[Au(dien)(N-heterocycle)]³⁺: Reactivity with Biomolecules and Zinc Finger Peptides

The reaction of [Au­(dien)­(N-heterocycle)]³⁺ (AuN₄) coordination compounds with simple amino acids and zinc finger proteins is reported. Compared to [AuCl­(dien)]²⁺, NMR studies show that the presence of a more substitution-inert N-donor as the putative leaving group slows the reaction with the sulfur-containing amino acids <i>N</i>-acetylmethionine (NAcMet) and <i>N</i>-acetylcysteine (NAcCys). Lack of ligand dissociation upon reaction with NAcCys indicates, to our knowledge, the first long-lived N-heterocycle–Au–S species in solution. Reactions with zinc finger proteins show a higher reactivity with the Cys₃His zinc finger than with Cys₂His₂, likely due to the presence of fewer aurophilic cysteines in the latter. Of the Au­(III) compounds studied, [Au­(dien)­(DMAP)]³⁺ (DMAP = 4-dimethylaminopyridine) appears to be the least reactive, with ESI-MS studies showing the presence of intact zinc fingers at initial reaction times. These results, in combination with previously reported characterization and pH dependency studies, will further aid in optimizing the structure of these AuN₄ species to obtain a substitution-reactive yet selective compound for targeting zinc finger proteins

Platinated DNA Affects Zinc Finger Conformation. Interaction of a Platinated Single-Stranded Oligonucleotide and the C-Terminal Zinc Finger of Nucleocapsid Protein HIVNCp7

This paper describes for the first time the intimate molecular details of the association between a platinated oligonucleotide and a zinc finger peptide. Site-specific platination of the guanine in a single-stranded hexanucleotide gave {[Pt­(dien)­d­(5′-TACGCC-3′)], Pt­(dien)­(6-mer)} (<b>II</b>) characterized by mass spectrometry and ¹H nuclear magnetic resonance (NMR) spectroscopy. The work extends the study of platinum–nucleobase complex–zinc finger interactions using small molecules such as [Pt­(dien)­(9-EtGua)]²⁺ (<b>I</b>). The structure of the (34–52) C-terminal finger of HIV nucleocapsid protein HIVNCp7 (<b>ZF1</b>) was characterized by ¹H NMR spectroscopy and compared with that of the N-terminal single finger and the two-finger “intact” NCp7. Interaction of <b>II</b> with <b>ZF1</b> results in significant changes in comparison to the “free” uncomplexed hexanucleotide; the major changes occurring for Trp37 resonances that are broadened and moved upfield, and other major shifts are for Gln45 (Hε21, Hγ3, Qβ), Met46 (NH, Hγ2), Lys47 (NH, Qγ), and Glu50 (Hγ2, Hγ3). The Zn–Cys/His chemical shifts show only marginal deviations. The solution structures of <b>ZF1</b> and the 6-mer–<b>ZF1</b> and <b>II–ZF1</b> adducts were calculated from the nuclear Overhauser effect spectroscopy-derived distance constraints. The DNA position in the <b>II–ZF1</b> adduct is completely different than in the absence of platinum. Major differences are the appearance of new Met46–Cyt6 H5 and Trp37–Cyt5 H5 contacts but severe weakening of the Trp37–Gua4 contact, attributed to the steric effects caused by Gua4 platination, accompanied by a change in the position of the aromatic ring. The results demonstrate the feasibility of targeting specific ZF motifs with DNA-tethered coordination compounds, such as Pt compounds and Co macrocycles, with implications for drug targetting and indeed the intimate mechanisms of DNA repair of platinated DNA

Comparison of Metal–Ammine Compounds Binding to DNA and Heparin. Glycans as Ligands in Bioinorganic Chemistry

