Enthalpy-driven nuclease-like activity and mechanism of peptide-chlorambucil conjugates.

We report the results of attaching the anticancer drug chlorambucil (CLB) to two high-affinity DNA binding peptides: Met-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyM-10) and Gln-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyQ-10). These CLB-peptide conjugates cleave DNA very effectively and sequence-selectively without the use of chemicals, heat, or UV irradiation. Polyacrylamide gel electrophoresis identifies the sites where CLB-HyM-10 and CLB-HyQ-10 attack a complementary pair of 5'-(32)P-labeled duplexes derived from pBR322 in the absence of piperidine or other chemical additives. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has confirmed the preferential cleavage sites as well as a novel stepwise cleavage mechanism of sequence-selective DNA cleavage. Resembling restriction endonucleases, the CLB-peptide conjugates appear to be capable of producing double strand DNA breaks. Circular dichroism studies show that CLB-HyM-10 and CLB-HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven. The maintenance of a conserved negative binding free energy in DNA-conjugate interactions is a crucial feature of the universal enthalpy-entropy compensation phenomenon. The strongly enthalpy-driven binding of CLB-peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.

Affinity of synthetic peptide fragments of MyoD for Id1 protein and their biological effects in several cancer cells.

MyoD is a DNA-binding protein capable of specific interactions that involve the helix-loop-helix (HLH) domain. The HLH motif of MyoD can form oligomers with the HLH motif of Id1 (the inhibitor of DNA-binding proteins) that folds into a highly stable helical conformation stabilized by the self-association. The Id family consists of four related proteins that contain a highly conserved dimerization motif known as the HLH domain. In signaling pathways, Id proteins act as dominant negative antagonists of the basic helix-loop-helix (bHLH) family of transcription factors which play important roles in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by binding as dominant negative HLH proteins to form high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. The goal of this study is to design and synthesize peptide fragments of MyoD with high affinity for Id1 to interrupt the interactions among Id1, MyoD, and other bHLH DNA-binding proteins and to inhibit the proliferation of cancer cells. Affinity of each peptide for Id1 was determined by surface plasmon resonance (SPR) technology. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in several cancer cells such as breast and colon cancer cells were analyzed. Results demonstrated that the peptide 3C (H-Tyr-Ile-Glu-Gly-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-NH(2)) not only showed high affinity for Id1 but also exhibited antiproliferative effects in HT-29 and MCF-7 cancer cells; the IC(50) value of 3C was determined as 25 microM in both cells. The percentage of sub-G1 in the cell cycle of the cancer cells treated with 5 microM of 3C was increased, indicating the induced apoptosis of cancer cells by 3C. Taken together, the peptide 3C is a promising lead compound for the development of antiproliferative agents.

Effects of peptidic antagonists of Grb2-SH2 on human breast cancer cells.

The growth factor receptor-bound protein Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, the antagonist of Grb2-SH2 has become a potential target for developing anticancer agents. Recently, we discovered a peptide 1 (Fmoc-Glu-Tyr-Aib-Asn-NH(2)) with high affinity for the Grb2-SH2 domain by using surface plasmon resonance (SPR)-biosensor technology. Herein, we report the further design of the lead peptide 1 by addition of an Arg-Gly-Asp sequence to 1 to enhance binding to Grb2-SH2 and inducing apoptosis in cancer cells. Both the linear and cyclic analogs of the newly designed compound were prepared along with an analog in which the N(alpha)-Fmoc group was removed. These peptide analogs were assayed for their affinity for the Grb2-SH2, their antiproliferative effect on human breast cancer cells, their specificity for cancer cells, and their effects on cytotoxicity and the cell cycle. MCF-7 and MDA-MB-453 breast cancer cells were treated with various concentrations of each peptide. The cell viability and cytotoxicity of peptide-treated cells were determined by using the cell proliferation kit (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-tetrazolium bromide, MTT) and cytotoxicity kit (lactate dehydrogenase, LDH), respectively. Effects of peptides on the cell cycle progression of cancer cells and apoptosis were analyzed by using flow cytometry. Results demonstrated that the peptide analog 2 (H-Arg-Gly-Asp-Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH(2)) had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with an IC(50) of 45.7 microM and 47.4 microM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells when cells were treated with higher concentration of the Arg-Gly-Asp-containing peptide 2. These results provide important information for the development of anti-cancer agents.

Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay.

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.