Covalent targeting of non-cysteine residues in PI4KIIIβ

The synthesis and characterisation of fluorosulfate covalent inhibitors of the lipid kinase PI4KIIIβ is described. The conserved lysine residue located within the ATP binding site was targeted, and optimised compounds based upon reversible inhibitors with good activity and physicochemical profile showed strong reversible interactions and slow onset times for the covalent inhibition, resulting in an excellent selectivity profile for the lipid kinase target. X-Ray crystallography demonstrated a distal tyrosine residue could also be targeted using a fluorosulfate strategy. Combination of this knowledge showed that a dual covalent inhibitor could be developed which reveals potential in addressing the challenges associated with drug resistant mutations.

High-resolution mass spectrometry -High-resolution mass spectra were recorded on a micromass Q-Tof Ultima ® time-of-flight mass spectrometer, and analytes were separated on an Agilent ® 1100 Liquid Chromatograph equipped with a Penomenex ® Luna C18 (2) reversed phase column (100 mm x 2.1 mm, 3 μm packing diameter).Conditions used were 0.5 mL/min flow rate at 35 °C, and an injection volume of 2-5 μL, using a gradient elution made up of water with 0.1% volume formic acid (v/v) and 0.1% formic acid (v/v) in MeCN.Elution conditions began with 5% MeCN mixture, and increased linearly to 100% over 6 min, before remaining at 100% for 2.5 min, decreasing back to 5% MeCN mixture for 1 min and equilibrating for 2.5 min prior to the next injection in the machine.Mass to charge (m/z) ratios are shown in Daltons.

Mass directed auto preparation (MDAP) chromatography -Mass directed auto preparation
was carried on a Waters ® ZQ MS using alternate scan positive and negative electrospray ionisation and a summed UV wavelength of 210-350 nm.According to the following methods:

Physicochemical Assays
Kinetic solubility -The kinetic aqueous solubility at pH 7.4 was determined by measuring the concentration of solute in solution after precipitation from DMSO stock solution.The DMSO stock solution was diluted 20-fold with PBS pH 7.4 and the solubility of the compound is measured after 1 h equilibration at room temperature.In this procedure quantification was done using a Charged Aerosol Detector (CAD).The DMSO concentration of the stock solution was also determined using this technique.Calibration parameters generated for CAD response of two calibrants (Primidone and Ketoconazole) are used to calculate the solubility of solutes considering the density of the compound and ion-pairing effects.

Artificial Membrane Permeability assay
The permeability rate was measured across a phospholipid bilayer system.The lipid was egg phosphatidyl choline (1.8%) and cholesterol (1%) dissolved in n-decane.This was applied to the bottom of the microfiltration filter inserts in a Transwell plate.Phosphate buffer (50 mM Na2HPO4 with 0.5% 2-hydroxypropyl--cyclodextrin), pH 7.4 was added to the top and bottom of the plate.The lipids were allowed to form bilayers across the small holes in the filter.
Permeation experiment was initiated by adding the compound to the bottom well and stopped at a pre-determined elapsed time.The compound permeated through the membrane to enter the acceptor well.The compound concentration in both the donor and acceptor compartments was determined by liquid chromatography after 3 h incubation at room temperature.The permeability (logPapp) measured how fast molecules pass through the black lipid membrane was expressed in nm/s.

Lipophilicity: ChromlogD assay
The Chromatographic Hydrophobicity Index (CHI) values are measured using reversed phase HPLC column (50 x 2 mm 3 µM Gemini NX C18, Phenomenex, UK) with fast acetonitrile gradient at starting mobile phase of pH 2, 7.4 and 10.5.CHI values were derived directly from the gradient retention times using calibration parameters for standard compounds.The CHI value approximates to the volume % organic concentration when the compound elutes.CHI was linearly transformed into ChromlogD by least-square fitting of experimental CHI values to calculated ClogP values for over 20K research compounds using the following formula: ChromlogD = 0.0857CHI-2.00.