We present spectroscopic and biophysical approaches to examine the affinity of metal–ammine coordination complexes for heparin as a model for heparan sulfate (HS). Similar to nucleic acids, the highly anionic nature of heparin means it is associated in vivo with physiologically relevant cations, and this work extends their bioinorganic chemistry to substitution-inert metal–ammine compounds (M). Both indirect and direct assays were developed. M compounds are competitive inhibitors of methylene blue (MB)–heparin binding, and the change in the absorbance of the dye in the presence or absence of heparin can be used as an indirect reporter of M–heparin affinity. A second indirect assay uses the change in fluorescence of TAMRA-R₉, a nonaarginine linked to a fluorescent TAMRA moiety, as a reporter for M–heparin binding. Direct assays are surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The <i>K</i>_d values for TriplatinNC–heparin varied to some extent depending on the technique from 33.1 ± 2 nM (ITC) to 66.4 ± 1.3 nM (MB absorbance assay) and 340 ± 30 nM (SPR). The differences are explained by the nature of the technique and the use of heparin of differing molecular weight. Indirect probes using the displacement of ethidium bromide from DNA or, separately, fluorescently labeled oligonucleotide (DNA-Fl) can measure the relative affinities of heparin and DNA for M compounds. These assays showed essentially equivalent affinity of TriplatinNC for heparin and DNA. The generality of these methods was confirmed with a series of mononuclear cobalt, ruthenium, and platinum compounds with significantly lower affinity because of their smaller overall positive charge but in the order [Co­(NH₃)₆]³⁺ > [Ru­(NH₃)₆]³⁺ > [Pt­(NH₃)₄]²⁺. The results on heparin can be extrapolated to glycosoaminoglycans such as HS, emphasizing the relevance of glycan interactions in understanding the biological properties of coordination compounds and the utility of the metalloglycomics concept for extending bioinorganic chemistry to this class of important biomolecules

Probing the HIV‑1 NCp7 Nucleocapsid Protein with Site-Specific Gold(I)–Phosphine Complexes

In this work, we examined a series of thiophilic Au­(I) compounds based on [Au­(L)­(PR₃)] (L = Cl^–, 4-dimethylaminopyridine (dmap); R= ethyl (Et), cyclohexyl (Cy)) for chemoselective auration of the C-terminal HIV nucleocapsid protein NCp7 F2 and the “full” HIV NCp7 (NC, zinc finger (ZnF)) as probes of nucleocapsid topography. The choice of phosphine allowed electronic and steric effects to be considered. The use of the heterocycle “leaving group” allowed us to study the effect of possible π-stacking with the essential tryptophan residue of NC on the reactivity and selectivity, mimicking the naturally occurring interaction between the zinc finger and nucleic acids. We also examined for comparison the “standard” gold–phosphine compound auranofin, which contains an S-bound glucose coordinated to the {Au­(PEt₃)} moiety. Both the nature of the phosphine and the nature of L affect the reactivity with the C-terminal NCp7 F2 and the “full” NC. ³¹P NMR spectroscopy showed the formation of long-lived {Au­(PR₃)}–ZnF species in all cases, but in the case of NCp7 F2, a selective interaction in the presence of the dmap ligand was observed. In the case of auranofin, an unusual Au–His (rather than Au–Cys) coordination was indicated on NC. The overall results suggest that it is useful to consider three aspects of zinc finger structure in considering the profile of chemical reactivity: (i) the zinc-bound cysteines as primary nucleophiles; (ii) the zinc-bound histidine as a “spectator” ligand; and (iii) ancillary groups not bound to Zn but essential for ZnF function such as the essential tryptophan in NCp7 F2 and NC. Modification of fully functional NC zinc finger by the Cy₃P-containing species confirmed the inhibition of the NC–SL2 DNA interaction, as evaluated by fluorescence polarization

Heparan Sulfate Proteoglycan-Mediated Entry Pathway for Charged Tri-Platinum Compounds: Differential Cellular Accumulation Mechanisms for Platinum

