Cleavage of heparan sulfate proteoglycans (HSPGs) by the enzyme heparanase modulates tumour-related events including angiogenesis, cell invasion, and metastasis. Metalloshielding of heparan sulfate (HS) by positively charged polynuclear platinum complexes (PPCs) effectively inhibits physiologically critical HS functions. Studies using bacterial P. heparinus heparinaseII showed that a library of Pt complexes varying in charge and nuclearity and the presence or absence of a dangling amine inhibits the cleavage activity of the enzyme on the synthetic pentasaccharide, Fondaparinux (FPX). Charge-dependent affinity of PPC for FPX was seen in competition assays with methylene blue and ethidium bromide. The dissociation constant (K-d) of TriplatinNC for FPX was directly measured by isothermal titration calorimetry (ITC). The trend in DFT calculated interaction energies with heparin fragments is consistent with the spectroscopic studies. Competitive inhibition of TAMRA-R-9 internalization in human carcinoma (HCT116) cells along with studies in HCT116, wildtype CHO and mutant CHO-pgsA745 (lacking HS/CS) cells confirm that HSPG-mediated interactions play an important role in the cellular accumulation of PPCs.

Physicochemical properties of coordination compounds can be exploited for molecular recognition of biomolecules. The inherent π-π stacking properties of [Pt(chelate)(N-donor)]2+ ([PtN4]) complexes were modulated by systematic variation of the chelate (diethylenetriamine and substituted derivatives) and N-donor (nucleobase or nucleoside) in the formally substitution-inert PtN4 coordination sphere. Approaches to target the HIV nucleocapsid protein HIVNCp7 are summarized building on (i) assessment of stacking interactions with simple tryptophan or tryptophan derivatives to (ii) the tryptophan-containing C-terminal zinc finger and (iii) to the full two-zinc finger peptide and its interactions with RNA and DNA. The xanthosine nucleoside was identified as having significantly enhanced stacking capability over guanosine. Correlation of the LUMO energies of the modified nucleobases with the DFT π-stacking energies shows that frontier orbital energies of the individual monomers can be used as a first estimate of the π-stacking strength to Trp. Cellular accumulation studies showed no significant correlation with lipophilicity of the compounds, but all compounds had very low cytotoxicity suggesting the potential for antiviral selectivity. The conceptual similarities between nucleobase alkylation and platination validates the design of formally substitution-inert coordination complexes as weak Lewis acid electrophiles for selective peptide targeting.

Department of Chemistry, Virginia Commo Richmond, VA 23284-2006, USA. E-mail: np Department of Science, Technology and M Beach, Virginia 23464, USA Department of Chemistry and Biochemistry 23529, USA. E-mail: CBayse@odu.edu Massey Cancer Center, Virginia Commonw † Electronic supplementary information experimental and characterization, mol studies and control gel shis. See DOI: 10 Cite this: Chem. Sci., 2017, 8, 1269

The high affinity of highly charged polynuclear platinum complexes for glycans such as heparan sulfate results in modulation of the biomolecule signaling functions leading to inhibition of angiogenesis.

Department of Chemistry, Virginia Commo Virginia, USA. E-mail: 5.npfarrell@vcu.edu The Massey Cancer Center, Virginia Comm Virginia, USA Institute for Glycomics, Griffith Univer Queensland 4222, Australia John Curtin School of Medical Research Canberra, Australia † Electronic supplementary information platinum complex–oligosaccharide in cytotoxicity data and wound heali 10.1039/c6sc02515c Cite this: Chem. Sci., 2017, 8, 241

Heparan sulfate is identified as a ligand receptor for polynuclear platinum anti-cancer agents through sulfate cluster binding. We present a new biological role for platinum and coordination compounds and a new target for metal-based drugs while presenting a new chemotype for heparanase and growth factor inhibition through modulation (metalloshielding) of their interactions. Masking of extracellular (ECM)-resident heparan sulfate (HS) through metalloshielding results in very effective inhibition of physiologically critical HS functions including enzyme (heparanase, HPSE) and protein growth factor recognition. The interaction of the highly cationic polynuclear platinum complexes (PPCs) with the highly sulfated pentasaccharide Fondaparinux (FPX, in this case as a model HS-like substrate) results in inhibition of its cleavage by the HS-related enzyme heparanase. Binding of the fibroblast growth factor FGF-2 to HS is also inhibited with consequences for downstream signalling events as measured by a reduction in accumulation of phospho-S6 ribosomal protein in human colon tumor HCT-116 cells. The end-point of inhibition of HPSE activity and growth factor growth factor signaling is the prevention of cell invasion and angiogenesis. Finally these events culminate in inhibition of HCT-116 cell invasion at sub-cytotoxic concentrations and the process of angiogenesis. A competition assay shows that Fondaparinux can sequester the 8+ TriplatinNC from bound DNA, emphasising the strength of PPC-HS interactions. Altering the profile of platinum agents from cytotoxic to anti-metastatic has profound implications for future directions in the development of platinum-based chemotherapeutics.

