Cisplatin Analogs Confer Protection against Cyanide Poisoning

Abstract

Cisplatin holds an illustrious position in the history of chemistry most notably for its role in the virtual cure of testicular cancer. Here we describe a role for this small molecule in cyanide detoxification in vivo. Cyanide kills organisms as diverse as insects, fish, and humans within seconds to hours. Current antidotes exhibit limited efficacy and are not amenable to mass distribution requiring the development of new classes of antidotes. The binding affinity of the cyanide anion for the positively charged metal platinum is known to create an extremely stable complex in vitro. We therefore screened a panel of diverse cisplatin analogs and identified compounds that conferred protection from cyanide poisoning in zebrafish, mice, and rabbits. Cumulatively, this discovery pipeline begins to establish the characteristics of platinum ligands that influence their solubility, toxicity, and efficacy, and provides proof of concept that platinum-based complexes are effective antidotes for cyanide poisoning.

Keywords: antidote; cisplatin; cyanide; phenotypic screening; platinum; zebrafish.

Figure 1. Platinum complexes act as antidotes to cyanide poisoning by binding the cyanide anion

Chemical structure of A) cisplatin, B) carboplatin, and C) hydroxocobalamin. Compounds were dissolved in DMSO for the assays in this figure. D) The effects of compounds on the survival of zebrafish exposed to 100 μM KCN. E) UV-VIS spectral shift data demonstrating the binding of the cisplatin to cyanide. F) Plot of the change in absorbance of 1 mM cisplatin over increasing concentrations of cyanide. G) Analysis by mass spectrometry demonstrates that cisplatin binds 4 cyanide anions. Also see Table S1.

Figure 2. Structure activity relationships of cisplatin analogs

A panel of 35 cisplatin analogs was grouped into the following classes: platinum (IV) (1-6), square planar (7-13), FDA approved (14-19), pyridine (20-24), triphenylphosphine (25-28), alkene (29-32), and sulfur-containing complexes (33-35). A ten point dose curve ranging from 1-1000 μM was tested. The doses that rescued 100% of zebrafish (EC₁₀₀) from a challenge with 100 μM KCN were determined. The EC₁₀₀ was determined in both DMSO and PBS solvents (blue and red, respectively). In a separate assay, the doses that caused 100% lethality (LD₁₀₀) in the absence of KCN were determined for complexes dissolved in DMSO (black). NA indicates instances in which the complex did not induce any toxicity or did not rescue cyanide lethality at any of the doses tested.

Figure 3. Identification of cis-diamminechloro(dimethylsulfoxide) platinum(II) as a potent cyanide antidote in zebrafish

A) Depiction of the associative substitution reaction of water with cisplatin (7), and cyanide with the aquated form of cisplatin (37). B) Spectral shift data demonstrating minimal binding of the aquated form of cisplatin to increasing concentrations of cyanide. C) Depiction of the solvation effect of PBS on cisplatin, and the associative substitution reaction of cyanide with cisplatin (7). D) Spectral shift data demonstrating minimal binding of cisplatin to increasing concentrations of cyanide. E) Depiction of the associative substitution reaction of DMSO with cisplatin generating complex 36, and cyanide with complex 36. F) Spectral shift data demonstrating binding of complex 36 to increasing concentrations of cyanide. G) Graph of the binding of 1 mM complex 37 (black), complex 7 (gray), complex 36 (red), and cisplatin dissolved in DMF (green) to 5 mM cyanide over time, demonstrating the rapid and increased binding rate of complex 36 to cyanide. H) Survival assay in zebrafish demonstrating that complex 36 (red) is a cyanide antidote while cisplatin (gray) is not an antidote. I) Mass spectrometry identification of cis-diamminechloro(dimethylsulfoxide)platinum(II) (complex 36) as the species created by the associative substitution reaction between DMSO and cisplatin. See also Table S2.

Figure 4. A subset of cisplatin analogues solvated in DMSO exhibit decreased cytotoxicity in human cells

A) Images of H1975 cells treated with vehicle, 34, 36, or cisplatin. B) Cell viability over increasing concentrations of 36 (red), cisplatin (gray) and 34 (black). Data represented as the mean ± SD. C) Western blots for phospho-p38 MAPK on lysates from cells treated with vehicle, 34, 36, or cisplatin. Complexes from each structural class (D) were dissolved in PBS (gray) or DMSO (color) and cell viability over increasing concentrations was determined (E). Also see Figure S1.

Figure 5. Cisplatin analogs protected mice exposed to a lethal dose of cyanide

Mice were exposed to cyanide gas for 15 min, injected with the indicated complex (Inj) and placed back in the gas chamber for another 25 min. The data are shown as percent survival versus time. The animals injected with vehicle (purple) consistently died between 30-35 minutes however A) 83% of mice treated with 20 μmol of 36, B) 100% of mice treated with 20 μmol of 34, and C) 100% of mice treated with 5 μmol of 3 survived exposure to a lethal dose of cyanide. n=6. Also see Figure S2.

Figure 6. Cisplatin analogs reversed cyanide induced changes in oxidative metabolism in rabbits

A representative rabbit injected with cyanide demonstrating A) increased concentration of hemoglobin in the oxygenated state (red) compared to the deoxygenated state (blue) in the CNS and B) decreased cytochrome oxidase c redox ratio in the muscle (black). Injection of 36 (C-D) or 3 (E-F) after the cyanide infusion results in rapid reversal of cyanide induced pathophysiologic changes. n=5.