We examined the mechanism of accumulation of charged polynuclear platinum complexes (PPCs) based on analogy of polyarginine interactions with the cell surface heparan sulfate proteoglycan (HSPG) family of protein-linked glycosoaminoglycan polysaccharides (GAGs). GAGS such as heparan sulfate (HS) and chondroitin sulfate (CS) mediate the cellular entry of many charged molecules. Fluorescence microscopy and flow cytometry showed that PPCs, but not the neutral cisplatin or oxaliplatin, blocked the cellular entry of TAMRA-R₉ (a nonarginine peptide, R₉) coupled to the TAMRA fluorescent label 5-(and 6-)­carboxytetramethylrhodamine) in Chinese hamster ovary (CHO), human colon carcinoma (HCT116), and osteosarcoma (SAOS-2) cells. Furthermore, detection of platinum accumulation in wt CHO, mutant CHO-pgsD-677 (lacking HS), and CHO-pgsA (lacking HS/CS) cells confirms that HSPG-mediated interactions are an important mechanism for PPC internalization but not so for uncharged cisplatin and oxaliplatin. Endocytosis inhibitor studies show that macropinocytosis, a mechanism of cell entry for heparan sulfate GAGs and arginine-rich peptides, is important in the cellular accumulation of noncovalent TriplatinNC and, to a lesser degree, the covalently binding BBR3464. Clathrin-mediated endocytosis, however, was not involved in either case. Overall, the results suggest a new proteoglycan-mediated mechanism for cellular accumulation of PPCs not shared by cisplatin or oxaliplatin. The results have significant implications for the rational design of platinum antitumor drugs with distinct biological profiles in comparison to those of the clinically used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors

Interaction of the HIV NCp7 Protein with Platinum(II) and Gold(III) Complexes Containing Tridentate Ligands

The human immunodeficiency virus (HIV) nucleocapsid protein (NCp7) plays significant roles in the virus life cycle and has been targeted by compounds that could lead to its denaturation or block its interaction with viral RNA. Herein, we describe the interactions of platinum­(II) and gold­(III) complexes with NCp7 and how the reactivity/affinity of potential inhibitors can be modulated by judicious choice of ligands. The interactions of [MCl­(N₃)]^<i>n</i>+ (M = Pt²⁺ (<i>n</i> = 1) and Au³⁺ (<i>n</i> = 2); N₃ = tridentate chelate ligands: bis­(2-pyridylmethyl)­methylamine (Mebpma, <b>L</b>^<b>1</b>) and bis­(2-pyridylmethyl)­amine (bpma, <b>L</b>^<b>2</b>) with the C-terminal zinc finger of NCp7 (ZF2) were investigated by electrospray ionization-mass spectroscopy (ESI-MS). Mass spectra from the incubation of [MCl­(Mebpma)]^<i>n</i>+ complexes (<b>PtL</b>^<b>1</b> and <b>AuL</b>^<b>1</b>) with ZF2 indicated that they were more reactive than the previously studied diethylenetriamine-containing analogues [MCl­(dien)]^<i>n</i>+. The initial product of reaction of <b>PtL</b>^<b>1</b> with ZF2 results in loss of all ligands and release of zinc to give the platinated apopeptide {PtF} (F = apopeptide). This is in contrast to the incubation with [PtCl­(dien)]⁺, in which {Pt­(dien)}–peptide adducts are observed. Incubation of the Au³⁺ complex <b>AuL</b>^<b>1</b> with ZF2 gave Au_<i>x</i>F^<i>n</i>+ species (<i>x</i> = 1, 2, 4, F = apopeptide) again with loss of all ligands. Furthermore, the formally substitution-inert analogues [Pt­(N₃)­L]²⁺ (L = 4-methylpyridine (4-pic), 4-dimethylaminopyridine (dmap), and 9-ethylguanine (9-EtGua)) were prepared to examine stacking interactions with <i>N</i>-acetyltryptophan (N-AcTrp), the Trp-containing ZF2, and the “full” two-finger NCp7 itself using fluorescence quenching titration. Use of bpma and Mebpma gave slightly higher affinity than analogous [Pt­(dien)­L)]²⁺ complexes. The dmap-containing complexes (<b>PtL</b>^<b>1</b><b>a</b> and <b>PtL</b>^<b>2</b><b>a</b>) had the greatest association constants (<i>K</i>_a) for N-AcTrp and ZF2 peptide. The complex <b>PtL</b>^<b>1</b><b>a</b> had the highest <i>K</i>_a when compared with other known Pt²⁺ analogues: [Pt­(dien)­(9-EtGua)]²⁺ < [Pt­(bpma)­(9-EtGua)]²⁺ < [Pt­(dien)­(dmap)]²⁺< <b>PtL</b>^<b>2</b><b>a</b> < <b>PtL</b>^<b>1</b><b>a</b>. A <i>K</i>_a value of ca. 40.6 ± 1.0 × 10³ M^–1 was obtained for the full NCp7 peptide with <b>PtL</b>^<b>1</b><b>a</b>. In addition, the mass spectrum of the interaction between ZF2 and <b>PtL</b>^<b>1</b><b>a</b> confirms formation of a 1:1 <b>PtL</b>^<b>1</b><b>a</b>/ZF2 adduct. The reactivity of selected complexes with sulfur-containing amino acid <i>N</i>-acetylcysteine (N-AcCys) was also investigated by ¹⁹⁵Pt and ¹H NMR spectroscopy and ESI-MS. The precursor compounds [PtCl­(N₃)]⁺ <b>PtL</b>^<b>1</b> and <b>PtL</b>^<b>2</b> reacted readily, whereas their [Pt­(N₃)­L]²⁺ analogues <b>PtL</b>^<b>1</b><b>a</b> and <b>PtL</b>^<b>2</b><b>a</b> were inert to substitution

