Abstract 5241: Using affinity probes to explore the nephrotoxicity of aristolochic acid

Abstract

Abstract Aristolochic acid, AA, is a natural compound found in Aristolochia plants used in Chinese Traditional Medicine and is a component of many herbal remedies used worldwide. In humans, exposure to AA is associated with kidney toxicity and otherwise rare upper urinary tract cancer (UTUC). AA undergoes bioactivation creating reactive species of AA that bind covalently to proteins and DNA. Formation of mutagenic AA-DNA adducts leads to AA-induced cancers. We identified the mutational signature associated with AA exposure in patients with UTUC in Taiwan, as well as in AA-initiated cancers in liver, bladder and kidneys. Meanwhile, the mechanism of AA nephrotoxicity remained a mystery, with covalent and non-covalent binding of AA or its metabolites to proteins hypothesized to be involved in its nephrotoxic effects. As millions of people in Asia have ingested AA and no effective therapies are known, establishing the molecular mechanism(s) of AA nephrotoxicity is a topic of major importance. A series of AA analogs were used to design affinity probes for target identification. Structure activity relationship studies in human cells aided in identifying a position in AA suitable for introducing biotinylated, aminopropyl and alkyne linkers. In addition, we have developed and validated monoclonal antibodies against AA-bovine serum albumin and/or AA-DNA covalent adducts. Combined with proteomics techniques, AA-affinity probes and monoclonal antibodies provide powerful tools for defining AA targets in human kidneys. We attached the aminopropyloxy derivative of AA to magnetic beads coated with N-hydroxysuccinimido acyl linkers. These probes, in parallel with unmodified, “control” beads were incubated with renal cortex lysates obtained from C3H mice selected for their sensitivity to the effects of AA. Bound proteins were digested "on bead", then identified by mass spectroscopic peptide analysis, with a total of 316 bound proteins detected. Spectral counts for 128 proteins bound to AA-beads were higher than those for control beads. After correction for nonspecific binding, 18 of these proteins had spectral counts of 10 or higher. These proteins are localized to various organelles, indicating high coverage of the major subcellular compartments. Among these proteins, Cryzl2, quinone oxidoreductase-like 2, was the leading candidate. Based on homology, Cryzl2 belongs to the family of the quinone oxidoreductase-like protein and bears a mitochondrial localization sequence. This protein retains conserved domains for both quinone oxidoreductase activity and NAD(P) binding sites, which is of particular interest since other oxidoreductases, such as NQO1, are known to catalyze nitroreduction, the initial step in the bioactivation of AA. In summary, our studies provide guidelines for design of AA-affinity probes and reveal an enzyme that may be responsible for AA activation in mitochondria, by generating active intermediates and inducing mitochondrial damage in renal tissue. Citation Format: Viktoriya S. Sidorenko, Kathleen G. Dickman, Thomas Rosenquist, Radha Bonala, Sivaprasad Attaluri, Irina Zaitseva, Charles Iden, Francis Johnson, Arthur P. Grollman. Using affinity probes to explore the nephrotoxicity of aristolochic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5241. doi:10.1158/1538-7445.AM2017-5241