Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, Principle of the CANDDY technology. Each CANDDY molecule includes a target interactor (burgundy hexagon) and a CANDDY tag (yellow oval). The target protein is bound by the CANDDY molecule via the target interactor and is directly degraded by proteasome via the CANDDY tag. b, The CANDDY tag was synthesized by chemically inactivating the inhibitor site of MLM2238 (MLN). c, Structures of the RAS-SOS inhibitor (ref. 5), RAS-SOS-NH 2 , and TUS-007. d, KRAS G12D was mixed with DMSO, RAS-SOS inhibitor (4 µM) or TUS-007 (4 µM) and incubated under heating from 25 °C to 99 °C. The denature of KRAS G12D was monitored by the fluorescence. The typical curve of each group was shown. e, The correlation between relative amount of KRAS G12D and concentration of TUS-007 were approximated with Rodbard using the means of 3 independent examinations. The DC50 was estimated about 4 µM. f, TUS-007 induced KRAS G12D chemical knockdown by 26S proteasome in a cell free assay. KRAS G12D was incubated with 26S proteasome and agents as indicated for 3 h.

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Synthesized, Incubation, Fluorescence, Concentration Assay, Cell-Free Assay

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: The levels of chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) proteasome activities was monitored by Suc-LLVY-AMC, Bz-VGR-AMC, and Z-LLE-AMC, respectively. AMC fluorescence was monitored by a plate reader with excitation and emission filters of 360 and 460 nm, respectively (DMSO, 30 min = 1) (mean ± SEM; n = 2–3).

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: Successful KRAS G12V degradation by TUS-007. KRAS G12V protein was incubated with TUS-007 at the indicated concentrations in the presence of 26S proteasome for 1 h (mean ± SEM; n = 3). *P < 0.05 vs. DMSO.

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, TUS-007 induced chemical knockdown was independent of ubiquitination. GFP fused KRAS G12D and DsRed were transiently expressed in HeLa cells. The cells were treated with TUS-007 in the presence of a proteasome inhibitor (MLN), a ubiquitination inhibitor (NAEi) and a molecular chaperon inhibitor (HSP70 inhibitor: VER-155008) for 24 h. b & c, RAS-less MEFs expressing KRAS G12D, G12V or G12C were incubated with TUS-007 for 72 h. Representative blots of KRAS are shown (b). The relative amounts of KRAS mutants normalized to GAPDH are shown in the bar graph (mean ± SEM; n = 3–4). The cell viability was also measured (mean ± SEM; n = 3–4) (g). *P < 0.05 and ** P < 0.01 vs. DMSO. TUS-007 induced chemical knockdown did not cause non-specific cytotoxicity.

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: In vivo KRAS G12D/V degradation mediated by CANDDY using a modified proteasome inhibitor

doi: 10.1101/2021.04.23.441075

Figure Lengend Snippet: a, KRAS G12D chemical knockdown induced by TUS-007 in SW1990 cells. SW1990 cells were treated with TUS-007 for 72 h and analyzed by immunoblotting. The KRAS G12D band intensity was normalized to that of GAPDH (mean ± SEM; n = 4). *P < 0.05 vs. DMSO. b, The proportion of annexin V-positive apoptotic SW1990 cells treated with DMSO, RAS-SOS-NH 2 or TUS-007 for 72 h (mean ± SEM; n = 3). *P < 0.05 vs. DMSO or RAS-SOS-NH 2 . c, TUS-007 increased the caspase 3/7 activity in SW1990 cells after treatment for 96 h (mean ± SEM; n = 3). *P < 0.05 and **P < 0.01 vs. DMSO. d, Caspase 3/7 activation by TUS-007 was proteasome dependent and ubiquitination independent. SW1990 cells were treated with the agents for 96 h (mean ± SEM; n = 3). **P < 0.01 vs. DMSO, ## P < 0.01 vs. TUS-007 (100 µM) only, and † no statistically significant difference compared with TUS-007 (100 µM) only. e, Tumor volume in mice with SW1990 cells transplanted subcutaneously and treated with p.o. administered TUS-007 or vehicle (mean ± SEM; n = 6–9). The agents were administered every 3 days. **P < 0.01 vs. vehicle alone. f, Body weight changes in mice with SW1990 cells transplanted subcutaneously (mean ± SEM; n = 6–9).

Article Snippet: The chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1) activities of the 20S proteasome were measured using a 20S Proteasome Activity Kit GOLD (StressMarq Bioscience Inc., Victoria, British Columbia, Canada).

Techniques: Western Blot, Activity Assay, Activation Assay