Discovery and Characterization of Clinical Candidate LXE408 as a Kinetoplastid-Selective Proteasome Inhibitor for the Treatment of Leishmaniases

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.

Section 6 Chemistry S10 Section 6.1 Chemistry procedures S10 Section 6.2 LCMS spectra S16 Section 7 In vitro pharmacology profiling for 1 S18

Section 1: Dissolution studies
Compound powder 1 or 2 (5 mg of free base equivalent) was weighed into a glass vial containing a stir bar. 5 mL of SGF was added with continuous stirring at 75 rpm. At 60 min, 5 mL of FaSSIF was added to the vessel. Aliquots (0.1 mL) were removed and filtered at specified time points (15,30,45, 60 min). Concentration of the filtrates was determined by UV absorbance with reference to a calibration curve.

S5
in parasite proliferation assays were determined following the protocols were published previously. 1 Growth inhibition high throughput screens on L. donovani axenic amastigotes, T. brucei bloodstream form trypomastigotes and intracellular T. cruzi amastigotes proliferating inside 3T3 cells are summarized in the Supplementary Information section and in Supplementary Tables 5, 6, and 7, respectively. In vitro potency on intramacrophage L. donovani MHOM/ET/67/HU3 (ATCC) was determined using primary mouse peritoneal macrophages infected with L. donovani splenic amastigotes. Primary mouse macrophages were elicited in female BALB/c mice for 72 hours following the injection of sterile 2% starch solution into the mouse peritoneal cavity. The protocol used for isolation of peritoneal macrophages was described in detail previously. 2 The isolated macrophages were re-suspended in the assay medium (0.8 x 10 6 cells/mL in RPMI-1640 with 2.05 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 10 mM sodium pyruvate, and 100U penicillin/ 10 µg/ mL streptomycin), and 25 µL of macrophage suspension were added to clear-bottom, black 384-well plates (Greiner Bio-One) containing 25 µL of the assay medium per well. Following overnight incubation at 37 °C/ 5% CO2, plate wells were washed with the assay medium to remove non-adherent cells. L. donovani splenic amastigotes were isolated from spleens of female BALB/c mice infected for 50-60 days as described elsewhere. 3 Isolated amastigotes were re-suspended in the assay media at a concentration of 6 x 10 7 cells/ mL, and 10 µL of the suspension were added to assay plates containing adherent macrophages. After a 24-hour infection period at 37 °C/ 5% CO2, plates were washed with the assay medium to remove residual extracellular parasites. L. donovani-infected macrophages were subsequently treated with compounds in dose-response for 120 hours. Following a wash step with the phosphate-buffered saline buffer (PBS) supplemented with 0.5 mM magnesium chloride and 0.5 mM calcium chloride, macrophages were fixed in 0.4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with SYBR Green I nucleic acid stain (Invitrogen, 1:100,000 dilution) overnight at 4 °C. Image collection and enumeration of macrophage cells and intracellular L. donovani amastigotes was performed using the OPERA Acapella software (Evotec Technologies) and 20x water immersion objective as described previously. 1 All reported kinetoplastid parasite EC50 values were calculated from at least 3 technical replicates (n= 3 or n= 4; specified in relevant Figure captions). Each technical replicate experiment was performed on a different day with freshly prepared reagents.
Mammalian cell cytotoxicity assay NIH/3T3 fibroblast cell (ATCC) viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) exactly as described previously. 1 Primary macrophage cell viability was determined on mouse peritoneal macrophages infected with L. donovani using the ratio of the number of macrophage cells in wells treated with a compound to those in wells treated with DMSO for calculation of CC50 values. The number of macrophage cells in wells was determined by high content microscopy as described previously. 1 All reported mammalian cell CC50 values were calculated from 4 technical replicates (n= 4; also specified in Figure 1 and Extended Data Figure 3 captions). Each technical replicate experiment was performed on a different day with freshly prepared reagents

