| Assay Method Information | |
| | Determination of Kinetic Parameters Ki App, kon App, koff, and tR |
| Description: | For each compound, the progress curves displayed a nonlinear profile of H10MbtAopt inhibition with the characteristic three phases of a time-dependent, slow-onset mechanism of inhibition (FIGS. 5A-C); i.e., an initial linear phase that extrapolates to a slope corresponding to a pre-equilibrium initial velocity (vps) at t=0; a final linear phase with a slope representing the equilibrium, steady-state velocity (vs): and an exponential phase that connects the two linear phases with a pseudo-first order rate constant (kobs) for the approach to the steady state (Morrison. J. F., et al. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 201-301; Copeland, R. A. (2013) Evaluation of enzyme inhibitors in drug discovery, pp 203-244, John Wiley & Sons, Inc.). In contrast, the progress curves for uninhibited reactions (DMSO controls) showed the expected linear profile of the steady-state kinetics (FIGS. 5A-C). Thus, the results demonstrated that salicyl-AMS (1) and its analogs including salicyl-6-MeO-AMSN (6) are slow-onset inhibitors of H10MbtAopt. Encouragingly, the results also provided the first indication of the potent activity of salicyl-AMSNMe (4b) and salicyl-6-MeO-AMSN (6) against MbtAtb.Interestingly, salicyl-6-MeO-AMSN (6) remains a fairly potent inhibitor of MbtAtb, despite the lack of a hydrogen-bond donor on the C6-substituent and the presence of the sulfamide linker. |
| Affinity data for this assay | |
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