Extended Data Figure 7: MS analysis of ribavirin and Ara-C glucuronidation.

a, MS/MS collision-induced fragmentation analysis indicates that a breakdown product of the ribavirin glucuronide missing the ribose ring (exact mass 288.07) was further fragmented into a fragment of this glucuronide (exact mass 244.08, red asterisk) and to the triazole ring, the key moiety of ribavirin (exact mass 112.04). No ribose-glucuronide or ribose fragment was detected in our experiments, suggesting that this is not a major glucuronidation site in these cells. However, we cannot rule out that this exists and could not be detected. b, Microsomes expressing UGT1A1, UGT1A4, UGT1A6 and UGT1A9 were treated with RTP, underwent hydrophilic interaction liquid chromatography (HILIC) and the resulting extracted ion chromatogram (EIC) is shown. The Rib-Glu peak is clearly present and fragmentation analysis as in a confirms that this is glucuronidated ribavirin. We note that microsomes only expressing UGT1A1 do not glucuronidate RTP; and that RTP, but not ribavirin, is glucuronidated in microsomes. These studies suggest that UGT1A4, UGT1A6 and/or UGT1A9 are required for glucuronidation, as is some phosphorylation event before glucuronidation. c, Using HILIC chromatography, we isolated the fraction containing the Rib-Glu peak in b. A portion of this was re-assessed by MS/MS to be sure that the correct peak was isolated. This material was used in the ³H-ribavirin competition assay in Fig. 4m. Material was quantified using a standard curve of ribavirin (see Methods). d, Western blot demonstrating equal loading of eIF4E–GST in the ³H-ribavirin pulldown assay shown in Fig. 4m. All results are representative of at least three independent experiments. e, AraC is glucuronidated (AraC-Glu) in FRII cells but not parental FaDu cells where AraC-TP (triphosphate) is observed. AraC-TP is also observed in FRII cells, but at much lower levels than AraC-Glu. Treatment of FRII cells with GDC-0449 results in the loss of the AraC-Glu peak and causes no alteration to the parental FaDu cells. Fragmentation strongly suggests that the cytosine is the major site of glucuronidation (data not shown). We did not observe masses consistent with an arabinose breakdown product or an arabinose-glucuronide but cannot rule out that they are present at low levels or that our isolation procedure precluded their detection. f, Structures of AraC and AraC-TP are shown. The red arrow indicating the most likely glucuronidation site, as per our mass spectrometry data. Note that no glucuronides were observed when reactions were incubated in the absence of UDP-glucuronic acid (data not shown).