Purification of L. tarentolae proteasome
Purification of L. tarentolae wild-type proteasome carried out as previously described with some minor modifications. 4 The parasites were lysed in lysis buffer by dounce homogenizer and the lysates were centrifuged. The resulting supernatant was fractionated through ammonium sulfate precipitation. Precipitated protein fraction was re-suspended and further purified by an anion exchange chromatography (HiTrapQ), followed by a size-exclusion chromatography (Superose 6) and finally by another anion exchange chromatography (MonoQ). The final proteasome was stored in 20 mM HEPES (pH 7.5), 150mM NaCl and 1mM TCEP. The L. tarentolae expressing beta 4 Phe24Leu FLAG protein was generated by expressing the beta 4 protein sequence (LtaP36.0310) with the 3x FLAG tag added to the C-terminus followed by replacing phenylalanine residue at position 24 by leucine (F24L). The entire gene sequence was then codon optimized and cloned into pLEXSY neo expression vector (Jena Bioscience). The vector was transfected into L. tarentolae and selected for clones with the best expression using anti-FLAG antibody. Purification of L. tarentolae beta 4 FLAG tagged Phe24Leu mutant was accomplished via affinity purification. Briefly, after the cells were lysed and cleared as previously described, the resulting supernatant was incubated with ANTI-FLAG M2 antibody column (Sigma-Aldrich) for 3 hours and eluted using 3x FLAG peptide (Sigma-Aldrich) with overnight incubation. The purified fractions were further subjected to an anion exchange chromatography (MonoQ) and size-exclusion chromatography (Superose 6). The final proteasome was stored in 20mM HEPES (pH 7.5), 150mM NaCl and 1mM TCEP.

Biochemical chymoptrypsin activity
Chymotrypsin activity of purified proteasome from both wild type and Phe24Leu were carried out as described previously (Khare et al., 2016)

Generation of T. brucei strains ectopically expressing proteasome β4 and β5 subunit variants
All the T. brucei proteasome mutants were generated by following protocols described earlier (Khare et al., 2016). Briefly, PSMB5 (Tb927.10.6080) was assembled into pHD1034 vector using GIBSON assembly technology (NEBuilder high fidelity DNA assembly cloning kit). The sense (5′-caccaaaaagtaaaattcacaagcttATGTTGGCAGATTTTGAAAG-3′) and antisense (5′-ccgtcgtggtccttgtagtcgaattctctagaTGCACTCTGAGAAGAATAAC-3′) primers were used. The PSMB5 Y113F gene was generated from the wild-type PSMB5 construct by site-directed mutagenesis using mutagenic sense (5′-TCCCTTTATTtcgtaGACGACAAGGGC-3′), antisense (5′-CGGCCCAAACTGATCCCA-3′) primers and QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The sequences of the wild-type and mutant PSMB5 genes were verified by sequencing and both gene versions were cloned into the T. brucei expression vector pHD1034 under control of a ribosomal RNA promoter. Transfected T. brucei Lister 427 cells were selected in medium supplemented with puromycin at 1 μg/ml. Susceptibility of transfected T. brucei cell lines to compounds was assessed after 2 days of compound treatment. Parasite viability was determined with CellTiter-Glo. All assays were carried out in three biological replicates. Reported EC50 values for T. brucei parasites ectopically expressing PSMB5 proteins were calculated from three technical replicates. Figure S4. Y113F mutation in proteasome β5 subunit confers selective resistance to LXE408. Growth inhibition of T. brucei bloodstream form constitutively overexpressing wild-type PSMB5 or PSMB Y113F protein by LXE408 and bortezomib. EC50 values for each strain/compound pair are listed inside plot panels next to their corresponding strain/compound symbol (defined in plot legends); Data shown represents mean ± s.e.m. from one of the representative experiment from three independent biological replicates. RU (relative units) corresponds to parasite growth relative to the DMSO control (%).

