All N-terminally capped FLIPs were synthesized and purified using standard Fmoc chemistry by GenScript (Piscataway, NJ). HPLC and MS were used to confirm the purity and structure of each peptide. Peptides were assembled by using standard solid- phase synthesis methods. A sample procedure is given as follows: 5 equivalents of the C- terminal amino acid were coupled to Rink resin at the first place using 1 DIEA (0.082 mL) and HBTU (189.6 mg) in 2 mL of DMF for 1 h. The Fmoc of the C-terminal amino acid was removed using 20% piperidine in 3 mL of DMF for 10 min before addition of 5 equiv of the next amino acid using DIEA (0.082 mL) and HBTU (189.6 mg) in 5 mL of DMF. Wash cycles (5 x 10 mL of DMF + 5 x 10 mL of DCM) were applied to each step in between coupling and deprotection of Fmoc. Upon completion of assembly, side chain protecting groups were removed, and peptides were cleaved from Rink resin using 90:5:5 mixtures of TFA/H2O/TIPS. Crude peptides were purified using reverse-phase flash chromatography and semi preparative reverse-phase HPLC methods. Pure compounds were lyophilized and characterized using mass spectrometry and analytical HPLC (see Supplementary Table 3).
99
4.4.2 Energy minimization and Interaction energy calculation
Energy minimization was performed in Discovery studio 3.0 in the Cylin A (PBD ID: 1OKV) at the cyclin binding groove. Prior to minimization, the heavy atoms of the protein were set as constraints, CHARMm force field was applied to the receptor ligand- complex. The minimization protocol was used for calculating the energy minimization. The parameters used were Smart minimizer Algorithm with 2000 steps and 0.1 as RMS gradient, GBSW (Generalized Born with a Simple Switching) implicit solvent model using Dielectric constant as 1 and implicit solvent dielectric constant as 80. The other parameters non-bonded radius and electrostatics were set as default.
The interaction energy between the protein and each ligand was calculated with interaction energy protocol in Discovery studio 3.0. Default parameters were used for (i) In-Situ Ligand Minimization, (ii) Ligand Conformational Entropy (iii) Non-bonded radius (iv) Electrostatics (v) Advanced and (vi) Parallel processing. The implicit solvent model was set to GBSW (Generalized Born with a Simple Switching) along with dielectric constant as 1 and implicit solvent dielectric constant as 80.
4.4.3 Cell culture74
U2OS osteosarcoma cells and DU145 human prostate cancer cells were obtained from ATCC (Manassas, VA). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM , Fisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS, USA Scientific) and 100µg/ml of Streptomycin (strep)/ 100 U/ml of Penicillin (pen). Cells were incubated at 37ºC with 5% CO2.
100 4.4.4 Viability assay
Cells were seeded in 96-well plates at (2 × 103 cells/ml) and allowed to adhere overnight in DMEM growth media containing 10% NU serum and 1% Pen/Strep. The compounds 5962, 5963, and 5964 were dissolved in DMSO and added in a dose dependent manner (1 µM to 100 µM). The diluted compounds were added to the cells in triplicate and incubated for 72 hours at 37oC with 5% CO2. The cell viability was
determined using MTT (3-(4,5-Dimethylthiazol-2YI)-2,5-Diphenyl tetrazolium Bromide) assay4. Absorbance readings were obtained using a DTX880 multimode detector fitted with 595nm filter. Cell viability results were represented as the ratio of the absorbance reading in treated vs. untreated cells. IC50 values were obtained using a
nonlinear regression line with 5.0 Graghpad Prism. Experiments performed in triplicates.
4.4.5 Cell Cycle Analysis
Cells were plated at 150,000/ml overnight in six well plates. CDK2 inhibitors were added in a dose dependent fashion for 48 hrs. The cells were then fixed with 70% Ethanol overnight at -20 degrees. The cells were centrifuged and ethanol was removed. The cells were washed twice with 1X PBS following staining with DAPI(4’,6’ diamino- 2-phenylindole) and analyzed using BD LSR II (Becton Dickenson) with a blue filter. Experiments were performed in triplicates.
4.4.6 Fluorescence Polarization Binding Assay
CDK4D1 FP assay: This assay was performed using black 384-well plates using a previously described procedure57 with the following modifications. To each well were added: 5 µl CDK4D1 (37 µg/ml purified recombinant human kinase complex from Invitrogen), 5 µl compound solution, 5 µl 12 nM fluoresceinyl-Ahx-Pro-Val-Lys-Arg-
101
Arg-Leu-(3ClPhe)-Gly tracer peptide. Compounds and kinase complexes were diluted using assay buffer (25 nM HEPES pH 7, 10 nM NaCl, 0.01% Nonidet P-40, BSA 1mg/ml, 20mM dithiothretiol (DTT). Plate was centrifuged for 1 min at 500 rpm and then incubated with shaking for 45 mins at room temperature. Fluorescence polarization was read on DTX880 multimode detector (Beckman Coulter, Brea, CA) fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein. Relative mp was calculated for each concentration tested using the equation showing below. IC50 values were determined by logarithmic regression by correlating relative mps
and testing concentrations.
Relative mp = mP(compound)-mP(DMSO, protein, tracer)
mP( DMSO, protein)-mP(DMSO, protein, tracer)
CDK2A2 FP assay :This assay was performed using black 384-well plates. To each well were added: 5 µl CDK2A2 (18 µg/ml purified recombinant human kinase complex), 5 µl compound solution, 5 µl 12 nM fluoresceinyl-Ahx-Pro-Val-Lys-Arg-Arg- Leu-(3ClPhe)-Gly tracer peptide. Compounds and kinase complexes were diluted using assay buffer (25 nM HEPES pH 7, 10 nM NaCl, 0.01% Nonidet P-40, BSA 1mg/ml, 20mM dithiothretiol (DTT). Plate was centrifuged for 1 min at 500 rpm and then incubated with shaking for 45 mins at room temperature. Fluorescence polarization was read on DTX880 multimode detector (Beckman Coulter, Brea, CA) fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein. Relative mp was calculated for each concentration tested using the equation showing below. IC50 values were determined by logarithmic regression by correlating relative mps
102 4.5 Acknowledgement
I thank Sandra Craig for all the cell based experiments and Dr. Wyatt for his help in the development of FP assay.
This chapter is written based on the following article
Padmavathy Nandha Premnath, Sandra Craig, Shu Liu, Erin Anderson, Asterios I. Grigoroudis, George Kontopidis, Tracy Perkins, Michael D. Wyatt, Douglas L. Pittman and Campbell McInnes, Iterative conversion of cyclin binding groove peptides into drug- like CDK inhibitors with anti-tumor activity, article submitted to Journal of medicinal chemistry.
103 CHAPTER 5
OPTIMIZATION OF BENZOIC ACID DERIVED INHIBITORS OF THE