Noticing that many false positive predictions involved specific ligands that are potent binders but present relatively low ΔGQB values, we analyze their dissociation pathways, comparing them with the more standard case (high potency, high ΔGQB). We reproduced the simulations by Kokh et al. , and monitored the intermolecular hydrogen bond used as a dissociation coordinate in DUck. While most ligands follow a one-step dissociation (bound-unbound) with short, undetectable transition states (Figure .A), some show a delay between rupture of the main H-bond and full dissociation (Figure .B). All ligands with a delayed dissociation had false activity cliff predictions. Thus, we hypothesize that DUck can only deliver good predictions when ligands dissociate from the protein in one step. Under these circumstances, the quasi-bound state may be close to the real transition state (Figure .C). Conversely, the appearance of intermediate (metastable) states makes the first barrier in the dissociation process irrelevant from a thermodynamic perspective (Figure .D), rendering DUck ineffective. If confirmed, this would provide a rationale for the unexpected ability of DUck to predict activity cliffs and, at the same time, it would allow us to define its applicability domain. The applicability of ΔGQB to predict activity cliffs is defined by the ligands' dissociation mechanism. To confirm the correspondence between DUck's efficacy and one-step dissociation, we analyzed a Cyclin Dependent Kinase 2 (CDK2) inhibitor data set using the same procedure. CDK2 is a wellstudied kinase with more than 8000 activity entries in the ChEMBL database, of which 35% are IC50 values. We subdivided the CDK2 activity cliff data set into four distinct congeneric series (CDK2-A, CDK2-B, CDK2-C, and CDK2-D), each using the scaffolds from the ligands in the PDB entries 2R3K, 1FVT, 2VTO, and 1JSV as a reference, respectively (Figure ). This procedure minimized the structural differences between members of each set and identified the change responsible for each cliff. Combining the 497 CDK2 active ligands, we obtained 1164 pairs, 121 of which were activity cliffs. The reference ligands' binding mode was used as a template to generate binding poses for the analogs. Per series, a representative pair was selected to explore the dissociation model a priori by τRAMD. Inhibitors from series A and C showed metastable intermediate states, where the ligand no longer formed the H-bond with the hinge region but preserved the remaining interactions (Figure ). By contrast, the representatives from series B and D featured a one-step dissociation, similar to Figure .A, where rupture of the H-bonds resulted in immediate expulsion of the ligands from the binding site (Figure ). Following the hypothesis generated for HSP90α, we predicted that DUck should have some predictive power for series B and D, but not for series A and C.