Conversion of a False Virtual Screen Hit into Selective JAK2 JH2 Domain Binders Using Convergent Design Strategies

Abstract

The Janus kinase 2 (JAK2) pseudokinase domain (JH2) is an ATP-binding domain that regulates the activity of the catalytic tyrosine kinase domain (JH1). Dysregulation of JAK2 JH1 signaling caused by the V617F mutation in JH2 is implicated in various myeloproliferative neoplasms. To explore if JAK2 activity can be modulated by a small molecule binding to the ATP site in JH2, we have developed several ligand series aimed at selectively targeting the JAK2 JH2 domain. We report here the evolution of a false virtual screen hit into a new JAK2 JH2 series. Optimization guided by computational modeling has yielded analogues with nanomolar affinity for the JAK2 JH2 domain and >100-fold selectivity for the JH2 domain over the JH1 domain. A crystal structure for one of the potent compounds bound to JAK2 JH2 clarifies the origins of the strong binding and selectivity. The compounds expand the platform for seeking molecules to regulate JAK2 signaling, including V617F JAK2 hyperactivation.

Insights on JAK2 Modulation by Potent, Selective, and Cell-Permeable Pseudokinase-Domain Ligands

“…In the pyrrolopyrimidine series, we identified a phenethyl group as a viable substituent to project into the W718 pocket as exemplified by the ligand JAK292 (Figure C, X = CH 2 ). To consider if the addition of a similar group into the diaminotriazole series is feasible, the crystal structures of 6 (Figure , cyan) and the pyrrolopyrimidine ligand JAK292 (Figure , magenta) bound to WT JAK2 JH2 were overlaid.…”

“…These efforts have led to the development of several series of novel JAK2 JH2 binders: indoloxytriazines, diaminotriazoles, and pyrrolopyrimidines (Figure ). , …”

“…These efforts have led to the development of several series of novel JAK2 JH2 binders: indoloxytriazines, 15 diaminotriazoles, 16 and pyrrolopyrimidines (Figure 1). 17,18 An initial step in these studies was the identification of JNJ7706621, a known Aurora A/B kinase and pan-CDK inhibitor, 19,20 as a non-selective JAK2 JH2 ligand (K d = 0.67 ± 0.18 μΜ for JH1 and 0.46 ± 0.12 μΜ for JH2) through a highthroughput fluorescence polarization (FP) screen (Figure 2, compound 1). 21 We then developed an FP assay with a fluorescein conjugate of JNJ7706621 as the tracer, which facilitated rapid binding affinity measurements with a lower detection limit.…”

“…22 Leveraging structural data and computeraided design, we previously optimized JNJ7706621 into a Representative structures of JAK2 JH2 binders discovered previously: (A) indoloxytriazines with single-digit μΜ affinity for JH2 and up to 14-fold selectivity over JH1, 15 (B) diaminotriazoles with low triple-digit nΜ affinity for JH2 and up to 19-fold selectivity over JH1, 16 and (C) pyrrolopyrimidines with double-digit nΜ affinity for JH2 and up to 360-fold selectivity over JH1. 18 Figure 2. Lead optimization studies on diaminotriazole ligands.…”

“…16 However, the affinity and properties of the ligands were not sufficient for testing in cellular assays. Newly discovered insights on essential pharmacophore elements of JH2 leads 18 prompted us to further optimize the diaminotriazole urea series, aiming to achieve higher potency and selectivity, and enable cell testing. Toward this goal, we report here affinity and permeability optimizations toward the development of potent, selective, and cell-permeable lead compounds (Figure 2).…”