Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of β-Coronaviruses

Abstract

From a designed library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe (17) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of inhibitor selectivity confirmed that this PIKfyve probe demonstrates excellent kinome-wide selectivity. A structurally related indolyl pyrimidinamine (30) was characterized as a negative control that lacks PIKfyve inhibitory activity and exhibits exquisite selectivity when profiled broadly. Chemical probe 17 disrupts multiple phases of the lifecycle of β-coronaviruses: viral replication and viral entry. The diverse antiviral roles of PIKfyve have not been previously probed comprehensively in a single study or using the same compound set. Our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has a non-overlapping kinase off-target profile with known PIKfyve inhibitors. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology.

Identification of a chemical probe for lipid kinase phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ)

“…12-(cyclopropylethynyl)-6,7,8,9-tetrahydro-5H-pyrimido[4’,5’:3,4]cycloocta[1,2-b]indol-2-amine ( 4 ) . The analytical data for 4 matches that previously published (Drewry et al ., 2022). 1 H NMR (400 MHz, CD 3 OD- d 4 ) δ 8.09 (s, 1H), 7.83 (s, 1H), 7.21 (dd, J = 8.3, 0.7 Hz, 1H), 7.10 (dd, J = 8.3, 1.5 Hz, 1H), 3.05 – 3.01 (m, 2H), 2.65 – 2.61 (m, 2H), 1.86 – 1.81 (m, 4H), 1.42 (dt, J = 8.2, 5.0 Hz, 1H), 0.87 – 0.80 (m, 2H), 0.71 – 0.66 (m, 2H).…”

“…Based on our published and some unpublished work, the indolyl pyrimidinamine core is a privileged lipid-targeting scaffold. We previously carried out an extensive study to establish structure–activity relationships (SAR) for this core in our development of a chemical probe targeting the lipid kinase PIKfyve (Drewry et al ., 2022). Upon screening a small library of this scaffold at Eurofins DiscoverX in their scan MAX panel, we discovered that many of the compounds in that set bound to PI5P4Kγ, suggesting that we could design a cell-active PI5P4Kγ-targeting chemical probe by modifying this scaffold.…”

“…2 and 3, we see that PIKfyve and RIPK5 are two off-target kinases within the S 35 (1 µM) for these two compounds, but the follow-up data indicated only very weak inhibition of RIPK5 by both the chemical probe and the negative control. With respect to PIKfyve, for compound 4 , which was recently published as compound 28 in a PIKfyve manuscript from our lab (Drewry et al ., 2022), the enzymatic inhibition IC 50 value (75 nM) indicated enhanced potency versus the corresponding NB inhibition IC 50 value (700 nM, Fig. S4).…”

“…Specific assay details can be found on the Eurofins website: https://www.eurofinsdiscoveryservices.com. We used a previously developed ADP-Glo assay (Promega) in dose–response (10-pt curve) format in duplicate at SignalChem to evaluate the enzymatic inhibition of PIKfyve (Drewry et al ., 2022). Radiometric HotSpot kinase assays from Reaction Biology Corp. (RBC) were carried out at the K m value for ATP in dose– response (10-pt curve) format for MYLK4, RIPK5, and YSK4.…”

“…A mixture of 1 (Drewry et al ., 2022) (192 mg, 0.58 mmol), 2-(3,6-dihydro-2 H -pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (122 mg, 0.87 mmol), and K 2 CO 3 (161 mg, 1.16 mmol) in a mixture of dioxane/water (2.0/0.3 mL) was degassed, followed by addition of Pd(PPh 3 ) 4 (34 mg, 29 µmol). The reaction mixture was heated to 95 °C and allowed to stir overnight.…”