Abstract Heparanase is an endo-β-D-glucuronidase that cleaves heparan sulfate glycosaminoglycans (HS-GAGs) in the extracellular matrix and basement membrane. Cancer cells that aberrantly express heparanase potentiate tumor progression, invasion and metastasis in two distinct ways; (1) cleavage of HS-GAGs releases growth-factors to directly activate growth receptors and (2) degradation of the heparan sulfate structural component of the extracellular matrix (ECM) allows metastatic spread of cancer cells. Recently, we determined that accumulation and cytotoxicity of polynuclear platinum compounds (PPCs), including the Phase II clinical trial compound, BBR3464 (+4), are dependent on the presence of cell-surface GAGs. Here, we demonstrate that high affinity PPC-GAG binding provides a new approach to glycan-based targeting by protection against enzymatic cleavage by heparanase. It was determined by NMR spectroscopy, that PPCs, especially the higher charged compound, TriplatinNC (+8), inhibit heparanase cleavage of the oligosaccharide, Fondaparineaux. Further, using the human umbilical primary cell line, HUVEC, it was determined that PPCs reduce heparanase cleavage of the GAG-bound growth factor, bFGF, from ECM. The end result of inhibition of heparanase cleavage is a reduction in tumor invasion and angiogenesis. Using the matrigel invasion assay, we show that sub-cytotoxic doses of PPCs, but not cisplatin, reduces serum-induced invasion of HCT116 cells through basement membrane. In an ex vivo rat aorta model, PPCs exhibit antiangiogenesis activity, measured by the inhibition of new blood vessel growth sprouting from the original aortic ring. Together, these encouraging results support the potential to combine anti-metastatic and cytotoxic activity in the development of dual-function platinum-based drugs. Citation Format: Erica J. Peterson, Susan J. Berners-Price, Anna Bezos, Lisa Bohlman, Samantha J. Katner, A. Gerard Daniel, Chih-Wei Chang, Mark von Itzstein, Christopher R. Parish, Nicholas P. Farrell. Antiangiogenic platinum through glycan targeting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4486. doi:10.1158/1538-7445.AM2015-4486

Zn(2+) inhibits the action of several of the caspases and thus may act as a regulator of apoptosis. Reversal of this inhibition is one possible approach for the development of apoptosis-based therapies. Few studies describe the molecular details of the Zn(2+)-caspase interaction, the understanding of which is essential for the success of any therapeutic strategies. Enzyme kinetics and biophysical studies have shown that the inhibition is of mixed type with prominent (ca. 60 % of inhibition) uncompetitive characteristics and an IC50 of 0.8 μM under the conditions used. Fluorescence-based techniques confirmed that, during inhibition in the sub-micromolar range, substrate binding remains unaffected. A new zinc binding site composed of the catalytic histidine and a nearby methionine residue, rather than the catalytic histidine and cysteine dyad, is proposed based on the experimental observations. DFT models were used to demonstrate that the proposed site could be the preferred inhibitory zinc binding site.

Zink- und Kupferionen spielen entgegengesetzte Rollen in der Apoptose, wobei Zink das proapoptotische Protein Kaspase-3 hemmt. In der Zuschrift auf S. 4182 ff. weisen N. P. Farrell et al. nach, dass diese Kaspase-3-Inhibierung über eine Wechselwirkung der Zinkionen mit dem Histidinrest im aktiven Zentrum – und nicht mit der katalytischen Diade, wie zuvor angenommen wurde – erfolgt.

The functional role assumed by zinc in proteins is closely tied to the variable dynamics around its coordination sphere arising by virtue of its flexibility in bonding. Modern experimental and computational methods allow the detection and study of previously unknown features of bonding between zinc and its ligands in protein environment. These discoveries are occurring just in time as novel biological functions of zinc, which involve rather unconventional coordination trends, are emerging. In this sense coordination sphere expansion of structural zinc sites, as observed in our previous experiments, is a novel phenomenon. Here we explore the electronic and structural requirements by simulating this phenomenon in structural zinc sites using DFT computations. For this purpose, we have chosen MPW1PW91 and a mixed basis set combination as the DFT method through benchmarking, because it accurately reproduces structural parameters of experimentally characterized zinc compounds. Using appropriate models, we show that the greater ionic character of zinc coordination would allow for coordination sphere expansion if the steric and electrostatic repulsions of the ligands are attenuated properly. Importantly, through the study of electronic and structural aspects of the models used, we arrive at a comprehensive bonding model, explaining the factors that influence coordination of zinc in proteins. The proposed model along with the existing knowledge would enhance our ability to predict zinc binding sites in proteins, which is today of growing importance given the predicted enormity of the zinc proteome.

