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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: ACS Chem Biol. 2012 Dec 11;8(3):578–587. doi: 10.1021/cb300568r

Figure 3. TCH-013 inhibits the 20S proteasome non-competitively and binds to a site other than the CT-L active site.

Figure 3

(A) Kinetic analysis of 20S inhibition by TCH-013 was accomplished using purified human 20S proteasome treated with either: vehicle, 10, 5, 2.5, 1.25 or 0.6 μM TCH-013. Km and Vmax values were calculated from Michaelis-Menton analysis using a range of Suc-LLVY-AMC substrate from 1 to 20 μM. (Km values were 5.35 ±0.28, 5.40 ±0.81, 7.37 ±0.59, 5.66 ±0.49, 6.02 ±0.36, and 5.43 ± 0.52 for vehicle, 10, 5, 2.5, 1.25, and 0.6 μM treatment respectively, while Vmax values were 0.99 ±0.03, 0.02 ±.001, 0.24 ±0.01, 0.72 ±0.03, 0.85 ±0.03, and 0.82 ±0.04 U/sec for vehicle, 10, 5, 2.5, 1.25, and 0.6 μM treatment respectively). A representative double reciprocal Lineweaver-Burk plot in which all of the treatments result in intersecting on the same point of the X-axis (1/[S]) illustrates non-competitive inhibition. (B) The biotinylated non-reversible covalent and competitive inhibitor Ada-Lys(biotinyl)-(Ahx)3-(Leu)3-vinyl sulfone is not blocked from binding to the catalytic binding sites of the 20S proteasome. Purified human 20S proteasome particles were pretreated for one hour with vehicle, 10, 5, 2.5, 1.3 or 0.6 μM TCH-013a. Ada-Lys(biotinyl)-(Ahx)3-(Leu)3-vinyl sulfone was then used to probe for catalytic binding sites that were not occupied by TCH-013a. The proteasome particles were then resolved by 12.5% SDS-PAGE and transferred to PVDF. Immunoblotting was performed using an avidin-HRP conjugate. (C) TCH-013 inhibition of the 20S proteasome is not restored after extensive washing. 1 nM 20S proteasome was treated with vehicle, 10 μM TCH-013 or 1 μM MG-132 and tested for CT-L activity. The treated samples were washed extensively 500 volumes of assay buffer and tested for CT-L activity. (D) The effect of TCH-013 on CT-L activity of the 20S proteasome is additive to the effect of bortezomib. Dose response curves of bortezomib in combination with vehicle or varying amounts of TCH-013. (E) TCH-013 and bortezomib retain constant relative potencies as demonstrated by the linear isobole created using the dose of bortezomib necessary to obtain 50% inhibition of CT-L activity with varying amounts of TCH-013.