to be 31 ± 4 μM.Analogous CD titration experiments with the G-quadruplexforming sequence showed no significant changes in topology(Fig. S3†). Indicating that the ligand is G-quadruplex stabilis-ing and has both i-motif destabilising and unfolding pro-perties. Taken together, the biophysical data illustrate thedynamic interplay of the two higher order DNA structures.To further compare the affinity of QN-302 for the S100Pi-motif and G-quadruplexes, UV titrations were performed(Fig. S8–S13 and Table S2†) and the dissociation constants (Kd)were determined. The Kd for the G-quadruplex (Kd = 2.0 ±0.3 µM) was found to be about six times lower than for thei-motif (Kd = 11.7 ± 2.9 µM), indicating that QN-302 has higheraffinity for G-quadruplex compared to i-motif. This was notunexpected, given QN-302 was designed to target G-quadruplexstructures.Numerous studies have shown that high expression of theS100P gene is correlated with pancreatic cancer progression inhumans.18–21 The proposed mode of action involves the stabil-isation of the G-quadruplex sequence in the promoter.17,22This stabilisation would inhibit transcription factor bindingand the progression of RNA polymerase, resulting in directdownregulation of S100P gene expression at the transcriptionallevel analogous to other ligands such as pyridostatin.16,17,30 Inthis study we have further examined the mechanistic details ofQN-302 interacting with the higher-order structures that canbe formed in this promoter region of the S100P gene and inparticular have examined the potential role of the i-motifformed by the C-rich strand. QN-302 has a strong destabilisingeffect on the S100P i-motif as it is illustrated by CD meltingand titration experiments. Therefore, QN-302 by stabilizing theG-quadruplex structure and destabilizing the i-motif structure,has a dual role and may exert a synergistic effect on the inhi-bition of transcription of the S100P gene. Ligand-inducedG-quadruplex stabilization inhibits gene expression whereasstabilization of i-motifs could activate transcription.31,32 Thishighlights the importance of evaluating the effects of a com-pound on both the i-motifs and the G-quadruplexes potentiallyformed from a duplex region of appropriate sequence. This isparticularly important given the fact that G-quadruplex andi-motif formation in cells are interdependent.33 In the case ofS100P, there is biological evidence that QN-302 can switch offgene expression17 which may be a consequence of both thestabilisation of the G-quadruplex and the destabilisation of thei-motif. This suggests how these two alternative structuresoperate together in the S100P promoter.The findings reported here demonstrate that QN-302 bothstrongly stabilizes the S100P promoter G-quadruplex andstrongly destabilizes the complementary i-motif in vitro. Thesedata are consistent with and supportive of previous con-clusions16 that QN-302 down-regulates the expression ofnumerous cancer-related genes by pan-quadruplex targeting.This is particularly important given the recent analysis ofTCGA PanCancer Atlas PDAC datasets that indicate poor prog-nosis in patients with high S100P expression.34 QN-302 exhi-bits exceptional combined synergistic effects compared tomany other G-quadruplex and i-motif interacting compounds.Overall, this work further emphasises the importance of con-sidering these two alternative DNA structures as one dynamicsystem and as one target.Data availabilityData is available on Figshare: 10.6084/m9.figshare.24476551.Author contributionsS. N. and Z. A. E. W. conceived the study, E. A, D. G. andZ. A. E. W. designed the experiments E. A. andD. G. performed the experiments E. A., D. G., S. N. andZ. A. E. W. wrote the paper, contributed to the manuscript revi-sion, and approved the final version.Conflicts of interestS. Neidle is a paid consultant and Advisory Board member ofQualigen Inc.AcknowledgementsD. G. is supported by the Biotechnology and BiologicalSciences Research Council grant BB/W001616/1.Fig. 4 Left: CD titration of the C-rich S100P sequence (10 µM) andQN-302 (0–110 µM) in 10 mM lithium cacodylate and 100 mM KClbuffer at pH 5.5. Right: plot of ellipticity at 288 nm against QN-302 con-centration and corresponding Hill 1 fitting.Organic & Biomolecular Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2023 Org. Biomol. Chem.Open Access Article. Published on 06 November 2023. Downloaded on 11/21/2023 3:53:58 PM.  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QN-302 demonstrates opposing effects between i-motif and G-quadruplex DNA structures in the promoter of the S100P gene

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QN-302 demonstrates opposing effects between i-motif and G-quadruplex DNA structures in the promoter of the S100P gene

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