Brij 35

Perchloric acid was obtained from Loba Chemie (Mumbai, India). The standard of biogenic amines, putrescine dihydrochloride and spermidine trihydrochloride, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Water, methanol, and acetonitrile of HPLC grade were obtained from RCl Labscan (Bangkok, Thailand). The other reagents for synthesis were: sodium acetate anhydrous, purchased from QReC (Selangor, Malaysia); sodium 1-octanesulfonate monohydrate, purchased from Sigma-Aldrich (St. Louis, MO, USA); boric acid and glacial acetic acid, obtained from RCl Labscan (Bangkok, Thailand); potassium hydroxide, purchased from Kemaus (Cherrybrook, New South Wales, Australia); Brij 35, purchased from Merck (Billerica, MA, USA); and 2-mercaptoethanol, purchased from Merck (Darmstadt, Germany). OPA was obtained from Himedia (Mumbai, India).

OP was kindly provided by the Nile Co. for Pharmaceuticals and Chemical Industries, Cairo, Egypt. Trehalose (coarse grade) was provided by Cargill Deutschland GmbH (Krefeld, Germany). Mannitol 60 (coarse grade) and glucose monohydrate (coarse grade) were provided from Roquette, Ludwigshafen, Germany. Lactose inhalation fine grades were of two types; Respitose^® ML006 was kindly provided by DMV-Fronterra Excipients, Nörten-Hardenberg, Germany, while Lactohale^® LH 300 was kindly provided by Friesland Foods Domo, Borculo, The Netherlands. Calu-3 (HTB-55) cells were purchased from the American type culture collection (ATCC, Manassas, VA, USA). MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), Hank’s buffered salt solution (HBSS), HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid) buffer solution, minimum essential medium (MEM) with Earle’s salt, fetal bovine serum, penicillin-streptomycin, nonessential amino acids, and sodium pyruvate were all supplied from Biochrom AG, Berlin, Germany. All reagents (Brij35, ethanol, glycerol, sodium dodecyl sulfate (SDS), dimethylformamide (DMF), trypsin, and Ethylenediaminetetraacetic acid (EDTA) used were of analytical grade and supplied by Merck KGaA (Darmstadt, Germany).

Daclatasvir (DAC) was kindly gifted by Marcyrl for pharmaceutical industries (Cairo, Egypt). β-sitosterol β-D-glucoside (Sito-G), Rhodamine B isothiocyanate (Rh B), stearylamine (SA), phosphate buffer saline (PBS) and dialysis membrane with 12000–14000 molecular weight cut-off were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Egg phosphatidyl choline 90% (EPC) was obtained from Fisher Chemical (UK). Cholesterol 95% was purchased from Acros Organics (New Jersey, USA). Sodium deoxycholate (SDC) was purchased from BASF Co. (Florham Park, New Jersey, USA). Triton X-100 was purchased from Fluka. Potassium dihydrogen orthophosphate, sodium hydroxide and Brij 35 were obtained from Merck (Darmstadt, Germany). Chloroform HPLC grade, Methanol HPLC grade, Acetonitrile HPLC grade and ethanol 75% were purchased from CARLO ERBA Reagents (France), Deionized water from Ultrapure (Type 1) water system (Direct–Q 3 UV) was used for the preparation of all buffer and water based solutions.

Enzyme reactions were performed in Proxiplate Plus 384-well microplates (PerkinElmer) at 8 μL per well. Reaction buffer used contained 50 mM HEPES (pH 7.5), 0.01% Brij35 (EMD Millipore), 10 mM MgCl₂, and 2 mM TCEP (Sigma-Aldrich). Purified HPK1 kinase domain protein (active mutant wild-type, L221 active mutant, or inactive mutant, in-house) enzyme was prepared at a 10 nm concentration in reaction buffer and added to the plate along with the substrate mix containing 1 mM ATP and 100 nM Biotin-SLP76 (generated in-house, full-length SLP76 M1-P533, expressed in E. coli, containing N-terminal His- and Avi-tags) and the reaction allowed to proceed at room temperature for 1 h along with a no kinase control. Detection of phosphorylated Biotin-SLP76 was achieved with an ɑphospho-S376 antibody (in-house), followed by Europium labeled secondary antibody (PerkinElmer), with fluorescence read using a LANCE Ultra TR-FRET protocol on an EnVision instrument (PerkinElmer). The assay was run with 16 replicates per condition. Acceptor-to-donor TR-FRET ratio was calculated for each replicate well and plotted in Prism (GraphPad).

Isopropanol, acetonitrile (HPLC grade), and polyoxyethylene lauryl ether (Brij 35) (for synthesis) were supplied from Merck (Lublin, Poland). Citric acid and Na₂HPO₄ (both pure) were purchased from POCh (Lublin, Poland). Deionized water was produced using the Direct-Q3 UV system (Millipore, Warsaw, Poland).

TA was a gift sample from PHARCO Pharmaceuticals Inc. (Alexandria, Egypt). Oleic acid, castor oil, olive oil, corn oil, and soya bean oil were purchased from Alpha CHEM Co. (Cairo, Egypt). Cremophor EL (polyoxy 35 castor oil) was a gift sample from BASF (Ludwigshafen, Germany). Tween 80 was obtained from El-Naser Pharmaceutical Chemicals Co. (Cairo, Egypt). Brij 35 was purchased from E. Merck (Darmstadt, Germany). PEG 400, PG, and glycerol were purchased from Iso-Chem Co. (Cairo, Egypt). Bovine serum albumin (BSA) was obtained from (Sigma-Aldrich, St. Louis, MO, USA).