Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate.

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 heparinase II 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 (Kd ) 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-R9 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.

Antiangiogenic platinum through glycan targeting.

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.

Dinuclear platinum complexes containing planar aromatic ligands to enhance stacking interactions with proteins.

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)2 Cl(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)}2 µ-H2 N(CH2 )6 NH2 ](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.

The bioinorganic chemistry of apoptosis: potential inhibitory zinc binding sites in caspase-3.

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.