An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine‐56

Abstract The stress‐inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine‐targeted irreversible inhibitor. Using rational design, we adapted a validated 8‐N‐benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine‐56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.


General Chemistry Experimental
Unless otherwise stated, all reactions were conducted in oven-dried glassware under an inert atmosphere of nitrogen using anhydrous solvents. All commercially obtained reagents and solvents were used as received. Oridonin 3 was purchased from Sigma-Aldrich.
Microwave reactions were performed using a Biotage Initiator Microwave Synthesizer. Thinlayer chromatography (TLC) was performed on precoated aluminium sheets of silica (60 F254 nm, Merck) and visualized using short-wave UV light. Column chromatography was performed on a Biotage SP1 purification system using Biotage Flash silica cartridges. All Chemical shifts are quoted to 0.01 ppm, using the following internal references: CDCl 3 (δC 77.0), MeOD (δC 49.0) and DMSO-d 6 (δC 39.5). High-resolution mass spectra were recorded on an Agilent 1200 series HPLC and diode array detector coupled to a 6210 timeof-flight mass spectrometer with dual multimode APCI/ESI source. Analytical separation was carried out on a Merck Purospher STAR RP-18, 30 mm x 4 mm column using a flow rate of 1.5 mL/min in a 4 min gradient elution, UV detection was at 254 nm. Infrared spectra were recorded on a Bruker Alpha-p FT-IR spectrometer. Only structurally important absorption peaks are quoted, absorption maxima (V max ) are quoted in wavenumbers (cm -1 ). All compounds were >95% purity by HPLC analysis unless otherwise stated.
DIBAL-H (1 M in toluene, 43.3 mL, 43.3 mmol) was added dropwise to the solution at -78 °C and stirred for 2 hours, then quenched with MeOH (20 mL) and warmed to room temperature. A sat. solution of Rochelle's Salt (150 mL) was added and the resulting suspension stirred overnight. The mixture was extracted with EtOAc (3 x 50 mL) and the combined organic layers were washed with sat. brine (3 x 50 mL) and dried over Na 2 SO 4 .

Purification of WT HSP72-NBD
The coding sequence for residues 1 to 380 HSPA1A (HSP72) was amplified by PCR from

General experimental
The aqueous buffer contained 50 mM TRIS base pH 7.4, 150 mM KCl, 5 mM CaCl 2 and 0.1% (w/w) CHAPS. The assay was conducted using 384 Plus F ProxiPlates (PerkinElmer) with a final assay volume of 10 μL. Plates were centrifuged at 1000 rpm for 1 minute prior to incubation and read using a 2103 Envision Multilable Plate Reader. Excitation and emission wavelengths used were 620 nm and 535 nm, respectively. Fluorescence polarization was measured in units of millipolarization (mP). All experiments were performed in triplicate. Data were plotted and analysed using GraphPad Prism 6, graphical data represents the arithmetic mean ± curve fitting standard error of the mean for a single representative experiment.

K D determination
To each well, 5 μL of probe molecule S5 (20 nM in assay buffer) and increasing concentrations of HSP72-NBD protein (5 μL, two-fold dilution series) were added.
Fluorescence polarization values for tracer control wells (10 nM probe S5 in assay buffer only) were subtracted from each data point prior to data analysis. K D determination was performed using non-linear regression analysis (GraphPad Prism 6, one site-specific binding model). pK D values are quoted as geometric mean ± standard error of the mean from 3 independent experiments.

Competitive binding experiments
Compounds (0.2 μL at 50 x screening concentration in DMSO) were dispensed using an ECHO 550 Liquid Handler (Labcyte Inc.). To the corresponding wells was added, 5 μL of probe molecule S5 (20 nM in assay buffer) and 5 μL of protein (two times their final concentration in assay buffer) to give a 50% bound fraction. Tracer controls (10 nM probe molecule S5 only) and bound tracer controls (10 nM probe in presence of appropriate protein concentration) were included on each assay plate. IC 50 determination was performed using non-linear least squares curve fitting (GraphPad Prism 6, log(inhibitor) vs. responsevariable slop (four parameters)). K i values were calculated using Huang's equation below.
pK i values are quoted as geometric mean ± standard error of the mean from 3 independent experiments.

Huang's Equation describes the relationship between IC 50 and K i
Huang's equation was used to calculate K i values from the measured IC 50 s. See equation below: The equation states that the IC 50 for a ligand that is competitive for binding with the assay probe is related to the binding affinity of the ligand (K i ), the bound fraction of the probe (f 0 ), the binding affinity of the probe (K d ) and the concentration of the probe (L 0 ). For competition experiments, it is recommended that a protein concentration giving a bound fraction between 0.5 and 0.8 be selected. A bound fraction below 0.5 will often result in an assay that is not statistically robust due to the decreased size of the binding window, however as the bound fraction approaches 1 the relationship between K i and IC 50 deviates from linear and the resolvable range of the assay decreases. For these reasons, a bound fraction of 0.5 was used for all assays.  acetonitrile. These were then reduced with 5 mM TCEP, alkylated with 10 mM chloroacetamide, and the proteins digested by addition of 20 ng trypsin and incubation for 1 h at 37 °C, followed by a second addition of 20 ng trypsin and incubation for a further 3 h at 37 °C. The digestion solution was purified in its entirety by C18 ZipTip cleanup and dried in S36 vacuo. The dried eluate was reconstituted in 10 uL of 1% acetonitrile/0.1% formic acid for LC-MS/MS.

Representative Fluorescence Polarisation K D determination curves with ATP-ATTO
Reversed phase chromatography was performed using an HP1200 platform (Agilent, Wokingham, UK). Approximately 50% of the digested sample was analysed as a 6 µL injection. Peptides were resolved on a 75 µm I.D. 15 cm C18 packed emitter column (3 µm particle size; NIKKYO TECHNOS CO. LTD, Tokyo, Japan) over 30 min using a linear gradient of 96:4 to 50:50 buffer A:B (buffer A: 1% acetonitrile/3% dimethyl sulfoxide/0.1% formic acid; buffer B: 80% acetonitrile/3% dimethyl sulfoxide/0.1% formic acid) at 250 nL/min. DMSO was used based on the methods of Hahne et al. [1] Peptides were ionised by electrospray ionisation using 1.8 kV applied immediately pre-column via a microtee built into the nanospray source. Sample was infused into an LTQ Velos Orbitrap mass spectrometer