In an approach to design drugs with higher affinity for π–π stacking and electrostatic interactions with targeted biomolecules, complexes of the type [{cis‐Pt(A)2(L)}2‐μ‐{trans‐1,4‐dach}](NO3)4 ((A)2=(NH3)2 or ethylenediamine (en), L=quinoline (quin) or benzothiazole (bztz), dach=trans‐1,4‐diaminocyclohexane) were synthesized. The quinoline complex, [{cis‐Pt(en)(quin)}2‐μ‐(dach)](NO3)4 (9) was synthesized from the precursor K[PtCl3(quin)] (1), while the benzothiazole complexes, [{cis‐Pt(A)2(bztz)}2‐μ‐(dach)](NO3)4 ((A)2=(NH3)2 (10) and (A)2=en (11)) were synthesized from the precursors cis‐[Pt(A)2Cl(bztz)] ((A)2=(NH3)2 (7) and (A)2=en (8)). Their interactions with N‐acetyltryptophan and a model pentapeptide (N‐Ac‐WLDSW‐OH), modeled on the pentapeptide recognition sequence (FSDLW) of p53–mdm2 interaction, were examined by fluorescence spectroscopy. The dinuclear complexes were found to be significantly stronger at quenching the fluorescence of tryptophan than their mononuclear Pt‐based analogues indicating stronger binding. Molecular modeling suggests a “sandwich” mode of binding, and the flexibility of the dinuclear motif can allow the design of more selective and stronger‐binding complexes. Based on these results a further prototype, [{Pt(en)(9‐EtGua)}2μ‐H2N(CH2)6NH2]4+, incorporating the purine 9‐ethylguanine (9‐EtG) as a stacking moiety, was prepared which showed good cytotoxicity in A2780 and OsACL tumor cell lines.

Zink- und Kupferionen spielen entgegengesetzte Rollen in der Apoptose, wobei Zink das proapoptotische Protein Kaspase-3 hemmt. In der Zuschrift auf S. 4182 ff. weisen N. P. Farrell et al. nach, dass diese Kaspase-3-Inhibierung über eine Wechselwirkung der Zinkionen mit dem Histidinrest im aktiven Zentrum – und nicht mit der katalytischen Diade, wie zuvor angenommen wurde – erfolgt.

Zinc ions and copper ions are known to play important and contrasting roles in apoptosis, with zinc inhibiting the pro-apoptotic protein caspase-3. In their Communication on page 4098 ff., N. P. Farrell and co-workers present evidence that zinc inhibits caspase-3 through interacting with the active-site histidine residue rather than with the catalytic dyad as had previously been assumed.

ZINC ENVIRONMENT IN PROTEINS: THE FLEXIBLE AND REACTIVE CORE OF HIV-1 NCP7 AND THE INHIBITORY SITE OF CASPASE-3 By Amalanayagame Gerard Daniel, M. Phil. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry at Virginia Commonwealth University. Virginia Commonwealth University, 2013. Major Director: Nicholas P. Farrell, Ph. D., Professor, Department of Chemistry Zinc is an essential cofactor of several proteins. The roles of zinc in these proteins are classified as catalytic, structural or regulatory. Zinc present in structural sites is considered to be a chemically inert, static structural element. On the contrary, previous studies on a C2H2 type zinc finger model compound and the C3H type HIV-1 NCp7 C-terminal zinc knuckle have shown that zinc at these sites can undergo coordination sphere expansion under the influence of a Pt based electrophile. The pentacoordination observed around zinc in these experiments raises an important question: are the structural zinc motifs found in the proteins susceptible to coordination sphere expansion? Through DFT modeling, the existence and nature of the five coordinate zinc species was investigated. mPW1PW91 was chosen as the DFT method by benchmarking against the experimental parameters of a molecule that closely resembles those to be modeled. The results suggest that the observed coordination sphere expansion is due to the flexible nature of thiolate and chloride ligands that are part of the structure. However, if certain conditions are not met, the same flexibility can lead to the destabilization of these rather fragile structures. Unlike the stable three or four coordinate catalytic and structural zinc sites, at regulatory sites, zinc is typically bound to one or two protein ligands. Zinc inhibition of caspases which are central to the process of apoptosis is one such scenario. Several of the caspases are inhibited by zinc at low micromolar range. Regulation of caspases is a strategy for drug development toward apoptosis related diseases; thus it is important to know the molecular details of zinc inhibition of caspases. Currently, it is speculated that zinc binds to the active site His and Cys residues of caspases thus competing with the substrate. However our studies on caspase-3, using enzyme kinetics and biophysical methods, imply more than one zinc binding sites. Contrary to current beliefs, more than 50% of the inhibition is achieved by zinc without affecting substrate binding. Using DFT models, an alternative inhibitory zinc binding site, which better fits our experimental observations, is predicted.