Targeted Covalent Inhibition of Plasmodium FK506 Binding Protein 35

FK506-binding protein 35, FKBP35, has been implicated as an essential malarial enzyme. Rapamycin and FK506 exhibit antiplasmodium activity in cultured parasites. However, due to the highly conserved nature of the binding pockets of FKBPs and the immunosuppressive properties of these drugs, there is a need for compounds that selectively inhibit FKBP35 and lack the undesired side effects. In contrast to human FKBPs, FKBP35 contains a cysteine, C106, adjacent to the rapamycin binding pocket, providing an opportunity to develop targeted covalent inhibitors of Plasmodium FKBP35. Here, we synthesize inhibitors of FKBP35, show that they directly bind FKBP35 in a model cellular setting, selectively covalently modify C106, and exhibit antiplasmodium activity in blood-stage cultured parasites.


Supplemental Tables and Figures
The concentration of residual FKBP35 as a function of time. Data was fit to an exponential "one phase decay" model which was used to calculate the rate (K) and half-life (t 1/2 ) of the reaction. The loss of FKBP35 in the DMSO and 1b controls is likely due to adsorption onto the vial. B. Concentration of covalent adduct as a function of time. Data was fit to an "exponential plateau" regression model, which was used to calculate the rate of formation (K'). Compound 1b, which has no electrophilic warhead, does not form any adduct. C. Tabulated rates calculated from the regressions in A and B. The rates of FKBP35 consumption correlate strongly with the rates of adduct formation.  A B C D Figure S5. Structure of GPI-1046. GPI-1046 is an FKBP12 inhibitor that entered Phase I clinical testing for Parkinson's Disease. The proline core of GPI-1046 binds with much weaker affinity to FKBP12 than the pipecolate core of the ligands in this study and others and as such serves as a negative control for the NanoBRET assay. Figure S6. P. falciparum strain NF54 growth-inhibition and plasma stability assays. A. NF54 parasites were incubated with 2c or 2d. Proliferation was measured by luminescence after a complete life-cycle and normalized to DMSO (100%) and chloroquine (100 nM, 0%). Compound IC50s (μM) are included. B. Plasma stability assay of investigated compounds tested in the antiplasmodium assay. Compound Plasma Stability (human) 1% 0% Figure S7. Cell viability in HEK293T cells. Antiplasmodial compounds were tested for cytotoxicity against HEK293T cells over a 72 hour treatment period. The protease inhibitor carfilzomib (IC50 = 15 nM) was included as a positive control. All compounds tested failed to achieve significant cell death except 1c and Rapamycin, which had IC50s of 75 and 63 μM, respectively.

Computational Modeling
The CovDock procedure was used in pose prediction mode with default settings in Schrodinger Release 2019-1. 1 The starting receptor was created from a high-resolution (1.44Å) FKBP35 crystal structure downloaded from the RCSB PDB (4QT2). The Protein Preparation Wizard in Schrodinger was used to inspect the structure, protonate at neutral pH, and run a restrained minimization (OPLS3). An FKBP51 structure downloaded from the PDB with a relevant ligand (4DRK, 1.50A resolution) was overlaid with the prepared 4QT2 structure. The water molecules and rapamycin were deleted from 4QT2 and from 4DRK only the ligand was retained. The Protein Preparation Wizard was run again with restrained minimization to yield the FKBP51 receptor with the 4DRK ligand. Glide SP was able to reproduce the pose of the 4DRK ligand. Design molecules were run through Schrodinger ligprep to generate reasonable tautomer and protonation states. Covalent complexes from the CovDock procedure were checked that they reproduced the pose seen in 4DRK.

Plasmid Generation
Plasmodium falciparum FKBP35 Q5-A128 (the FKBP35 binding domain, FBD35) and Human FKBP12 were generated by ordering gDNA block of codon-optimized DNA fragments (IDT) which were incorporated into pETHSUL, an N-terminal 6His-SUMO backbone vector, using the In-Fusion ligation-independent cloning kit (Takara). The mixture was transformed into E. coli XL10-gold competent cells for ligation and amplification of the plasmids. The sequences of pETHSUL-FBD35 and pETHSUL-FKBP12 were confirmed by DNA sequencing.

