Figure 1. (a) Simulation showing the percent enzymatic inhibition F as a function of the ratio [S]/K M for the three inhibitory mechanisms. The K I and the concentration of the inhibitor [I] were set to 300 and 200 µM, respectively. The horizontal dashed line drawn at 20% inhibition represents the detection limit. (b) Simulation showing the percent enzymatic inhibition F as a function of the ratio [S]/K M and of the K I of the inhibitor. The concentration of the inhibitor [I] was 200 µM. Only the graph above the plane taken at F = 20% is displayed.

Figure 2. Schematic diagram of the n‐FABS NMR methodology applied to the screening of biological systems with different levels of complexity:
  1. one substrate and one enzyme,
  2. one substrate and multiple enzymes,
  3. multiple substrates and one enzyme, and
  4. multiple substrates and multiple enzymes. S and P indicate the substrates and the products, respectively, whereas n indicates the number of fluorine atoms used for the NMR signal detection.


Figure 3. Influence of serum albumin on the IC 50 measurement: spectra in the absence (left) and presence (right) of BSA. The reactions were performed in the absence (top) and presence (bottom) of 100 nM of a strong inhibitor. S and P correspond to the signals of the substrate (displayed in the figure) and of the product (peptide phosphorylated at the Ser position), respectively. The incubation times were 200 (left) and 80 min (right) so that the amount of phosphorylated peptide was the same in the presence and absence of BSA for the top spectra. The activated protein AKT1, the peptide, ATP, and BSA concentrations were 25 nM, 30 µM, 131 µM, and 3 µM, respectively. The chemical shift values are expressed in ppm with CF 3COOH as the reference signal. (Reprinted with permission from [ 40]. © ACS.)

Figure 4. One substrate with multiple enzymes: multiple screening performed with 3‐FABS. The first enzymatic reaction was performed with AKT1 and quenched with the potent staurosporine inhibitor. Trypsin was subsequently added to the solution and the enzymatic reaction was quenched with 0.5 mM phenylmethylsulfonylfluoride (PMSF). The enzymes AKT1, trypsin, the peptide, ATP and compound concentrations were 25 nM, 1 nM, 30 µM, 250 µM and 20 µM, respectively. S, P K, , , and indicate the signals of the substrate (displayed structure), product of the kinase, products of the protease, and products of both enzymatic reactions, respectively. H89 and leupeptin are inhibitors of AKT1 and trypsin, respectively. (Reprinted with permission from [ 49]. © Elsevier.)

Figure 5. Multiple substrates and one enzyme: three substrates for trypsin (displayed in the figure) were tested with 6‐FABS. The NMR spectra of the three substrates were acquired after 4 (lower trace) and 260 min (upper trace) from the addition of trypsin. The concentrations of the substrates and of the enzyme were 20 µM and 440pM, respectively. The enzymatic reactions were performed at room temperature in 50 mM Tris, pH 7.5, 0.001% Triton X‐100, and 8% D 2O for the lock signal. S and P are the substrates and the products of the enzymatic reaction, respectively. (lower right) Plot of the enzymatic velocity (product/min) as a function of the substrate concentration. The reactions were performed in Eppendorf vials in the presence of 50 mM Tris, pH 7.5, 0.001% Triton X‐100, and 1 nM trypsin, and quenched after a defined delay with 80 µM leupeptin. After the quenching of the reaction, the solutions were transferred to 5‐mm tubes and spectra were recorded. Curves A, B, and C correspond to substrates S A, S B, and S C, respectively. K M and k cat values derived from the best fit of the experimental data with the Henri–Michaelis–Menten equation are reported in the table. (Reprinted with permission from [ 50]. © ACS.)

Figure 6. Multiple substrates and multiple enzymes: multiple screening performed with 3‐FABS. The CF 3‐tagged substrates Z‐Gly‐Pro‐Phe‐4‐(CF 3)‐NH 2 of POP and Gly‐Pro‐7‐amino‐4‐trifluoromethylcoumarin of DPP IV are displayed above the respective resonances. The signals of the respective products are indicated with an asterisk. The spectra are for the reference sample (no inhibitor present) (upper trace), for the sample in the presence of 10 µM selective POP inhibitor Z‐prolyl‐prolinal (middle trace), and for the sample in the presence of 10 µM selective DPP IV inhibitor (2S,3S)‐2‐amino‐3‐methyl‐1‐(1,3‐thiazolidin‐3yl)pentan‐1‐one (P32/98) (lower trace). (Reprinted with permission from [ 47]. © Wiley.)

Figure 7. Application of 3‐FABS to functional genomics with NMR spectra of the displayed substrate in the presence of four different proteins, AKT1, trypsin, PKA and PAK‐4. S, P K, , and indicate the signals of the substrate, product of the kinases, and products of the protease, respectively. The reaction performed in the presence of the p21‐activated protein PAK‐4 results in the appearance of a signal at the chemical shift of the phosphorylated peptide as observed in AKT1 and PKA.

Figure 8. Screening performed with 12‐FABS using 10 µM of the tetrapeptide PFG‐ARA‐NH 2 (PFG = polyfluorinated glycine) substrate of trypsin and the cryoprobe for detection. The enzymatic reaction was performed with only 200 fM trypsin in 50 mM Tris, pH 7.5. The spectra were recorded on a Bruker 600‐MHz NMR spectrometer operating at a Larmor frequency of 564 MHz at 20 °C with an acquisition time of 12 min. The substrate and product of the reaction (cleaved peptide) are indicated with the letters S and P, respectively. The tiny amount (500 nM) of product is clearly visible in the upper spectrum, whereas in the presence of 20 µM of the inhibitor leupeptin (lower spectrum), no product formation is observed. (Reprinted with permission from [ 53]. © ACS.)

Figure 9. (Left) K M calculation of ATP, (middle) screening of chemical mixtures and deconvolution, and (right) IC 50 calculation of the active ingredient H89 of the mixture performed with 6‐FABS against the kinase PKA. The experiments were performed with the CF 3‐containing substrate displayed in the figure. S and P correspond to the signals of the substrate (displayed in the figure) and of the product (peptide phosphorylated at the Ser position), respectively. The asterisks indicate the tiny amount of phosphorylated peptide. The chemical shift values are expressed in ppm with CF 3COOH as the reference signal. (Reprinted with permission from [ 52]. © Wiley.)