(B), SDS-PAGE analysis under non-reducing {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| and reducing conditions of purified hDM-αH-C6.5 MH3B1 visualized by Coomassie Blue staining. Lanes 1, 4, 5, and 8, MW markers in kDa (Invitrogen); lanes 2 & 3, hDM-αH-C6.5 MH3B1 at 1 and 2 μg, respectively, not reduced; lane 6 & 7, hDM-αH-C6.5 MH3B1 at 1 and 2 μg, respectively, reduced. (C), Size exclusion chromatography of hDM-αH-C6.5 MH3B1 under non-reducing condition using a Sepharose-6 column. For comparison, molecular weight standards were analyzed under identical conditions. hDM-αH-C6.5 MH3B1 unlike hPNP-αH-C6.5 MH3B1 converts the non-toxic prodrug F-dAdo to the cytotoxic drug, F-Ade The activity of hDM-αH-C6 MH3B1 was examined
in a spectrophotometeric assay in which conversion of F-dAdo to
F-Ade was followed by a decrease in absorbance at 260 nm and a concurrent increase in absorbance at 280 nm. The fusion protein had a K M of 264 μM and a k cat of 0.155 s-1 with an overall efficiency of 586 M-1s-1 (Fig. 2A, Table 1). When compared to the enzymatic activity of hDM fused to a short a nti- H ER2/n eu p eptide called AHNP [5, 15], hDM-αH-C6.5 MH3B1 showed a two-fold reduction in K M with a two-fold increase in k cat , with the NVP-BSK805 nmr overall efficiency of the enzyme remaining unchanged with respect to F-dAdo. Unlike the wild-type PNP, enzymatic activity of hDM-αH-C6.5 MH3B1 with respect to guanosine was weak (data not shown). A cell based assay confirmed that the fusion protein converts F-dAdo to a cytotoxic TCL agent. First, a concentration
of F-dAdo was determined that was not toxic to cells, but if converted to F-Ade, would inhibit cellular proliferation; this concentration was 1.5 μM for CT26 or CT26HER2/neu and 6 μM for MCF-7HER2 cells. CT26 or CT26Her2/neu cells grew normally when either 1.5 μM of F-dAdo or 0.2 μM of hDM-αH-C6.5 MH3B1 was added (Fig. 2B), but when added together, F-dAdo was converted to F-Ade by hDM and cell proliferation was inhibited (Fig. 2B). In a similar experiment using MCF7-HER2 cells, addition of 6 μM F-dAdo or 0.1 μM of hDM-αH-C6.5 MH3B1 did not affect cell proliferation (Fig. 2C); however, addition of hDM-αH-C6.5 MH3B1 in the presence of 6 μM F-dAdo inhibited cell proliferation in a dose https://www.selleckchem.com/products/Vorinostat-saha.html dependent manner with half-maximum inhibition of proliferation at 0.6 nM, and complete inhibition of cell proliferation at 2 nM (Fig. 2C). Since no toxicity was seen with 6 μM of F-dAdo or 0.1 μM of hDM-αH-C6.5 MH3B1 (Fig. 2C), the observed cytotoxicity must be the result of the conversion of F-dAdo to F-Ade through the enzymatic activity of hDM. In summary, F-dAdo is toxic to cells only when cleaved to the cytotoxic drug, F-Ade by hDM-αH-C6.5 MH3B1. Significantly, F-Ade inhibits proliferation of a variety of cell types including the murine colon carcinoma CT26 or CT26HER2/neu and the human breast cancer line MCF7-HER2, as well as melanoma tumor cell line, B16 and murine B-cell tumor cells, 38C13 (data not shown).