FKBP Expression and Purification
The pETHSUL-FBD35 plasmid was transformed into E. coli strain BL21 DE3 (Invitrogen) and grown in 1 L of TB media (6 g tryptone, 12 g yeast extract, 1.15 g KH2PO4(monobasic), 6.25 g K2HPO4 (dibasic), 20 mL glycerol) containing 100 μg/mL kanamycin at 37˚C until OD600=1.0. The temperature was reduced to 20 ˚C, and expression was induced after 40 minutes by addition of isopropyl-β-D-thiogalactopyranose (IPTG) to a final concentration of 0.4 mM. The cultures were incubated 16 hr at 20 ˚C and cells were harvested by centrifugation at 3,000 x g.
All purification steps were carried out at 4˚C. Cell pellets were resuspended in 30 mL lysis buffer (25 mM HEPES pH 7.8, 500 mM NaCl, 20 mM imidazole, 5% glycerol), lysed by sonication, and centrifuged at 27,000 x g for 45 minutes at 4°C. The supernatant was loaded onto a 5-mL HiTrap chelating column (GE Healthcare) pre-equilibrated with lysis buffer. The column was washed with 30 mL lysis buffer. The protein was eluted with a linear gradient of 20-500 mM imidazole in lysis buffer. Fractions containing FKBP35, as determined by 12% SDS-PAGE, were pooled and dialyzed 1 hr against 1 L dialysis buffer (20 mM HEPES pH 7.8, 500 mM NaCl, 5% glycerol).
The 6His-SUMO fusion was removed by incubating the protein at 4˚C overnight with His-tagged ULP1-hydrolase at a final concentration of 1:1000 (protease:protein) and dialysis was continued with fresh buffer for 16 hr. The proteolysis mixture was loaded on a 5-mL HiTrap column preequilibrated with lysis buffer, and cleaved protein (FBD35) was washed from the column with lysis buffer.

Mass Spectrometry
Prior to DSC experiments 400 µL of the reaction mixture was saved for mass spectrometry analysis. For intact mass analysis, 100 µL of the reaction mixture was diluted to 0.05 mg/mL and and injected onto a ZORBAX StableBond 300 C8 HPLC column [2.1 x 100 mm, 3.5 µm (Agilent)] on an Agilent HPLC binary pump system. Initial mobile phase conditions were 15% acetonitrile/85% water, both containing 0.1% formic acid. Protein was desalted for 2 minutes and eluted from the column with a gradient of 15% to 75% acetonitrile over 10 min. Intact mass measurement was performed on a Q Exactive mass spectrometer with an ESI source (Thermo Scientific). Source parameters: spray voltage, 3500 V; capillary temperature, 340°C; sheath gas, 35; auxiliary gas, 5; (gas flows in arbitrary units of the ESI source). MS detection was performed using a Full MS scan acquisition method, scanning over the range 800-2100 m/z. BioPharma Finder v 3.0 (ThermoFisher Scientific) was used for data acquisition and analysis. DSC and mass spectrometry experiments were performed in parallel.
For chymotrypsin digestion, 20 µL of the reaction mixture was added to 20 µL of fresh denaturing solution (0.1 M urea, 0.1 M NaCl, adjusted to 10 ml with 50 mM Tris Buffer pH 8.0). Then 10 µL of 10 mM TCEP was added to the mixture, which was then incubated at 37˚C for 60 minutes. To prevent modification of free thiol groups in FKBP proteins during digestion, 4 µL of 50 mM iodoacetamide (IAA, Sigma No. A3221) was added followed by incubation at room temperature in the dark for 30 minutes. After the incubation, the reaction mixture was brought up to 200 µL with 140 µL of 10 mM Trizma pH 7.5. The peptide fragments were generated by adding chymotrypsin at a molar ratio of 1 chymotrypsin : 100 FKBP protein and incubated overnight at 37˚C. The reaction was quenched by adding 10 µL of formic acid (~5% of total volume) followed by a few seconds of a vortex pulse.
While not a true kinetic analysis of the covalent ligands, we can compare relative rates of formation and consumption using simple mathematical models. The rate of FKBP35 consumption was fitted to a "one phase decay" exponential function: = ( 0 − ) * − + Likewise, the covalent adduct formation data was fit to an "exponential plateau" regression model: Parasitemia was determined every 48 h through microscopic examination of thin blood smears fixed in methanol, stained with 10% Giemsa solution, and sub-cultured to 0.5-1% parasitaemia.

Compound susceptibility assays
In 96-well round-bottom plates, sorbitol-synchronized cultures of ring-stage NF54 P. falciparum parasites were established in triplicate and treated with varying concentrations of the inhibitors (25-0.0038 μM) serially diluted in complete medium. Parasite proliferation rates were analyzed after 72 h using the Renilla-Glo Luciferase Assay, and luminescence was measured using the GloMax® Discover Microplate Reader. EC 50 values were obtained from corrected doseresponse curves using GraphPad Prism (version 5; GraphPad Software). Data represent the mean % luminescence relative to 5% DMSO-treated (vehicle, 100% growth) and 100 nM chloroquinetreated (no growth) controls.

