To ensure that SEEKR2 correctly replicated the rate constants as predicted in the original MMVT-SEEKR implementation, we repeated the host-guest and trypsin-benzamidine simulations in a nearly identical process (details listed in the Materials and Methods) to obtain kinetic and thermodynamic quantities, which are reported in this section. In recent years, there has been a great interest in studying the inhibitors of Janus kinase (JAK) -signal transducer and activator of transcription (STAT) pathway, especially in cancer therapy. The JAK-STAT *8 signaling pathway plays a critical role in regulating immune response, and any irregularities can lead to immune disorders. We demonstrate the ability of SEEKR in correctly estimating the extraordinarily long residence time of a novel ATP-competitive inhibitor of the JAK2-STAT5 signaling pathway. Several of our previous papers have run SEEKR calculations on the trypsin-benzamidine system. In Table , the rate constants for those calculations are listed alongside the values computed using the SEEKR2 program. SEEKR2 obtains a koff and a kon that are within an order of magnitude of the experimentally measured quantities. Compared to previous versions of SEEKR, and compared to the experimental values, SEEKR2 obtains a koff that is slightly too fast (990±130 s -1 from SEEKR2 compared to the experimental 600± 300 s -1 ). The kon obtained by SEEKR2 is very close relative to experiment ((2.4±0.2) ⨉ 10 7 M -1 s -1 from SEEKR2 compared to the 2.9 ⨉ 10 7 M -1 s -1 from experiment). Finally, the ΔGbind computed by SEEKR2 was off by a little more than 0.8 kcal/mol (-5.98 ± 0.09 kcal/mol from SEEKR2 compared to -6.71 ± 0.05 kcal/mol from experiment). Additional convergence analyses are reported in the SI for both the koff (Figure ), and the kon (Figure ). For the trypsin-benzamidine system, the total steered molecular dynamics (SMD) simulation time was 20 ns, the total MD MMVT simulation time was 5 µs, and two million BD trajectories were performed, in total. This is slightly morethan the 4.4 µs of MD simulation time in our previous MMVT study. The simulations in this study were lengthened in order to obtain a round 500 ns per anchor, a simple choice in contrast with our previous MMVT study which ended simulations as they satisfied a convergence metric resulting in arbitrary simulation lengths in each anchor. In addition, the SEEKR2 calculations were run with desolvation forces on for the BD stage. This required us to adjust the outermost milestones of the trypsinbenzamidine system, and even add a new milestone with a 16Å radius. Without making this *9 modification for SEEKR2, the desolvation forces and rigid body dynamics of BD prevented any reaction events from being observed, an issue that was not present for the MMVT-SEEKR version.