Chimeric Platinum-Polyamines and DNA Binding. Kinetics of DNA Interstrand Cross-Link Formation by Dinuclear Platinum Complexes with Polyamine Linkers

The first observation of a polyamine–DNA interaction using 2D [¹H, ¹⁵N] HSQC NMR spectroscopy allows study of the role of the linker in polynuclear platinum-DNA interactions and a novel “anchoring” of the polyamine by Pt–DNA bond formation allows examination of the details of conformational B → Z transitions induced by the polyamine. The kinetics and mechanism of the stepwise formation of 5′-5′ 1,4-GG interstrand cross-links (IXLs) by fully ¹⁵N-labeled [{<i>trans</i>-PtCl­(¹⁵NH₃)₂}₂{μ-(¹⁵NH₂(CH₂)₆¹⁵NH₂(CH₂)₆¹⁵NH₂)}]³⁺ (1,1/<i>t,t</i>-6,6, <b>1</b>) and [{<i>trans</i>-PtCl­(¹⁵NH₃)₂}₂{μ-(¹⁵NH₂(CH₂)₆¹⁵NH₂(CH₂)₂¹⁵NH₂(CH₂)₆¹⁵NH₂)}]⁴⁺ (1,1/<i>t,t</i>–6,2,6, <b>1</b>′) with the self-complementary oligonucleotide 5′-{d­(ATATGTACATAT)₂} (duplex <b>I</b>) are compared to the analogous reaction with 1,0,1/<i>t,t,t</i> (BBR3464) under identical conditions (pH 5.4, 298 K). Initial electrostatic interactions with the DNA are delocalized and followed by aquation to form the monoaqua monochloro species. The rate constant for monofunctional adduct formation, <i>k</i>_MF, for <b>1</b> (0.87 M^–1 s^–1) is 3.5 fold higher than for 1,0,1/<i>t,t,t</i> (0.25 M^–1 s^–1; the value could not be calculated for <b>1</b>′ due to peak overlap). The evidence suggests that several conformers of the bifunctional adduct form, whereas for 1,0,1/<i>t,t,t</i> only two discrete conformers were observed. The combined effect of the conformers observed for <b>1</b> and <b>1</b>′ may play a crucial role in the increased potency of these novel complexes compared to 1,0,1/<i>t,t,t</i>. Treated as a single final product, the rate of formation of the 5′-5′ 1,4-GG IXL, <i>k</i>_CH, for <b>1</b> (<i>k</i>_CH = 4.37 × 10^–5 s^–1) is similar to that of 1,0,1/<i>t,t,t</i>, whereas the value for <b>1</b>′ is marginally higher (<i>k</i>_CH = 5.4 × 10^–5 s^–1)

