Michael E. Friedman

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.

Platinum(II) complexes containing amine ligands

Abstract New Platinum(II) complexes of o - and p -phenylenediamine, 2-aminopyridine, and 2,3- 3,4-, and 2,6- diaminopyridine have been prepared and characterized by a number of techniques. The complexes of 2- aminopyridine are of the general formula Pt(2- AmPy) 2 X 2 ·nH 2 O where X is Cl or I and n is 1 or 2. In these complexes only the ring nitrogen is coordinated. Complexes of the diaminopyridines have complex stoichiometries Pt 3 L 2 X 6 ·6H 2 O (L + 3,4 DamPy, X = Cl, I), Pt 2 (2,3-DamPy) 3 Cl 3 OH, Pt 3 L 4 I 5 OH (L = 2,3 or 2,6 DamPy), and [Pt(2,6-DamPy) 3 Cl 4 ]·4H 2 O which contain both ring nitrogen and NH 2 groups coordinated to the metal. The phenylenediamine complexes have the general formula PtLX 2 (L = OPDA, PPDA; X = Cl, I) where the amine groups are coordinated. The biological activity of these complexes are reported elsewhere.

Enhanced inhibition of both cellular protein synthesis and malate dehydrogenase by aged aquoplatinum(II) complexes.

Previous studies have shown cis-diamminedichloroplatinum(II) (Cis) an effective anti-tumour agent in man and animals. Evidence is presented here that formation of aquo complexes of this platinum derivative will significantly enhance its inhibitory properties with respect to two separate biochemical functions, namely inhibition of protein synthesis in hamster medulloblastoma cells and in inhibiting the activity of L-malate dehydrogenase (MDH) in a cell free system. Inhibition of cell protein synthesis rises from 8% using freshly dissolved drug to 30% when aged solutions of drug are employed at an inhibitor concentration of 0.1 mM. The inhibitory enhancement seen using purified malic dehydrogenase increases from 16% (fresh) to 57% (aged) at an inhibitor concentration of 1 mM.

Inhibition of malate dehydrogenase by platinum(II) complexes.

Abstract 1. 1. The reversible inhibition of malate dehydrogenase ( l -malate:NAD oxido reductase, EC 1.1.1.37) by the tetrabromoplatinate(II) and tetrachloroplatinate(II) ions have been studied through a pH range 6.5–8.5. The equilibrium dissociation constants for both of those ions at low concentrations (1–4 moles of ions per mole of enzyme) were calculated over the given pH range. 2. 2. The relative rates of inhibition by these ions were studied at higher concentrations (100 moles of ions per mole of enzyme). 3. 3. It was shown that the inhibition decreased for both ions with increasing pH in the equilibrium and rate experiments. 4. 4. Several possible mechanisms of inhibition based on these findings have been proposed. The first is based upon the fact that the enzyme becomes more negative with increasing pH, and the repulsive effect with a negative inhibitor ion increases. Secondly, the tetrabromoplatinate inhibits the enzyme at a rate of 6–8 times that of the tetrachloroplatinate. This parallels previous evidence23 which showed that bromide ligands are more labile than chloride ligands in platinum(II) complexes by about the same factor. Thus, it has been postulated that the inhibition takes place by a replacement of the halide ligand by a nucleophilic group on the enzyme. This group may be less accessable as the pH increases causing a reduction in the inhibition.

The reactivities of isomers of dichlorodiammine-platinum (II) with dehydrogenase enzymes. Evidence for inhibition via cross-linkage.

Abstract The reversible inhibition of malate dehydrogenase ( l -malate:NAD+ oxidoreductase, EC 1.1.1.37), lactate dehydrogenase ( l -lactate:NAD+ oxidoreductase, EC 1.1.1.27) and horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) by both cis- and trans-dichlorodiammine-platinum (II) (Pt(NH3)2Cl2) were carried out at pH 7.1 and 25°C. Inhibition of both liver and yeast alcohol dehydrogenase were measured at 4°C due to the latters instability at higher temperature for long periods of time under the experimental conditions used in this study. The equilibrium constant (Ke) was calculated for each enzyme-platinum complex system. It was shown that inhibition of both malate dehydrogenase and liver alcohol dehydrogenase was independent of the particular platinum isomer, while the trans isomer was a significantly better inhibitor than the cis form when either yeast alcohol dehydrogenase or lactate dehydrogenase was used. Thus, it has been proposed that the two former enzymes are being inhibited by a monodentate chelation with the platinum derivatives while the latter enzymes are being inhibited by a bidendate chelation. It has also been proposed that absolute differences in inhibition of various enzymes by a specific platinum inhibitor is due to different goemetries about the inhibitor site while similar inhibition values are caused by similar geometries.