Section 3.4: CYP profiling with dog and human species
A panel of dog and human recombinant CYP enzymes (150 pmol CYP/mL) were incubated with 1 µM of compound 1 LXE408 in 100 mM phosphate buffer with 3 mM MgCl2 in the presence of NADPH (1 mM) for 30 min at 37ºC. After the incubation, the reactions were quenched with an equal volume of cold 50:50 MeOH/ACN containing glyburide as an internal standard (IS). The samples were centrifuged and an aliquot of the supernatant was diluted and analyzed by LC/MS/MS. The MS peak area ratio (area of parent peak/area of IS peak) was determined for each incubation and compared to the control CYP incubation. The data were expressed as percent of parent drug remaining with respect to the control incubation. Section 3.5: Metabolic Stability Metabolic Stability. The metabolic stability of drug candidates is determined in human, mouse, and rat liver microsomes using the compound depletion approach, quantified by LC/MS/MS. The assay measures the rate and extent of metabolism of chemical compounds by measuring the disappearance of the parent compound. The assay determines the compound's in vitro half-life (T1/2) and hepatic extraction ratios (ER) and predicts metabolic clearance in human, rat, and mouse species. [5][6][7][8] Section 4: In vivo biology Ethics statement for animal models. All procedures involving mice were performed in accordance with AAALAC standards or under UK Home Office regulations, and were reviewed and approved in accordance with the Novartis Animal Welfare Policy.
Formulation of study drugs for in vivo efficacy experiments. LXE408 administered to mice during efficacy experiments was formulated as a suspension in distilled water containing 0.5% methylcellulose and 0.5% Tween-80 (mouse model of visceral leishmaniasis) or hydroxypropylcellulose and Tween-80 (mouse models of cutaneous leishmaniasis). Miltefosine was formulated as a solution in distilled water containing 0.5% methylcellulose and 0.5% Tween-80. Amphotericin B (AmBisome) was purchased from Astellas Pharma and was formulated according to the manufacturer's instructions.

Mouse model of visceral leishmaniasis.
Female BALB/c mice (6-8 weeks old) were infected by tail vein injection with 4x 10 7 L. donovani MHOM/ET/67/HU3 splenic amastigotes. Seven days after infection, animals were orally dosed for 8 days with vehicle (0.5% MC/0.5% Tween-80 in distilled water), miltefosine (12 mg/kg once-daily), or LXE408 (twice-daily). At the end of treatment, liver samples were collected and L. donovani parasite burdens were quantified by qPCR as follows. L. donovani genomic DNA was extracted from drug-treated mice livers using the DNeasy Blood and Tissue Kit (Qiagen). Two types of DNA were quantified in parallel using the TaqMan assays for L. donovani major surface S8 glycoprotein gp63 (Ldon_GP63) and mouse GAPDH. L. donovani GP63 DNA was quantified using the following primers: TGCGGTTTATCCTCTAGCGATAT (forward primer), AGTCCATGAAGGCGGAGATG (reverse primer). Mouse GAPDH DNA was quantified using the following set of primers: GCCGCCATGTTGCAAAC (forward primer), CGAGAGGAATGAGGTTAGTCACAA (reverse primer). Each qPCR reaction (10 µL) included 5 µl of TaqMan Gene Expression Master Mix (Life Technologies), 0.5 µL of a 20X primer/probe mix (Life Technologies), and 4.5 µL (50 ng) of total DNA from liver samples. DNA amount was quantified using the Applied Biosystems 7900HT instrument. L. donovani parasite burden (RU: relative units) was expressed as the abundance of L. donovani GP63 DNA relative to the abundance of mouse GAPDH DNA.
Lesion cure model of Old World leishmaniasis (L. major). Female BALB/c mice (6 weeks old) were used in this model. Mice were infected intradermally at base of the tail with 100 µl parasite culture containing 1x10 7 L. major MHOM/IR/-/173 stationary phase promastigotes. Starting from the third week post-infection, the lesion induration diameters (length=D1 and width=D2) were measured using a caliper instrument (Fisher Scientific, USA) with 0.1 mm sensitivity. Length and width measurements were taken to account for asymmetrical lesions. Lesion size area was then calculated using the πR1*R2 formula (where R1=D1/2 and R2 = D2/2). Lesions were measured at a 10-day (+/-2 days) interval until the end of the study. Experimental endpoint is lesion cure or % lesion size reduction relative to lesion size in vehicle-treated group. Lesion cure is considered 100% re-epithelialization of the lesion (lesion size = 0 mm 2 ). Animals were dosed orally for 10 days with vehicle (twice-daily) or LXE408 (1 mg/kg, 3 mg/kg, 10 mg/kg and 20 mg/kg twice-daily). In a positive control treatment group, animals were dosed by intraperitoneal injection for 10 days with Ambisome (25 mg/kg once-daily).

