The synthesized compounds were tested against the panel of purified β-lactamases, i.e. AmpC, CTX-M-15, KPC-2, OXA-24, NDM-1 and VIM-2 by spectrophotometric assay, using a DU-640 spectrophotometer (Beckman Coulter)20 (link). Boronic acids were dissolved in dimethyl sulfoxide (DMSO) stock solutions at 10 mM; more dilute stocks were subsequently prepared as necessary by dissolving them in 50 mM phosphate buffer at pH 7.5 (DMSO ≤ 3% V/V, no inhibitory effect confirmed). Boronic acids isolated as pinacol-protected boronic acid were tested without further ester cleavage reaction; compounds spontaneously hydrolysed to the free acid form when dissolved in 50 mM phosphate buffer at pH 7.5. Assay conditions were as follows: 50 mM phosphate buffer, pH 7.5; 100 μM cephalothin (sodium salt, Sigma) as reporter substrate, reaction monitored at 265 nm, time course 300 seconds 25 °C. The background rate of cephalothin hydrolysis was found to be negligible under these conditions (approximately 1%). Reactions were initiated with addition of BL enzyme (the concentration values for each BL enzyme into the SI, Table S1 have been reported).
Each inhibitor compound was assayed at five different concentrations, in duplicate for calculating an error value with 95% confidence interval (ρ < 0.05). From the resulting inhibition percentages at each different inhibitor concentration and, assuming a competitive inhibition for the boronic compounds panel, it was possible to calculate the IC50 values from the Dixon plot 1/V vs [I], using the equation IC50 = ((1/0.5 ∙ v0) − m)/q61 (link), where v0 is the rate of hydrolysis of the reporter substrate (v0 being the rate measured in the absence of inhibitor), q the y axis intercept and m the slope of the resulting linear regression. As previously reported for other boronic acid inhibitors of BLs, tested compounds were competitive inhibitors and no incubation effect was detected21 (link),22 (link). A known inhibitor was included in the compounds panel for each β-lactamase group, i.e. 3APBA (3-aminophenylboronic-acid) for class A, C and D serine β-lactamases (IC50 = 25 ± 1.9 µM for AmpC_BL44 (link)) and L-Captopril for class B β-lactamases as reference compound (IC50 = 154 ± 1.7 µM for NDM-1)62 (link).
Each inhibitor compound was assayed at five different concentrations, in duplicate for calculating an error value with 95% confidence interval (ρ < 0.05). From the resulting inhibition percentages at each different inhibitor concentration and, assuming a competitive inhibition for the boronic compounds panel, it was possible to calculate the IC50 values from the Dixon plot 1/V vs [I], using the equation IC50 = ((1/0.5 ∙ v0) − m)/q61 (link), where v0 is the rate of hydrolysis of the reporter substrate (v0 being the rate measured in the absence of inhibitor), q the y axis intercept and m the slope of the resulting linear regression. As previously reported for other boronic acid inhibitors of BLs, tested compounds were competitive inhibitors and no incubation effect was detected21 (link),22 (link). A known inhibitor was included in the compounds panel for each β-lactamase group, i.e. 3APBA (3-aminophenylboronic-acid) for class A, C and D serine β-lactamases (IC50 = 25 ± 1.9 µM for AmpC_BL44 (link)) and L-Captopril for class B β-lactamases as reference compound (IC50 = 154 ± 1.7 µM for NDM-1)62 (link).
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