October 12, Tue 2010
1:00 pm, MRB 100 Conference Room
Dr. Robert P. Hanzlik
Professor, Department of Medicinal Chemistry, University of Kansas
Reactive Metabolite Target Proteomics. What Are We Learning?
The covalent binding (CVB) of xenobiotic metabolites to cellular proteins, first recognized in the 1940s, became firmly associated with acute cytotoxicity (and subsequent tissue/organ damage) in the 1970s. For many small molecules protein CVB correlates with cell injury, both in vivo and in isolated cells. Early studies emphasized adduct structure elucidation but target protein identification via C-14 labeling, 2D gels and mass spectrometry has recently accelerated. Unequivocal target identification requires the sequencing of adduct-bearing peptides but this is seldom achieved in in-life studies because the limits of detection are strained by proteins of low abundance, low fractional adduction and adduct heterogeneity. More commonly, when a single protein is identified in a radioactive spot it is assumed to be a covalently labeled target. Ironically, increased analytical sensitivity results in the identification of more proteins per spot, but fewer proteins that can confidently be called targets. This problem can be reduced by using protoxins that contain an isotopic signature, or that are amenable to pre-concentration by affinity capture via antibodies, streptavidin or click chemistry. Among >250 reported targets of >25 protoxins, few are common to ≥ 4 protoxins, and none appear to be an "Achilles heel" for the cell. Our overall knowledge of target proteins, and how they differ from non-target proteins, remains sparse. Global analysis of target lists has provided little insight into downstream mechanisms of toxicity, but bioinformaticpathway analysis of their interacting partner proteins reveals many to be involved in apoptosis, binding or response to unfolded proteins, and MAPK signaling. Future work should focus on 1) identifying more targets for more protoxins, 2) deeper bioinformaticanalysis leading to hypothesis testing, and 3) attempts to evaluate the cytotoxicity of adducted proteins directly (in the sense of Koch's postulates) by introducing them into cells without using small molecules or bioactivating enzymes. (Supported by NIH-GM-21784).