The blocking of the tetrachloroplatinate (II) inhibition of malate dehydrogenase by sulfur-containing amino acids

Abstract The inhibition of malate dehydrogenase ( l -malate:NAD oxidoreductase, EC 1.1.1.37) by the tetrachloroplatinate (II) complex, PtCl42−, in the presence of various concentrations of the amino acids d l -methionine and l -cysteine was measured. The relative concentrations of PtCl42− to malate dehydrogenase was 100:1. From the data, the half-life and the relative rate of inhibition in the presence and absence of the amino acids were calculated. Using these values it was possible to calculate a stability constant for each system. The stability constants for the malate dehydrogenase-PtCl42−- l -cysteine ( K c ), malate dehydrogenase-PtCl42−- d l -methionine ( K m ) and, malate dehydrogenase-PtCl42−( K E ) were 56 M−1, 917 M−1 and 8·105 M−1, respectively. The reversibility of the malate dehydrogenase-PtCl42− complex was also demonstrated, by addition of d l -methionine to the completely inhibited enzyme. About 40% of the enzyme activity was regenerated. Using the stability constants it was calculated that 27% of the enzymes activity should be regenerated. From the results it is suggested that the free platinum complexes may be maintained in solution for a longer period of time if some or all of the halide ligands were replaced by sulfur groups from molecules like cysteine or methionine.

The inhibition of malate dehydrogenase by chlorammine-platinum complexes

Abstract Equilibrium association constants have been calculated for various platinum (II) and platinum (IV) complexes. The association constants were greatest for the dinegatively charged state, regardless of the valence state of the platinum, and the constant decreased considerably as the charge increased. There were no measurable values for positively charged complex states. The conclusion is that the electrostatic charge of the platinum complex is the most important factor causing the inhibition of the enzyme, and the steric differences have only a minor effect, while geometric variation as in the comparison of cis and trans dichlorodiamine-platinum (II) isomers yields no differences in inhibition.

Inhibition of leucine aminopeptidase and malate dehydrogenase by aquoplatinum (II) complexes

Abstract Halide complexes of platinum have been previously shown to inhibit tumors and cell growth as well as to possess immunosuppresive activity. Evidence is presented here that the active species in Rb2PtBr4 solutions which inhibits both leucine aminopeptidase ( l -leucyl-peptide hydrolase, EC 3.4.1.1) and malate dehydrogenase ( l -malate:NAD+ oxidoreductase, EC 1.1.1.37) enzymes is PtBr3(H2O)−. The aquo complex earlier has been shown to be in equilibrium with PtBr42− in solution and the rate of formation is known. This is in accord with the rate of inhibition of enzyme activity by fresh solutions of Rb2PtBr4. This reagent should provide another method for studying the active site and function of enzymes.

Synthesis and properties of some new platinum blues derived from the reaction of cis-[Pt(NH3)2(OH2)2]2+ with pyrimidines and adenine, and their inhibition of two mitochondrial enzymes(L-malate dehydrogenase and fumarase) and DNA synthesis in E. coli

Abstract Several new platinum blue and green materials, with reproducible analyses and properties, have been synthesized form the reaction of cis[Pt(NH3)2(OH2)2]2+ when the pyrimidines uracil, thymine and cytosine. New blues have also been obtained from the reaction of [Pt(aden-H)2(aden)2][PtI4] (aden-H = monodeprotonated adenine) with dimethylformamide, dimethylsulphoxide, tetradrofuran, acetone and pyridine. Some physical properties of these compounds are described, as well as their liability to inhibit tow mitochondrial enzymes (L-malate dehydrogenase and fumarase) and DNA synthesis in E. Coli.

Interactions of cis- and trans-platinum(II) complexes with dehydrogenase enzymes in the presence of different mono- and polynucleotides: evidence for a ternary complex.

The inhibition of several dehydrogenase enzymes by cis- and trans-Pt(NH3)2Cl2 have been measured in the presence of baker yeast ribonucleic acid (RNA), calf thymus and salmon sperm deoxyribonuclic acid (DNA) and several mononucleotides (AMP and ATP). The binding constants for the interaction of the platinum complexes to the nucleotides have been calculated and a comparison of those values to the previously calculated platinum complex-enzyme binding constants strongly suggest that platinum compounds are more tightly bound to the enzymes. The binding of the platinum complexes to most of the enzymes was decreased in the presence of any nucleotide, yet it was observed that when using rabbit muscle (M4) lactate dehydrogenase the mononucleotides reduced the binding to a lesser degree while the polynucleotides actually enhanced the platinum-enzyme interaction. The implications of these interactions are discussed.