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

Abstract

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.

Advancement and applications of peptide phage display technology in biomedical science

“…Compared to ADCs, PDCs have additional advantages of having smaller molecular mass, higher tissue penetration, enhanced flexibility and well-defined conjugation chemistry, and faster and easier synthesis process when prepared in a homogenous form [130]. Several peptides included RGD motif peptides [131][132][133][134][135][136], cell penetrating peptides [137][138][139][140][141], and tumor cell specific peptides [142][143][144][145] are used for synthesizing PDCs for cancer therapy. After more than decades of research, lots of PDCs have been discovered, and some have been clinically evaluated, although none has yet received regulatory approval.…”

Protein– and Peptide–Drug Conjugates

“…There may also happen complete change in therapeutic outcome of drugs (i.e., release of drugs with linked amino acid residues may show different activity than free drug) or the conjugate itself may become highly active (Yang et al, 2014). Peptide-drug conjugates, with PEG chains as a linker have flexibility to allow interaction of targeting peptide with its receptors and also improve the hydrophilicity and provide longer circulation time to conjugates (Ravel et al, 2008;Tai, Shukla, Qin, Li, & Cheng, 2011).…”