ABSTRACT
Pyrrole-imidazole polyamide (PIP) can specifically bind in the B-DNA minor groove that has been used in several biological applications, such as anti-cancer activity, gene expression and translation control, and visualization of complex genomic areas. c-kit is a family member of the Tyrosine Kinase (RTK) type III receptor and plays a vital role in tumor growth, proliferation, differentiation, and cell apoptosis; however, its mutations and overexpression induce tumor dysfunction. Here, we designed and synthesized five matched PIPs that can recognize and bind to the DNA sequence in the oncogene c-kit promoter region, and evaluated their anti-cancer activity. The RTCA assay findings revealed that the PIPs would prevent the proliferation of cancer cells A549 and SGC-7901. EMSA assay showed that the PIPs were actively interacting with the c-kit gene target DNA. RT-PCR and Western blot assays have an effect on expression levels of the c-kit gene in the presence of PIPs. Flow cytometry and wound-healing assays showed that PIPs 4, 5 would cause apoptosis of cancer cells and inhibit the migration of cells, respectively. Overall findings indicate that PIP 5 has a relatively significant intracellular and extracellular impact on the target, contributing to migration and proliferation reduction, and cancer cell apoptosis. In addition, PIP has a certain ability to evolve into c-kit gene-targeting drugs.
Subject(s)
Antineoplastic Agents/pharmacology , Down-Regulation/drug effects , Imidazoles/pharmacology , Nylons/pharmacology , Proto-Oncogene Proteins c-kit/genetics , Pyrroles/pharmacology , A549 Cells , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Design , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Neoplasms/drug therapy , Neoplasms/genetics , Nylons/chemical synthesis , Nylons/chemistry , Promoter Regions, Genetic/drug effects , Pyrroles/chemical synthesis , Pyrroles/chemistryABSTRACT
In order to develop an optimal subunit as a T-recognition element in hairpin polyamides, 15 novel chirality-modified polyamides containing (R)-α,ß-diaminopropionic acid ((R) ß α-NH 2), (S)-α,ß-diaminopropionic acid ((S) ß α-NH 2), (1R,3S)-3-aminocyclopentanecarboxylic acid ((RS) Cp), (1S,3R)-3-amino-cyclopentanecarboxylic acid ((RS) Cp), (1R,3R)-3-aminocyclopentanecarboxylic acid ((RR) Cp) and (1S,3S)-3-amino-cyclopentanecarboxylic acid ((SS) Cp) residues were synthesized. Their binding characteristics to DNA sequences 5'-TGCNCAT-3'/3'-ACGN'GTA-5' (Nâ N'=Aâ T, Tâ A, Gâ C and Câ G) were systemically studied by surface plasmon resonance (SPR) and molecular simulation (MSim) techniques. SPR showed that polyamide 4, AcIm-(S) ß α-NH 2-ImPy-γ-ImPy-ß-Py-ßDp (ß/(S) ß α-NH 2 pair), bound to a DNA sequence containing a core binding site of 5'-TGCACAT-3' with a dissociation equilibrium constant (K(D) ) of 4.5×10(-8) m. This was a tenfold improvement in specificity over 5'-TGCTCAT-3' (K(D) =4.5×10(-7) M). MSim studies supported the SPR results. More importantly, for the first time, we found that chiral 3-aminocyclopentanecarboxylic acids in polyamides can be employed as base readers with only a small decrease in binding affinity to DNA. In particular, SPR showed that polyamide 9 ((RR) Cp/ß pair) had a 15-fold binding preference for 5'-TGCTCAT-3' over 5'-TGCACAT-3'. A large difference in standard free energy change for Aâ T over Tâ A was determined (ΔΔG(o) =5.9 kJ mol(-1) ), as was a twofold decrease in interaction energy by MSim. Moreover, a 1:1 stoichiometry (9 to 5'-TGCTCAT-3'/3'-ACGAGTA-5') was shown by MSim to be optimal for the chiral five-membered cycle to fit the minor groove. Collectively, the study suggests that the (S)-α-amino-ß-aminopropionic acid and (1R,3R)-3-aminocyclopentanecarboxylic acid can serve as a T-recognition element, and the stereochemistry and the nature of these subunits significantly influence binding properties in these recognition events. Subunit (1R,3R)-3-aminocyclopentanecarboxylic acid broadens our scope to design novel polyamides.