Plasma Stability Assay
Each compound was prepared in duplicate at 1 µM final concentration (0.2% DMSO) in human or mouse plasma (Aldrich). Samples were incubated at 37 °C for 5 hours with mixing at 350 rpm on an orbital shaker. Aliquots of each sample were taken at time zero and following 5 hours. Each sample was quenched by adding acetonitrile in a 3:1 ratio and further diluting with 50 µl of PBS. After quenching, samples were centrifuged to pellet precipitated particulates and an aliquot of supernatant was diluted 1:1 with water. The resulting solution was analyzed by UPLC-MS/MS with compounds detected by MRM detection on a triple quadrupole mass spectrometer. The ratio of compound peak areas at 0 and 5 hours were used to calculate the percent remaining.

General Information
All reactions were carried out under an atmosphere of N2 using flame-dried glassware. All reagents and solvents were purchased from commercial vendors and used without further purification. D44 and D44a-c were synthesized from WuXi AppTec and used as received. GPI-1046 was ordered from Toronto Research Chemicals (cat. D472690) and used as received.
Rapamycin was ordered from Carbosynth (cat. AE27685) and used as received. NMR spectra were recorded on a v Bruker (

Methyl (S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate (SI-2). A 3-neck 100ml
flask equipped with a stir bar, 50ml pressure-equalizing addition funnel, and sealed with rubber septa was flame dried and cooled under N2. SI-1 (3.12 g, 13.6 mmol) was syringed into the flask and dissolved in 35ml THF (0.4 M) and cooled to -78 °C in a dry ice/acetone bath. After equilibrating, 1,1-dimethylpropylmagnesium chloride (1.0 M in diethyl ether, 15.6 mL, 15.6 mmol) was syringed into the addition funnel and added to the reaction at a rate of roughly 1 drop/s. The reaction was stirred at -78 °C for two hours. The reaction was then quenched with saturated NH4Cl and warmed to room temperature. The reaction was poured into a separatory funnel and diluted with EtOAc (100 mL). The aqueous layer was separated and the organic layer was washed sequentially with water and brine, dried over MgSO4, filtered and concentrated to furnish SI-2 (3.02 g, 82% yield) as a colorless oil, which was of sufficient purity to advance to the next step.

2-oxo-2-(3,4,5-trimethoxyphenyl)acetic acid (SI-3).
1-(3,4,5-trimethoxyphenyl)ethan-1-one (2.0 g, 9.5 mmol, 1 equiv.) and SeO2 (1.58 g, 14.3 mmol, 1.5 equiv.) were added to a 20 mL pressure release vial with a stir bar. Dry pyridine (9.5 mL, 1.0 M) was syringed in and the reaction was heated to 110 °C for 3 hours. The reaction was allowed to cool to room temperature and was filtered through a pad of Celite. The Celite was washed with toluene and the filtrate was concentrated by rotary evaporation. Toluene was added to the residue and concentrated again and the residue was taken up in EtOAc and poured into a separatory funnel. The organics were washed with 0.5 M HCl to remove residual pyridine. The organics were then dried over MgSO4, filtered, and concentrated to furnish SI-3 (1.89 g, 83% yield) as a pale yellow solid. Characterization was consistent with previous reports.

General Procedure A: Synthesis of chalcones
In a round bottom flask with a stir bar, 3,4-dimethoxybenzaldehyde (1 equiv.) and ketone (1 equiv) were dissolved in a 10:1 mixture of ethanol:water (0.2-0.5 M). After dissolution, the reaction was cooled in an ice bath and KOH pellets (2 equiv.) were added. The reaction was stirred for the indicated time and quenched with 2N HCl. The resultant solid was extracted with hot EtOAc and washed with water and brine, dried with MgSO4, and concentrated. The crude mixture was purified by recrystallization to furnish the product.

General Procedure B: Hydrogenation.
A round bottom flask with a stir bar was sealed with a rubber septum, evacuated under vacuum, and backfilled with N2 3x. Chalcone (1 equiv.) and Pd/C (10% by weight) were added and the flask was purge with N2 twice more. Solvent (0.1 M) was syringed into the flask and a H2 balloon was affixed to the flask and the stirring rate was set to 750 rpm. A small outlet needle was inserted into the septum and the flask was purged with 1 balloon's worth of H2. A fresh balloon was attached and the outlet needle was removed and the reaction was stirred until monitoring by LC-MS showed conversion to product 5a-d. Upon completion, the reaction was filtered through silica, washed with EtOAc, and concentrated. The crude product was purified by automated flash chromatography (RediSep Gold columns, EtOAc in Hexanes) to furnish the pure product.

