Design, synthesis and antitumor evaluation of novel pyrazolo[3,4-d]pyrimidines incorporating different amino acid conjugates as potential DHFR inhibitors

Abstract The present study aimed to investigate the antitumor effect of simultaneous inhibition of dihydrofolate reductase (DHFR) enzyme. We designed some novel pyrazolo[3,4-d]pyrimidines bearing different amino acid conjugates as efficient antifolate agents attributable to their structural similarity with methotrexate (MTX) and MTX-related antifolates. All compounds were tested to screen their enzymatic inhibition against DHFR compared with the reference drug MTX and for their in vitro antitumor cytotoxicity against six MTX-resistant cancer cell lines. The flow cytometry indicated that the most potent compound 7f arrested MCF-7 cells in the S-phase and induced apoptosis. Western blot for visualisation proved the ability of compound 7f to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein. Molecular modelling studies concluded that compound 7f displayed better binding energy than that of the normal ligand MTX. HIGHLIGHTS New pyrazolo[3,4-d]pyrimidine derivatives 7a–m which are structurally similar to the classical methotrexate (MTX) and MTX-related antifolates were synthesised as antitumor agents. Novel N-acyl amino acid compound 7f exhibited marked DHFR inhibition activity that are parralel to both the molecular docking results and cytotoxic activity. Compound 7f could induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein. All prepared compounds obey Lipinski rule of five except compound 7f.


Principle:
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate proteins based on their size. When coupled with western blotting (immunoblotting), both are typically used to determine the presence and/or relative abundance of a target protein in a sample containing a complex mixture of proteins. In this technique, total protein in each sample is loaded and electrophoretically separated by applying an electric current which allows the proteins to migrate through the gel matrix. In order for the proteins to migrate through the gel, they are first denatured and negatively charged by exposure to a detergent such as SDS. A molecular weight marker that produces bands of known size is used to help identifying proteins of interest. After the protein components have been sufficiently separated, they can be transferred to a polyvinylidene fluoride (PVDF) membrane by applying an electric current to the gel so that the proteins migrate out of the gel onto the membrane. For detection of a specific protein on the membrane, a primary antibody against that protein is added to form a protein-antibody complex followed by the addition of a secondary antibody that binds to the complex through its antibody side. The secondary antibody is typically linked to an enzyme that produces luminescence upon the reaction with its substrate. The amount of the luminescence, directly proportional to the amount of the protein that reacted with the antibody, is captured by Biorad Imager Reagents preparation: • Tris-Glycine transfer buffer: 25 mM Tris; 192 mM glycine; 0.05% SDS; 15% methanol. Methanol was immediately added before the transfer.
• Secondary antibodies for the proteins to be detected • ECL TM western blotting detection chemiluminescent substrate (PerkinElmer, USA).

Procedure:
• The experiment was terminated by lysing the cells in cold lysis buffer.
The cells were then immediately frozen at −20 °C for 1 h for further lysis, and collected by cell scraper and sonicated 2×10s, followed by centrifugation at 4000 rpm for 10 min under cooling.
• Total protein concentrations were determined colorimetrically in the supernatant using Bradford method before proceeding to the western blotting.
• Western blotting • Equal amounts (20 µg) of protein samples were mixed and boiled with SDS Loading buffer for 10 min, allowed to cool on ice and then loaded into SDS-polyacrylamide gel and separated by Cleaver electrophoresis unit (Cleaver, UK), transferred onto polyvinylidene fluoride (PVDF) membranes (BioRad) for 30 min using a Semi-dry Electroblotter (Biorad, USA) at 2.5 A and 25 V for 30 min.
• The membrane was blocked with 5% nonfat dry milk in TBS-T for two hours at RT, in order to reduce non-specific protein interactions between the membrane and the antibody.
• The membrane was incubated overnight at 4°C with primary antibodies (Cell Signaling Technology) and β-actin (Sigma). The blots were then washed for three times (10 min each) with TBS-T.
• The membrane was then incubated with the corresponding horse radish peroxidase (HRP)-linked secondary antibodies (Dako) for another hour at room temperature, followed by washing for three times (10 min each) with TBS-T • The chemiluminescent Western ECL substrate (Perkin Elmer, Waltham, MA) was applied to the blot according to the manufacturer's recommendation. Briefly, the membranes were incubated for 1 min with a mixture of equal volumes from ECL solution A and ECL solution B.
• The chemiluminescent signals were captured using a CCD camera-based imager (Chemi Doc imager, Biorad, USA), and the bands intensities were then measured by ImageLab (Biorad) • Protein-sized markers were used in all gels to localize the gel transfer regions for specific proteins and determine the transfer efficiency.