Cellular uptake and binding of guanidine-modified phthalocyanines to KRAS/HRASG-quadruplexes

Abstract

Guanidino-modified phthalocyanines are evaluated in vitro (polymerase-stop assays and FRET) and in cultured cells as G4-DNA ligands and modulators of gene transcription.The hypothesis that G-quadruplex DNA (or G4-DNA) is involved in transcription regulation is gaining support. 1 Recent studies have shown that, in addition to the telomeres, G4-DNA motifs are found with a high frequency in the regions surrounding transcription start sites of many genes. 2 G-quadruplex structures have been identified in protooncogenes as well as in 5 0 -untranslated regions of mRNA. 3 Several studies have suggested that G4-DNA affects the transcription of several genes including c-MYC, 4 c-kit, 5 KRAS, 6 VEGF, 7 c-myb 8 and ILPR (insulin gene). 9 Due to its potential as a cancer-specific target, there is considerable interest in developing small ligands that stabilize G4-DNA. 10 Structure-selective G-quadruplex ligands typically have shape and charge complementarity with the stacked G-tetrads that constitute G-quadruplex DNA. For example, pyridinium and ammonium-containing porphyrazine derivatives exhibited improved G-quadruplex specificity as compared to the widely studied, yet non-selective ligand 5,10,15,20-tetra(N-methyl-4pyridyl) porphine (TMPyP4). 11,12 However, no information regarding the cellular uptake or promoter binding of these compounds was reported. Here, we report G4-DNA binding, cellular uptake, and promoter deactivation of a new class of cationic phthalocyanines called guanidino phthalocyanines (GPcs). 13 We have used polymerase-stop assays, CD spectroscopy, and a fluorescence quenching assay to characterize the G-quadruplex affinity and specificity of tetrakis-(diisopropylguanidine) phthalocyanine ''DIGP'' (1), and its Zn-containing derivative ''Zn-DIGP'' (2) (Scheme 1). To facilitate a direct comparison of porphyrin versus phthalocyanine scaffolds, a porphyrin containing four diisopropyl guanidinium groups at meso positions ''DIGPor'' (3) was synthesized and evaluated.Scheme 1 Structures of investigated compounds and common names.

The KRAS Promoter Responds to Myc-associated Zinc Finger and Poly(ADP-ribose) Polymerase 1 Proteins, Which Recognize a Critical Quadruplex-forming GA-element

“…9b shows that 10 M DIGP and Zn-DIGP weakly compete with the binding of MAZ to the GA-duplex. In a recent study we found that DIGP and Zn-DIGP interact with the murine KRAS quadruplex with a K D between 10 Ϫ7 and 10 Ϫ6 M (21). Polymerase stop assays showed that these ligands efficiently stabilize the G-quadruplex formed by the GA-element (21).…”

“…In a recent study we found that DIGP and Zn-DIGP interact with the murine KRAS quadruplex with a K D between 10 Ϫ7 and 10 Ϫ6 M (21). Polymerase stop assays showed that these ligands efficiently stabilize the G-quadruplex formed by the GA-element (21). Indeed, circular dichroism spectra obtained at various temperatures (20 -90°C) showed that the T M of the G-quadruplex in the presence of DIGP (r ϭ 4) increased from 68 to Ͼ90°C in 50 mM KCl (supplemental Fig.…”

“…S5). In light of these findings we used guanidine-modified phthalocyanines (GPc), DIGP and Zn-DIGP, a new class of G4-DNA ligands that specifically binds to G4-DNA and poorly competes with the binding of proteins to duplex DNA (21,29). Indeed, Fig.…”

Porphyrins in complex with DNA: Modes of interaction and oxidation reactions

“…Bulky porphyrin derivatives H 2 -TMPipEOPP, H 2 -TMPy2PP, H 2 -GP, H 2 -DIGPor and H 2 -TPyEtCAP can perfectly discriminate between quadruplex and double-stranded DNA [63,69,71,[116][117][118][119][120]. Their binding constants for quadruplex DNA is comparable to that of best G4 ligands [120].…”

“…These derivatives are cationic phtalocyanines/porphyrazines [111,118,[126][127][128][129] or core-expanded porphyrins [130,131]. It is striking to note that negatively charged compounds such as Nmethylmesoporphyrin IX (NMM) [44,45,132,133], protoporphyrin IX [134], hemin (Fe IIIprotoporphyrin IX) [135][136][137] with two carboxylic groups or tetraphtalocyanines with four sulfonate groups [138][139][140] are endowed with G-quadruplex binding capability.…”