General Procedure C: Asymmetric reduction of ketones
A round-bottom flask with a stir bar was fitted with a rubber septum and flame-dried under N2.
Aryl ketone (1 equiv.) was added to the flask and dissolved in 0.1 M dry THF. The flask was lowered into an acetonitrile bath and the temperature was lowered to -45 °C with slow addition of dry ice. (+)-B-Chlorodiisopinocampheylborane ((+)-DIP-Chloride, 1.6 M in hexane, 1.5 equiv.) was added dropwise to the solution and the reaction was allowed to warm up to room temperature in the acetonitrile bath overnight. The stir bar was removed and the reaction was concentrated under rotary evaporation. The residue was dissolved in diethyl ether (0.05 M), the stir bar returned, and diethanolamine (2.5 equiv.) was added at room temperature. The reaction was stirred at room temperature for 2 hours and was vacuum filtered through a pad of Celite. The Celite was washed with ethyl acetate and the filtrate concentrated by rotary evaporation. The crude residue was purified by automated flash column chromatography (RediSep Gold 24g to 40g, 0-100% EtOAc in hexanes). Enantiomeric excess (% ee) was confirmed by chiral SFC and absolute stereochemistry was inferred based on literature precedent. 5,12

General Procedure D: DCC Coupling
In a round-bottom flask or vial with a stir bar, carboxylic acid (1 equiv.), alcohol or amine (1.1 equiv.), and DMAP (0.1 equiv.) were dissolved in DCM (0.5 M). DCC (1.0 M in DCM, 1.1 equiv.) was syringed into the flask and the reaction was stirred at RT for 2 hours. The reaction was filtered through silica and washed with additional DCM. The filtrate was concentrated by rotary evaporation and purified by automated flash chromatography (RediSep Gold columns, EtOAc in hexanes for normal phase or C18 columns, MeCN in H2O + 0.1% formic acid for reverse phase separations) to furnish the coupled product.

tert-butyl (S)-1-(4-(N-(4-methoxybenzyl)propionamido)-3,3-dimethyl-2oxobutanoyl)piperidine-2-carboxylate (12b).
In a 2-dram vial with a stir bar, 11 (131 mg, 0.46 mmol) and 4-methoxybenzylamine (70 μL, 0.55 mmol) were dissolved in MeOH (2.5 mL). Formaldehyde (37% aqueous, 140 μL, 1.85 mmol) was syringed in and the reaction was heated to 50 °C. After 44 hours, the reaction was concentrated under reduced pressure and directly purified by automated reverse-phase chromatography (RediSep Gold C18 15g, 10-100% MeCN in water + 0.1% formic acid). The fractions containing the amine (as identified by LC-MS) were neutralized with saturated NaHCO3 and extracted with DCM. The combined organics were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The purified residue was then transferred to a new 2-dram vial and dissolved in DCM (2 mL). DIPEA (160 μL, 0.93 mmol) was added and the reaction was cooled in an ice bath. Propionyl chloride (80 μL, 0.93 mmol) was added and the reaction was stirred at 0 °C for 2 hours. The reaction was quenched with saturated NaHCO3 and extracted 3x with DCM. The organics were washed with brine, dried over MgSO4, and concentrated. The residue was purified by automated flash chromatography to furnish 12b (160 mg, 71% yield) as a colorless oil.  1-(tert-Butoxycarbonyl)piperidine-2-carboxylic acid (SI-18). In a 100 mL round bottom flask with a stir bar, pipecolic acid (2.0 g, 15.48 mmol) was dissolved in MeOH (30 mL). Boc2O (6.76 g, 30.97 mmol) and NEt3 (2.37 mL, 17.03 mmol) were added and the reaction was heated to 50 °C for 5 minutes. When the vigorous bubbling ceased, the reaction was cooled to RT and stirred for an additional hour. The reaction was then concentrated by rotary evaporation and the crude extract was dissolved in EtOAc and added to a separatory funnel with saturated NaHCO3. The organic was washed twice more with saturated NaHCO3 and the combined aqueous fractions were acidified with 2N HCl. The aqueous fractions were then extracted twice with EtOAc and the combined organics washed with 0.1 M HCl, dried over MgSO4, filtered, and concentrated to give SI-18 (3.23 g, 91% yield) as a white powder. Characterization was consistent with previous reports.  1-carboxylate (SI-19). A 40 mL pressure-release vial with a stir bar was flame dried under N2. Isatin (1.68 g, 11.45 mmol) and DMAP (70 mg, 0.57 mmol) were added to the vial and dissolved in dry THF (20 mL). Boc2O (2.75 g, 12.6 mmol) was dissolved in 5 mL THF and added to the reaction over 5 minutes. The reaction was stirred at RT for 6 hours. Upon completion, the reaction was poured into brine and extracted 3x with EtOAc. The combined organics were dried over MgSO 4 , filtered, and concentrated. The crude residue was recrystallized from hot hexanes and EtOAc to furnish SI-19 (2.73 g, 96% yield) as a yellow powder. Characterization was consistent with previous reports. 16 1 H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.2 Hz, 1H), 7.77 -7.73 (m, 1H), 7.73 -7.67 (m, 1H), 7.30 -7.26 (m, 1H), 1.65 (s, 9H).