The evaluation of inhibitive effectiveness of the tumour necrosis factor-α converting enzyme selective inhibitors by HPLC

Figures & data

Figure 1.  Absorption spectra of DNBF (□) and internal standard Ala-Dpa (Δ). The absorption maximum of DNBF and Ala-Dpa were 240nm and 360nm, respectively.

Figure 2.  The impact of reaction time on the extent of the enzymatic reaction for TACE (Left) and MMP-9 (Right). The ratios of the product peak area to the internal standard peak area (A_i /A_is) as determined by the HPLC method increased linearly with reaction time from 0–15min for TACE and 0–25min for MMP-9.

Figure 3.  The impact of TACE (Left) and MMP-9 (Right) concentrations on the extent of the enzymatic reaction. The ratios of the product peak area to the internal standard peak area (A_i /A_is) as determined by the HPLC method increased linearly with the enzyme concentration from 0.1–0.3 mg/L for TACE and 0.2–0.6 mg/L for MMP-9.

Figure 4.  The chromatograms of the eluted reacted substrate using (A) 55% acetonitrile solution, (B) 45% methanol solution, (C) 55% methanol solution, and (D) 70% methanol solution as mobile phases. The peaks in (C) are: (1) Dpa-SPLAQAVRSSSR, (2) Ala-Dpa, and (3) Dpa-SPLAQA.

Figure 5.  The chromatograms of the eluted reacted substrate using (A) 40% acetonitrile solution, (B) 50% methanol solution, (C) 55% methanol solution, and (D) 70% methanol solution as mobile phases. The peaks in (C) are: (1) Dpa- KPLGLAR, (2) Ala-Dpa, and (3) Dpa- KPLG.

Table 1.  The relative standard deviation (RSD) for the stability of the reacted TACE substrates at varying concentrations (n = 6).

Concentration of substrate	50 μM	150 μM	300 μM
within day RSD
RSD of Ai	7.48%	5.33%	6.32%
RSD of Ai/As	3.27%	3.60%	2.00%
between day RSD
RSD of Ai	7.57%	6.84%	5.89%
RSD of Ai/As	4.77%	3.18%	3.99%

Figure 6.  The inhibitive curve of GM6001 on TACE (left) and MMP-9 (right). The activity of TACE and MMP-9 as determined by HPLC method was decreased by the different concentrations of GM6001.

Table 2.  The IC₅₀ values of GM6001 and inhibitor A to TACE and MMP-9.

	GM6001	Inhibitor A
TACE	317 nM	175 nM
MMP-9	0.26 nM	537 nM

Figure 7.  The response curve of the logarithmic dose of GM6001 to TACE (right) and MMP-9 (left). There is a sigmoidal relationship between the inhibition ratio and the logarithmic concentration of GM6001. The value of IC₅₀ of GM6001 for TACE and MMP-9 are 317 nM and 0.26 nM (260 pM), respectively.