Pharmacokinetic studies
In Vivo PK Studies in Mice. 1 was formulated in solution using 25% dextrose 5% in distilled water (D5W) and 75% polyethylene glycol (PEG300) at 2.5 mg/mL. The solution was filtered using a 0.45 m syringe filter before dosing. Two male Balb/c mice were administered 1 intravenously via the lateral tail vein at 5 mg/kg with a dose volume of 2.0 mL/kg. 1 was also formulated in a suspension of 0.5% methylcellulose with 0.5% Tween 80 (0.5% MC/0.5% Tw80) in water at 2.5 mg/mL. A group of mice (n=3) were administered 1 orally via gavage at 20 mg/kg with a dose volume of 8.0 mL/kg. Six blood samples of 50 μL each were collected serially from each animal via the tail vein up to 24 h after dosing. The mouse blood samples were centrifuged to separate plasma and the plasma samples were frozen at -20 °C until analysis.
In Vivo PK Studies in Rats. 1 was formulated in solution using 25% dextrose 5% in distilled water (D5W) and 75% polyethylene glycol (PEG300) at 3.0 mg/mL. Two male Wistar rats were administered 1 intravenously via the lateral tail vein at 3 mg/kg with a dose volume of 1.0 mL/kg. 1 was also formulated in a suspension of 0.5% methylcellulose with 0.5% Tween 80 (0.5% MC/0.5% Tw80) in water at 2.0 mg/mL. The second group of rats (n=3) were administered 1 orally via gavage at 10 mg/kg in suspension with a dose volume of 5.0 mL/kg. Six blood samples of 100 μL each were collected serially from each animal via the saphenous vein up to 24 hours after dosing. The rat blood samples were centrifuged to separate plasma and the plasma samples were frozen at -20°C until analysis.
In Vivo PK Studies in Dogs. 1 was formulated in solution using 25% dextrose 5% in distilled water (D5W) and 75% polyethylene glycol (PEG300) at 3.0 mg/mL. The solution was filtered using a 0.45 m syringe filter before dosing. Three male Beagle dogs were administered 1 intravenously via saphenous vein at 0.3 mg/kg with a dose volume of 0.1 mL/kg. 1 was also formulated in a suspension of 0.5% methylcellulose with 0.5% Tween 80 (0.5%MC/0.5% Tween 80) in water at 1.0 mg/mL. The second group of male dogs (n=2) were administered 1-orally via gavage at 1.0 mg/kg with a dose volume of 1.0 mL/kg. Seven blood samples of 250 µL each were collected serially from each animal via the jugular vein up to 24 h after dosing. The dog blood samples were centrifuged to separate plasma and the plasma samples were frozen at -20 o C until analysis.
In Vivo Intravenous PK Study in Non-human Primates. The IV PK study in non-human primates was carried out at Covance research facility in Greenfield, Indiana. The research facility is accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care). The male Cynomolgus monkeys were approximately 4.5 years of age and weighed 4.2 to 5.0 kg. 1 was administered as a 3 mg/mL formulation in the vehicle control article (25% dextrose 5% in distilled water (D5W) and 75% polyethylene glycol (PEG300) as described in the earlier section. Doses were administered once by intravenous injection via the saphenous vein at a dose of 0.3 mg/kg with a dosing volume of 0.1 ml/kg. Clinical observations and food consumption determinations were performed on all animals. Blood samples for PK analysis were collected from all animals via the femoral vein approximately 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, and 72 hours post-dose.
In Vivo Oral PK Study in Non-human Primates. The PO PK study in non-human primates was conducted at a Novartis facility using Non-naïve male Cynomolgus monkeys weighing 3.8 to 4.0 kg. The monkeys were fasted overnight and fed 2 hours post-dose. 1 was formulated in a suspension of 0.5% methylcellulose with 0.5% Tween 80 (0.5%MC/0.5% Tw80) in water at 2.0 mg/mL. Monkeys were orally gavaged with 5 mL/kg of the suspension for a total dose of 10 mg/kg. The gavage tubing was flushed with 5 mL of water to ensure the gavage tubing was cleared of the formulation. Blood samples for PK analysis were collected from all animals via the saphenous veins approximately 0.25, 0.5, 1, 2, 4, 7, 24, 48, and 72 hours post-dose.