Gold(I)-Phosphine-N-Heterocycles: Biological Activity and Specific (Ligand) Interactions on the C‑Terminal HIVNCp7 Zinc Finger

The syntheses and the characterization by chemical analysis, ¹H and ³¹P NMR spectroscopy, and mass spectrometry of a series of linear triphenylphosphine gold­(I) complexes with substituted N-heterocycle ligands (L), [(PPh₃)­Au­(I)­(L)]⁺, is reported. The reaction of [(PPh₃)­Au­(L)]⁺ (L = Cl^– or substituted N- heterocyclic pyridine) with the C-terminal (Cys₃His) finger of HIVNCp7 shows evidence by mass spectrometry (ESI-MS) and ³¹P NMR spectroscopy of a long-lived {(PPh₃)­Au}-S-peptide species resulting from displacement of the chloride or pyridine ligand by zinc-bound cysteine with concomitant displacement of Zn²⁺. In contrast, reactions with the Cys₂His₂ finger-3 of the Sp1 transcription factor shows significantly reduced intensities of {(PPh₃)­Au} adducts. The results suggest the possibility of systematic (electronic, steric) variations of “carrier” group PR₃ and “leaving” group L as well as the nature of the zinc finger in modulation of biological activity. The cytotoxicity, cell cycle signaling effects, and cellular accumulation of the series are also reported. All compounds display cytotoxicity in the micromolar range upon 96 h continuous exposure to human tumor cells. The results may have relevance for the reported inhibition of viral load in simian virus by the gold­(I) drug auranofin

Luminescent Ruthenium Complexes for Theranostic Applications

The water-soluble and visible luminescent complexes <i>cis-</i>[Ru­(L-L)₂(L)₂]²⁺ where L-L = 2,2-bipyridine and 1,10-phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ and human serum albumin occurs via noncovalent interactions with <i>K</i>_b = 9.8 × 10⁴ mol^–1 L, Δ<i>H</i> = −11.5 ± 0.1 kcal mol^–1, and <i>T</i>Δ<i>S</i> = −4.46 ± 0.3 kcal mol^–1. High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ against proliferation of HCT116p53^+/+ and HCT116p53^–/– shows IC₅₀ values of 0.1 and 0.7 μmol L^–1. Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53^+/+. The complex activates proapoptotic PARP in p53^–/–, but not in p53^+/+. A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested

Luminescent Ruthenium Complexes for Theranostic Applications

The water-soluble and visible luminescent complexes <i>cis-</i>[Ru­(L-L)₂(L)₂]²⁺ where L-L = 2,2-bipyridine and 1,10-phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ and human serum albumin occurs via noncovalent interactions with <i>K</i>_b = 9.8 × 10⁴ mol^–1 L, Δ<i>H</i> = −11.5 ± 0.1 kcal mol^–1, and <i>T</i>Δ<i>S</i> = −4.46 ± 0.3 kcal mol^–1. High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ against proliferation of HCT116p53^+/+ and HCT116p53^–/– shows IC₅₀ values of 0.1 and 0.7 μmol L^–1. Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53^+/+. The complex activates proapoptotic PARP in p53^–/–, but not in p53^+/+. A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested