Kinase experiment:

Purified His6-Plx1 (250 ng) is added to a 20 μL reaction mixture containing 1× kinase buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM TT), 25 μM cold ATP, and 1 μCi [γ-32P]ATP in the presence of different concentrations of LFM-A13 ranging from 5 μg/mL (13.9 μM) to 100 μg/mL (278 μM). The reaction mixtures are incubated at room temperature for 15-30 min and autophosphorylation is stopped by addition of 2× SDS-PAGE reducing sample buffer. A parallel experiment is performed in the presence of cold ATP. The kinase reactions are then subjected to immunoblotting using the commercially available anti-Plk antibodies. The immunoblots confirmed that the same amount of Plx1 protein is present in each reaction. In addition, we also examined the effects of LFM-A13 on substrate phosphorylation by Plx1. In brief, 250 ng of purified Plx1 is first incubated at room temperature for one hour with different concentrations of LFM-A13. After one hour of incubation, the tubes containing the reaction mixtures are put on ice and the substrate, GST-Cdc25 peptide (254-316) (200 ng), kinase buffer, and [γ-32P]ATP are added and the kinase reaction allowed to proceed for 15 min at room temperature. Immunoblotting with anti-Cdc25 antibodies is used to confirm that equal amounts of the substrate peptide are present in each reaction mixture. Anti-Plk antibodies, the polyclonal antibodies to gluthathione-S-transferase (GST) and ECL kit are used in the assay. The mode of human PLK3 inhibition by LFM-A13 is examined in titration experiments using increasing concentrations of [γ-32P]ATP and purified N-terminal His6-tagged recombinant human PLK3, residues 19-301, expressed by baculovirus in Sf21 insect cells. In brief, in a final reaction volume of 25 μL, PLK3 (h) (5-10 mU) is incubated with 8 mM MOPS, pH 7.0, 0.2 mM EDTA, 2 mg/mL casein, 10 mM Mg acetate, and [γ-32P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 min at room temperature, the reaction is stopped by the addition of 5 μL of a 3% phosphoric acid solution. Ten microliters of the reaction is then spotted onto a P30 filtermat and washed three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting. The Ki of PLK3 by LFM-A13 is calculated from the reciprocal plots of the intensity of phosphorylation of the substrate (1/v) versus the concentration of the inhibitor (i) (viz., LFM-A13). From this Dixon plot, the Ki represents the dissociation constants of the EI complex, which is determined by the point of linear intersection[4].

Animal experiment:

Mice[4]Neu transgenic mice carrying one or more tumors are randomLy placed in the study. For the evaluation of tumor kinetics, tumor-bearing mice are randomLy assigned to either vehicle control or treatment groups. Tumor growth is determined by the measurement of tumors with a caliper in three dimensions three days a week and expressed as tumor volume in cubic millimeters (mm3). Tumor volumes are calculated using the formula for the volume of a prolate spheroid, V = 4/3 × 3. 14 × length/2 × width/2 × depth/2. Due to the large heterogeneity in transgenic tumor volumes on day 0, tumor growth for each mouse is normalized to the starting volume for that particular tumor. Therefore, each mouse also serves as its own control, and the tumor growth curves are generated to show the rate of change in tumor volumes. LFM-A13 (10 or 50 mg/kg) is administered by twice daily intraperitoneal injections on 5 consecutive days per week. Paclitaxel is administered intraperitoneally on days 1, 3, 5, 8, 10, and 12 at a dose level of 6.7 mg/kg. Gemcitabine is administered on days 1, 8, and 15 at a dose level of 33.7 mg/kg.Rats[4]Lewis rats are kept in microisolater cages containing autoclaved food, water, and bedding. Lewis rats are treated with i.v. injections of LFM-A13 at multiple dose levels. LFM-A13 is administered as a 0.5 mL bolus injection containing 10% DMSO as a vehicle. Animals are electively sacrificed on day 7 to determine the toxicity of LFM-A13 by evaluating multiple organs for the presence of toxic lesions. Blood is collected by intracardiac puncture following anesthesia with ketamine:xylazine and immediately heparinized. Blood counts (red blood cells [RBC], white blood cells [WBC], and platelets [Plt]) are determined using a HESKA Vet ABC-Diff Hematology Analyzer. Absolute neutrophil counts (ANC) and absolute lymphocyte counts (ALC) are calculated from WBC values after determining the percentages of neutrophils and lymphocytes by a manual differential count. Values for the laboratory parameters are pooled for vehicle controls and LFM-A13 treatments, and for each parameter differences between means are evaluated for statistical significance using Students t-test (vehicle vs LFM-A13 treatment, unequal variances, two-tailed). The calculations are performed in Excel spreadsheets. To determine significant effects, the p-values are adjusted using the Bonferroni method to control for random variation. For histopathologic studies, formalin fixed tissues are dehydrated and embedded in paraffin by routine methods. Glass slides with affixed 4-5 micron tissue sections are prepared and stained with hematoxylin and eosin.

Description:

IC50: 17.2 μM

Bruton’s tyrosine kinase (BTK), a member of the BTK/Tec family of protein tyrosine kinases, is a cytoplasmic PTK involved in signal transduction pathways regulating growth and differentiation of B-lineage lymphoid cells. BTK participates in signal transduction pathways initiated by the binding of a variety of extracellular ligands to their cell-surface receptors. LFM-A13 is a BTK-specific tyrosine kinase inhibitor.

In vitro: LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also found to be a highly specific inhibitor of BTK. Even at very high concentrations, LFM-A13 did not affect the activity of other protein tyrosine kinases [1].

In vivo: LFM-A13 exhibited a favorable pharmacokinetic behavior which was not adversely affected by the standard chemotherapy drugs and significantly improved the chemotherapy response and survival outcome of BCL-1 leukemia cells challenged mice. While only 14% of mice treated with the standard triple-drug combination treatment became long-term survivors, 41% of mice treated with this combination plus LFM-A13 survived long-term [2].

Clinical trial: Up to now, LFM-A13 is still in the preclinical development stage.

Reference:
[1] Mahajan S, Ghosh S, Sudbeck EA, Zheng Y, Downs S, Hupke M, Uckun FM.  Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide]. J Biol Chem. 1999 Apr 2;274(14):9587-99.
[2] Uckun FM, Zheng Y, Cetkovic-Cvrlje M, Vassilev A, Lisowski E, Waurzyniak B, Chen H, Carpenter R, Chen CL.  In vivo pharmacokinetic features, toxicity profile, and chemosensitizing activity of alpha-cyano-beta-hydroxy-beta- methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a novel antileukemic agent targeting Bruton's tyrosine kinase. Clin Cancer Res. 2002 May;8(5):1224-33.