Selective Targeting of the TPX2 Site of Importin-α Using Fragment-Based Ligand Design

Protein–protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2–importin-α interaction. Importin-α is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites—major and minor—to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment-based approaches were used to identify small molecules that bind importin-α, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure-guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein–protein interaction.


Supplemental protein expression and purification
Plasmids were transformed into E. coli BL21 (DE3) CodonPlus RIL competent cells [2] by heat shock at 42 °C, 1 min, 0 °C, 5 min and then incubation in 1 mL 2xTY media at 37 °C, 30 min. These were then plated on TYE (Kanamycin 25 µg/mL) agar plates and grown overnight at 37 °C. Single colonies were then grown in starter cultures of 2× 50 mL LB media (Kanamycin 25 µg/mL) on a shaker at 37 °C overnight. From these 20x 1 L 2xTY expression media (Kanamycin 25 µg/mL) were inoculated at 1:100 volume dilution and grown at 37 °C to an OD 600 of 0.6 -0.8. The temperature was lowered to 20 °C and overnight expression was induced with IPTG (1 mM final concentration). Cells were harvested by centrifugation (5,000 rpm, 15 min, 4 °C) and pellets were either frozen at -20 °C for future purification or were resuspended in lysis buffer (20 mM Tris.HCl pH 8.0, 200 mM NaCl, 2 mM β-mercaptoethanol).
To resuspended cell pellets were added PMSF (1 mM) and 2 anti-protease tablets (Roche) before lysis by sonication while keeping samples on ice. 0.04% Bovine DNase I (Sigma Aldrich), 0.1% Mn 2+ and 1% Mg 2+ were then added to the lysate on ice, before separation of the cell debris by centrifugation (20,000 rpm, 30 min, 4 °C).
Further clarification was performed through 0.22 µm syringe filters (Sartorius) and these clarified lysates were added to a glass column containing Nickel NTA agarose resin (Qiagen, 2.5 mL of 50% slurry per litre of culture) pre-equilibrated with buffer The purified protein fractions were collected and analysed for purity by SDS-PAGE (using standard PAGE protocols) and those deemed to be >95% pure were pooled and concentrated to 200 µM (as assessed by Nanodrop) using 30 kDa cut-off Amicon Ultra concentrator tubes (Millipore). Average protein yield was ~10 mg/L and mass spectrometry showed a molecular weight of 50435 ± 90 Da consistent with untagged ΔIBB-importin-α1. The protein was flash-frozen in liquid nitrogen in 100 or 200 µL aliquots in thin-wall PCR tubes before storing at -80 °C. Each aliquot was defrosted fully prior to use.
All experiments, competitive and direct, were run using the optimised experiment conditions: 20 injections (first volume 1 µL subsequent 2 µL with default durations), reference power 5 -7 µCal/sec, initial delay 60 sec, stirring speed 1000 rpm, spacing 90 sec and filter period of 2 sec. Experiment injection of 39 µL titrant into 250 µL of protein solution over 40 min.

Co-crystallisation attempts
A screen for co-crystallisation conditions of unliganded mouse ΔIBB-importin-α1 with fragments 1 -10 was set up in the MRC-LMB using in-house robotic nanolitre crystallisation methods on 96-well plates as described by Stock et al. [3] . The protein was used in the purification buffer at 200 µM supplemented with 10% DMSO and 20 -50 µM of the desired fragment (as determined by solubility). 200 nL drops were set up as a 1:1 protein to precipitant volume ratio on in-house 96 well plates with a range of crystallisation conditions. No crystal hits were seen in any case even after three months at 18 °C.

Standard Procedure A) Solid phase peptide synthesis
Solid phase peptide synthesis was performed in batches on NovaPEG Rink amide resin (Novabiochem, Merck Chemicals Ltd.) that was swelled with DCM and then DMF before deprotection and coupling steps. The following commercial protected amino acids were used: Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Thr( t Bu)-OH, and Fmoc-L-Phe-OH (AGTC Bioproducts or Merck Novabiochem). Removal of the Fmoc protecting group at the start of each coupling was performed with 3× 20% piperidine in DMF (v/v) for 5 min each. The resin was then washed with 5× DMF before addition of 5 eq. of Fmoc-protected amino acid, 5 eq. PyBOP and 10 eq. DIPEA in DMF. The vessel was then sealed and left on a shaker at room temperature for the time described. Couplings were repeated until a Kaiser Test on a small portion of the resin was negative. Further deprotections and couplings were repeated as above.
On coupling and deprotection of the final amino acid, peptides were either capped by N-terminal acetylation or coupled to a fragment containing monomer. N-terminal acetylation was affected by 1 eq. of acetic anhydride in DMF for 20 min while fragment coupling was performed using 5 eq. of the fragment containing monomer and either 5 eq. PyCLOCK or PyBOP and 10 eq. DIPEA in DMF or NMP under the conditions and times described.
Once capped, the finished peptide was washed with 5× DMF, 5× DCM and dried before cleaving with concomitant global deprotection with 4 mL 95% TFA in water (v/v) for 3 hr at room temperature. Resin was washed with a further 2 mL 95% TFA in water before the majority of TFA was evaporated under a nitrogen stream. The peptide was precipitated with ice-cold diethyl ether overnight and collected by centrifugation before washing with 3× cold diethyl ether to give the crude peptide which was then lyophilised overnight before purification by HPLC. Product peaks were isolated and lyophilised overnight giving the product.

Standard Procedure B) Suzuki couplings
1 eq. of each boronic acid and bromide starting material were combined in a microwave vial (Biotage) and dissolved in DME before the addition of potassium carbonate (2 eq.) dissolved in water (3:1 v/v DME/water). Nitrogen was bubbled through the stirring solution before the addition of Pd(dppf)Cl 2 (0.04 eq.) and sealing the vial. The reaction was heated in a Biotage Initiator Microwave Synthesizer at before addition of sat. sodium bicarbonate solution and extraction with 3× EtOAc.
The combined organics were then dried with anhydrous magnesium sulphate, filtered and the solvent was removed in vacuo to give the crude compound, which was then purified as described.

Tert-butyl (S)-(6-amino-6-oxo-5-((4-(pyridin-3-yl)benzyl)amino)hexyl)carbamate [5]
A solution of Methyl N 6 -(tert-butoxycarbonyl)-N 2 -(4-(pyridin-3-yl)benzyl)-L-lysinate (303 mg, 0.71 mmol) was made up in a microwave vial (Biotage) with methanol (4 mL) before cooling to 0 o C whereupon magnesium nitride (356 mg, 3.55 mmol) was added in one portion. The vial was sealed and allowed to warm to room temperature in a water bath for 1 hr. The reaction was then heated to 80 o C for 24 hr, carefully monitoring the state of the cap over the first 3 hr. CAUTION: these reactions must be carried out behind a blast shield. The reaction was cooled to room temperature and vented carefully before diluting with chloroform (25 mL) and water (25 mL). The reaction was neutralised with HCl (3N) before the layers were separated and the aqueous layer further extracted with chloroform (3× 25 mL). The combined organics were dried over anhydrous magnesium sulphate, filtered and reduced in vacuo. The crude product was purified by column chromatography (Biotage, eluent 0 -20% MeOH/DCM v/v) to give the pryridine lysine amide as a pale yellow solid (159 mg, 55% yield).

2-yl)hexanamide 13
Following Standard Procedure A for solid-phase peptide synthesis and starting with 100 mg Rink amide resin, the following amounts of amino acids were reacted for the times below:
Product containing fractions were reduced in vacuo to give the Fmoc-D-lysine [9] as a white solid (555 mg, 97% yield).   Data were consistent with those previously reported by Watts et al. [9] .
The crude product was dissolved in EtOAc (15 mL) and washed with sat. sodium bisulphite (15 mL) before the organic layer was dried over magnesium sulphate, filtered and reduced in vacuo to give the pyridine methyl lysinate as a yellow oil (415 mg, 65% yield).
CAUTION: these reactions must be carried out behind a blast shield. The reaction was cooled to room temperature and vented carefully before diluting with chloroform (20 mL) and water (20 mL). The reaction was neutralised with HCl 3 N before the layers were separated and the aqueous layer further extracted with chloroform (3× 20 mL). The combined organics were dried over anhydrous magnesium sulphate, filtered and reduced in vacuo. The crude product was purified by column chromatography (Biotage, eluent 0 -20% MeOH/DCM v/v) to give the pyridine lysine amide as a yellow solid (45 mg, 22% yield).