In Vitro Assays
Protein supply for biochemical assays and mass spectrometry studies Human PI4KIIIβ (residues 13-828, 316-330 deleted) was expressed with a 6H-TEV n-terminal tag by baculovirus in insect cells.The resulting lysate was purified by nickel affinity and hydroxyapatite chromatography.The tag was removed by TEV-cleavage, followed by nickel affinity chromatography and size exclusion chromatography to provide the purified protein at 1 mg/mL in 50 mM Tris-HCl pH7.5, 250 mM NaCl, 5 mM TCEP, 1 mM CHAPS, 10% Glycerol.
Dispense 3 µL of 2x substrate solution into all columns and add 3 µL of assay buffer (25 mM HEPES pH 7.5 (NaOH), 10 mM MgCl, 0.5 mM EGTA, 0.1% Triton X-100, 2 mM TCEP, 0.1 mg/mL BSA, 100 mM ATP (fresh)) to column 18 (no enzyme low control).Add 3 µL of 2x enzyme solution to columns 1-17 and 19-24 and centrifuge plates at 1000 rpm for 1 min, apply plate seal to each plate and incubate for 180 min at room temperature.Centrifuge plates at 1000 rpm for 1 min prior to removal of plate seal and add 6 µL of ADP-Glo 1 reagent +0.1% CHAPS to all wells.Centrifuge plates at 1000 rpm for 1 min and incubate for 60 min at room temperature.Add 12 µL of ADP-Glo 2 reagent +0.1% CHAPS to all wells, centrifuge plates at 1000 rpm for 1 min and incubate for 40 min at room temperature.Read plates on Viewlux or PHERAstar (signal stable for 30-90 min following Glo 2 incubation).In the enzymatic assay, the ADP product is detected through a 2-step coupled detection with a luminescent read-out.
In the first step the kinase reaction is quenched, and unused ATP substrate is converted to AMP, removing it from the detection system.In the second step, the ADP produced by the kinase reaction is converted back to ATP, and this is then detected via a luciferin/luciferase reaction, as shown in Figure S1.The luminescent signal produced is proportional to the quantity of ADP produced, therefore correlating with PI4KIIIβ activity.Remove the foil seals and pre-warm assay medium to 37 °C in sterile water bath.Wash confluent monolayer of HeLa cells with 10 mL of PBS and add 3 mL TryplE to cover cells.
Incubate 5 min at 37 °C and gently agitate flask to detach cells, then remove cells to 50 mL Falcon tube.Centrifuge 300 g for 5 min and re-suspend pellet in approx.50 mL of media, then count the cells using Vicell.Adjust concentration to 6.6 x 10 4 cells/mL, for the 96-well assay, cells and virus are pre-mixed prior to dispensing into the compound plates.Add virus to moi 1 and HRV type A strain 16 stock, or a pre-diluted stock, is added to the 6.6 x 10 4 cells/mL suspension at the appropriate pre-determined dilution for the current virus stock to achieve an MOI of 1, or a suitable MOI for the current virus stock.Add 150 µL of cell/virus suspension per well using a Multidrop to columns 1 to 11 and add 150 µL of cell suspension per well using a multichannel pipette to column 12. Incubate the plates at 33 °C, 5% CO2, for 2 days post infection.Prepare fresh, or thaw frozen, CellTiter-Glo reagent.Avoid repeated freeze thaw cycles.Remove the 96-well plates from the 33 °C, 5% CO2 incubator and equilibrate to room temperature.Dispense 60 µL CellTiter-Glo reagent into all columns of the 96-well plates using a Multidrop.Cell Titer Glo reagent can be stored at RT or 4 °C for 8 h.Once reconstituted can be stored at -20 °C for 21 weeks with ~3% loss of activity.Stable up to 10 freeze thaw cycles with <10% loss of activity.After approximately 20 minutes of room temperature incubation, read the 96-well plates on the Envision using the Cell Titre Glo for CPE protocol to measure luminescence.

Protein Mass Spectrometry
Intact protein mass spectrometry -Intact protein masses were recorded by liquid chromatography-mass spectrometry (LC-MS) using a 6224 TOF (Agilent) Accurate Mass Series mass spectrometer, interfaced with an Agilent 1200 liquid chromatography and sample handling system.The protein sample was injected using an Agilent 1200 series AutoSampler (Model No. G1367B) with a 10 µL injection volume and maintained at a temperature of 10 °C.
Chromatography was carried out on an Agilent Bio-HPLC PLRP-S (1000 Å, 5 µm × 50 mm × 1.0 mm, PL1312-1502) reverse phase HPLC column at 70 °C.Using an Agilent 1200 series binary pump system (Model No. G1312B) the sample was eluted at 0.5 mL/min using a gradient system from Solvent A (water, 0.2% (v/v) formic acid) to Solvent B (MeCN, 0.2% (v/v) formic acid) according to the following conditions: The eluent was injected directly into an Agilent TOF mass spectrometer (Model No. G6224A) using a dual ESI source and scanning between 600-3200 Da with a scan rate of 1.03 s in positive mode.The following MS parameters were used: Capillary voltage limit -4200; Desolvation temperature -340 °C; Drying gas flow -8.0 L/min.Data acquisition was carried out in 2 GHz Extended Dynamic range mode.Spectra were processed using Mass Hunter Qualitative Analysis™ B06.00 (Agilent) software with the Maximum Entropy method employed.The total ion chromatograms (TIC) were extracted (region containing protein) and the summed scans were deconvoluted over a m/z range with an expected mass range dependent on the protein (see below).

4 :
Ramachandran plots for each crystal structure.The key below the plot details what type of residue each point represents, and each point on the plot is coloured green for favoured, yellow for allowed and red for an outlier.The residue type and position of each outlier is indicated in blue using the three-letter residue code.(Images produced by PSILO 9 ).Compound Compound 8: