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The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide’s in vivoreaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum–sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5–DMSO and Na (NH3)2PtCl–DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitroand fully recapitulated in vivocyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Virtually all organisms seek to maximize fitness by matching fuel availability with energy expenditure. In vertebrates, glucose homeostasis is central to this process, with glucose levels finely tuned to match changing energy requirements. To discover new pathways regulating glucose levels in vivo, we performed a large-scale chemical screen in live zebrafish and identified the small molecule alexidine as a potent glucose-lowering agent. We found that alexidine inhibits the PTEN-like mitochondrial phosphatase PTPMT1 and that other pharmacological and genetic means of inactivating PTPMT1 also decrease glucose levels in zebrafish. Mutation of ptpmt1eliminates the effect of alexidine, further confirming it as the glucose-lowering target of alexidine. We then identified succinate dehydrogenase (SDH) as a substrate of PTPMT1. Inactivation of PTPMT1 causes hyperphosphorylation and activation of SDH, providing a possible mechanism by which PTPMT1 coordinates glucose homeostasis. Therefore, PTPMT1 appears to be an important regulator of SDH phosphorylation status and glucose concentration.

In addition to having a major role in energy homeostasis, leptin is emerging as a pleiotropic cytokine with multiple physiological effector functions. The recently discovered proangiogenic activity of leptin suggested the hypothesis that its production might be regulated by hypoxia, as are other angiogenic factors. To examine this proposal, the expression of leptin protein and mRNA was measured and found to be markedly up-regulated in response to ambient or chemical hypoxia (upon exposure to desferrioxamine or cobalt chloride), an effect that requires intact RNA synthesis, suggesting a transcriptional mechanism. Transient transfection of cultured cells with deletion constructs of the leptin gene promoter linked to a reporter gene revealed a functional hypoxiaresponse element (HRE) located at position –116 within the proximal upstream region. This putative HRE harbors a characteristic 5′-RCGTG-3′ core motif, a hallmark of hypoxia-sensitive genes and recognized by the hypoxia-induciblefactor 1 (HIF1), which consists of a HIF1α/HIFβ heterodimer. Constructs harboring this –116/HRE supported reporter gene expression in response to hypoxia but not when mutated. Expression of HIF1α cDNA in normoxic cells mimicked hypoxia-induced reporter gene expression in cells cotransfected with the wild type leptin –116/HRE construct but not with the mutant. Gel shift assays with a32P-labeled leptin promoter –116/HRE probe and nuclear extracts from hypoxia-treated cells indicated binding of the HIF1α/β heterodimer, which was blocked with an excess of unlabeled –116/HRE probe or a HIF1-binding probe from the erythropoietin gene enhancer. Taken together, these observations demonstrate that the leptin gene is actively engaged by hypoxia through a transcriptional pathway commonly utilized by hypoxia-sensitive genes.

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to ∼16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor α, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitrowas associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor α/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.