Beta-lactams as anti-pancreatic cancer agents: A pilot study
Introduction
Four-membered cyclic amides, commonly known as ß-lactams, are found in nature. Since its discovery, beta-lactam antibiotics have played a central role in fighting against bacterial infections. However, the ‘upgradation’ of ß -lactams from one generation to another is required for drug resistance which is predominantly due to bacterially produced ß-lactamase enzymes that hydrolyze the highly strenuous ß-lactam ring because of tremendous angular strain. Conversely, beta-lactam as an anticancer agent, is a newer field of anticancer drug discovery research, and not many reports are available in the literature. We hypothesize that as cancer cells do not produce ß-lactamase enzymes so the stability of the beta-lactam ring should be higher in tumor environments, and with appropriate chemical modifications, ß-lactams should inhibit the proteins that are responsible for the proliferation, angiogenesis, and metastasis of various cancers. On the other hand, hepatobiliary-pancreatic carcinomas (HPCs) are the most common cause of cancer-related death in the United States. HPCs include hepatocellular carcinoma (HCC), biliary tract cancers (BTCs), and pancreatic cancer (PanCa), which are highly difficult to treat and manage. Around 95% of pancreatic tumors are mainly driven by codon G12, G13, and Q61 mutations in the KRAS gene. Thus, there is a considerable unmet need in this area for the development of selective KRAS inhibitors. As a part of our ongoing research in developing small molecule inhibitors, we have successfully carried out design, multi-step synthesis, and in vitro anti-pancreatic cancer evaluation of a small series of ß-lactam inhibitors.
Methods
Considering high frequency, three main KRAS mutation subtypes (KRASG12D, KRASG12V, KRASG13C) were considered as lead biomolecular targets for HPCs. Prior to the synthesis, extensive docking studies and in silico validation were carried out. In all the steps, column chromatographic and subsequent flash chromatographic separations were conducted after each step to isolate the respective beta-lactams in pure form. In-depth spectral studies characterized all compounds. The next step involved in vitro anticancer evaluation against PANC-1 cell lines. Further, apoptosis assay, cell cycle analysis, gamma-H2AX (phospho-Ser139) straining, and BrdU incorporation studies were conducted in PANC-1 cell lines for one representative β-lactam inhibitor.
Results
From the docking studies, we selected only those beta-lactams that showed excellent docking scores and binding to the target biomolecules. During synthesis, a moderate to good yield of each pure beta-lactam was obtained. Extensive spectroscopic studies confirmed the structures of the small molecule inhibitors. The newly synthesized beta-lactams demonstrated hundreds to thousands-fold higher activity than the positive control Gemcitabine in PANC-1 cells. Comparison of the IC50 values in pancreatic cancer cells (PANC-1) and normal pancreatic epithelial cells (NPC) clearly indicates that almost all the compounds possess 3 to 359 times higher selectivity towards PANC-1 compared to NPC. Further, apoptosis assay, cell cycle analysis, gamma-H2AX (phospho-Ser139) straining, and BrdU incorporation studies were conducted in PANC-1 cell lines. The ‘best’ beta-lactam showed IC50 values in 2D-culture and 3D-tumorsphere assays at 2.55 nM and 2.33 nM, respectively, in PANC-1 cells.
Conclusion
The in silico and in vitro validated chemotherapeutic β-lactams could successfully serve as an entry point for clinical trials after appropriate in vivo evaluation. The work will significantly help in strengthening the battle against pancreatic cancer and will undoubtedly foster a collaborative environment for future research. In brief, this preclinical research on drug-resistant anti-pancreatic cancer ß-lactams may create an avenue of clinical trials and subsequent commercialization.