Selectivity and Ranking of Tight-Binding JAK-STAT Inhibitors Using Markovian Milestoning with Voronoi Tessellations

Abstract

Janus kinases (JAK), a group of proteins in the nonreceptor tyrosine kinase (NRTKs) family, play a crucial role in growth, survival, and angiogenesis. They are activated by cytokines through the Janus kinase-signal transducer and activator of a transcription (JAK-STAT) signaling pathway. JAK-STAT signaling pathways have significant roles in the regulation of cell division, apoptosis, and immunity. Identification of the V617F mutation in the Janus homology 2 (JH2) domain of JAK2 leading to myeloproliferative disorders has stimulated great interest in the drug discovery community to develop JAK2-specific inhibitors. However, such inhibitors should be selective toward JAK2 over other JAKs and display an extended residence time. Recently, novel JAK2/STAT5 axis inhibitors (N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives) have displayed extended residence times (hours or longer) on target and adequate selectivity excluding JAK3. To facilitate a deeper understanding of the kinase-inhibitor interactions and advance the development of such inhibitors, we utilize a multiscale Markovian milestoning with Voronoi tessellations (MMVT) approach within the Simulation-Enabled Estimation of Kinetic Rates v.2 (SEEKR2) program to rank order these inhibitors based on their kinetic properties and further explain the selectivity of JAK2 inhibitors over JAK3. Our approach investigates the kinetic and thermodynamic properties of JAK-inhibitor complexes in a user-friendly, fast, efficient, and accurate manner compared to other brute force and hybrid-enhanced sampling approaches.

Figure 1

JAK2–inhibitor 9 complex with interacting residues within a cutoff distance of 2.5 Å (center). The inhibitors with large residence times for JAK2 proteins are displayed.

Figure 2

A representative Voronoi diagram where V₁, V₂, V₃, .···, V_n represent the Voronoi cells, and a₁ ∈ V₁, a₂ ∈ V₂, a₃ ∈ V₃, .···, a_n ∈ V_n.

Figure 3

Residence times of JAK2 and JAK3 inhibitors as obtained from the experiments and the SEEKR2 milestoning method. The values of the residence times and the error bars for each JAK–inhibitor complex is the average of the three independent SEEKR2 calculations. (a) Residence times of the inhibitors for the JAK2 protein and (b) residence times of the inhibitors for the JAK3 protein are displayed. Error bars are present for the SEEKR2 residence time data, but they are sometimes too small be visible. An unpaired t test is carried out to measure the statistical significance of the difference between the experimentally determined residence times of JAK2 and JAK3 inhibitors and the SEEKR2-calculated residence times. The p-values obtained from the t test determined that there is no significant difference between the mean of the SEEKR2-calculated residence times and the experimentally determined residence times (Table S3).

Figure 4

Free energy profile (ΔG_i) obtained from the SEEKR2 milestoning method for the JAK proteins complexed with the inhibitors. Also shown are the dominant poses of inhibitor 9 as it unbinds from the ATP binding site of JAK complexes. These poses are obtained from the SEEKR2 trajectories for milestones with the local maximum values of ΔG_i. ΔG_i values obtained for each JAK–inhibitor complex is the average of the three independent SEEKR2 calculations. The additional X-axis at the bottom of the graph denotes the distance between the center of masses of the inhibitor and the α carbon atoms of the binding site for each milestone. (a) ΔG_i values for the JAK2 protein complexed with inhibitor 5 and inhibitor 9 along with (i) JAK2–inhibitor 9 complex at TS 1, (ii) JAK2–inhibitor 9 complex at TS 2 (pose 1), and (iii) JAK2–inhibitor 9 complex at TS 2 (pose 2). (b) ΔG_i values for the JAK3 protein complexed with inhibitor 6 and inhibitor 9 along with (i) JAK3–inhibitor 9 complex at TS 1, (ii) JAK3–inhibitor 9 complex at TS 2 (pose 1), and (iii) JAK3–inhibitor 9 complex at TS 2 (pose 2).

Figure 5

(a, b) Major hydrogen bond interactions formed during SEEKR2 simulations at transition states for the JAK2–inhibitor 9 complex displaying (a) TS 1 H-bond donor–acceptor pairs and (b) TS 2 H-bond donor–acceptor pairs. (c, d) Major hydrogen bond interactions formed during SEEKR2 simulations at transition states for the JAK3–inhibitor 9 complex displaying (c) TS 1 H-bond donor–acceptor pairs and (d) TS 2 H-bond donor–acceptor pairs.

Figure 6

Major hydrogen bond interactions formed during SEEKR2 simulations for the JAK2–inhibitor 9 complex at (a) TS 1 displaying H-bond acceptor residues (yellow), H-bond donor residues (green), and H-bond donor/acceptor residues (red) and (b) TS 2 displaying H-bond acceptor residues (yellow) and H-bond donor residues (green).

Figure 7

(a) Composition of inhibitor 9 and (b) molecular orbitals of inhibitor 9.

Figure 8

(a, b) Binding site of inhibitors for JAK2 complex showing important interactions with surrounding residues: (a) JAK2–inhibitor 5 complex and (b) JAK2–inhibitor 9 complex. (c) 2D formulas schemes for the JAK inhibitors indicating the location of modifications. In inhibitor 9, the substituted fluorine atom in the heteroaryl C-ring leads to the electrostatic pull of the hydrogen atom in the nearby serine residue, which contributes to the higher residence time in the kinase domain.

Figure 9

Principal component analysis for JAK2–inhibitor complexes from 2 μs of MD simulation trajectory: (a) First normal mode for JAK2–inhibitor 5 complex (47% of accounted variance). (b) First normal mode for JAK2–inhibitor 9 complex (46% of accounted variance).

Figure 10

Binding site (green mesh) obtained from minimum average inhibitor–residue distances from three independent 2 μs of MD simulation trajectories: (a) JAK2–inhibitor 9 complex and (b) JAK3–inhibitor 9 complex.

Figure 11

Residue fluctuation analysis for JAK2 and JAK3–inhibitor complexes obtained from three independent 2 μs of MD simulation trajectories: (a) JAK2–inhibitor 5 vs JAK2–inhibitor 9 complex and (b) JAK3–inhibitor 6 vs JAK3–inhibitor 9 complex