Section 5: Cryo-Electron microscopy
Purification of native L. tarentolae proteasome from cell pellet Frozen L.tarentolae P10 epimastigotes wet cell pellet was homogenized in 400 ml 50 mM Tris/HCl pH 7.5, 5 mM EDTA, 1 mM TCEP, 10 μM E-64 using an Ultra-Turrax ® T25 disperser. Subsequently, the homogenate was pressed through a syringe equipped with a 21 gauge needle prior to being frozen on dry ice and stored over night at -80 °C. After thawing, the cell debris was removed by centrifugation at 35000 x g for 2 h and then fractionated through ammonium sulfate precipitation. Protein fractions precipitated between 40 and 65% saturation was re-suspended in 25 mM Tris/HCL pH 7.5, 2.5 mM EDTA and 1mM TCEP, filtered using a 0.22 μm Stericup ® membrane filter device purified using anion exchange chromatography (Q Sepharose ® ) followed by size exclusion chromatography (Toyopearl ® HW-65S). The active fractions were pooled, adjusted to 1 M ammonium sulfate and loaded onto a 5 ml HiTrap ® Phenyl FF (high sub). The eluted fractions were further purified using a Mono Q column and final active fractions pooled, aliquoted, and stored at -80 ° degrees with 20% glycerol.

Preparation of vitrified specimen
Leishmania tarentolae 20S proteasome (4mg/ml) was incubated with 60µM concentration of LXE408 at 4°C for 20 minutes. 4 μL aliquots of complex were applied to glow-discharged 300-mesh Quantifoil R 1.2/1.3 grids (Quantifoil, Micro Tools GmbH, and Germany). The grids were glow-discharged for 90s at 15mA in a PELCO easiGlow TM glow discharger in the presence of pentylamine (Fluka 77060) right before use. Grids were blotted for 3 s and plunged into liquid ethane using Leica EM GP Plunger operated at 4°C and 85% humidity.
For the ternary complex, proteasome (4mg/ml) was incubated with 60 µM concentration of LXE408 and 180 µM of bortezomib at 4 °C for 20 minutes. The grids were prepared as described above

Data acquisition and image processing of cryo-EM data
High-resolution images were collected with a Cs-corrected FEI Titan Krios TEM operated at 300 kV equipped with a Quantum-LS Gatan Image Filter (GIF) and recorded on a K2-Summit direct electron detector (Gatan GmbH). Images were acquired in an automated fashion (with EPU, Thermo Fisher) in electron-counting mode (nominal post-GIF magnification of ×130,000 and calibrated pixel size of 0.86 S10 Å). Exposures of 7 s were dose-fractionated into 40 frames. The total exposure dose was ~40 e-/Å 2 . Defocus values varied from -0.8 to -2.5 m.
Micrographs were drift-corrected using UNBLUR before estimating CTF parameters using CTFFIND4. 9,10 Particle picking was carried out using Gautomatch. Picked particles were extracted into boxes of 300 × 300 pixels. Micrographs with severe drift or ice contamination were discarded based upon inspection of the power spectra. A total of 1,695 micrographs were acquired from which 207116 particles were extracted for processing using the Relion software package. 11 Particle sorting included two cycles of reference-free 2D classification. The 47515 particles in the best 2D classes were used for 3D refinement using C2-symmetry. The best looking class contained a total of 38786 particles which were subjected to 3D-refinement. We performed particle-based beam-induced movement correction and radiation-damage weighting (known as particle polishing on the first 25 frames (corresponding to a total dose of ~20 e -/Å 2 ). 12 The resulting polished particles gave rise to a reconstruction with an overall resolution of 3.44 Å.
The resolution values reported are based on the gold-standard Fourier shell correlation curve (FSC) at 0.143 criterion. The crystal structures of 20S proteasome was manually fitted into the final cryo-EM map using the program Coot. 13 The resultant atomic model was subjected to multiple cycles of model rebuilding using the program Coot and real space refinement against the map using the program Phenix. 14 This process resulted in an atomic model of the proteosome-1 complex that fit well into the cryoEM density. Structural illustrations were prepared with PyMOL (www.pymol.org). For the ternary complex, we collected 3021 micrographs. Following the same protocol as for the binary complex, a total of 127,053 particles were selected for 3D-refinement. After movie correction using the first 25 frames, the polished particles gave rise to a reconstruction with an overall resolution of 3.18 Å. Model building was performed as described for the binary complex.