Replacement of L-amino acid peptides with D-amino acid peptides mitigates anti-PEG antibody generation against polymer-peptide conjugates in mice.

The generation of anti-PEG antibodies in response to PEGylated proteins, peptides, and carriers significantly limits their clinical applicability. IgM antibodies mediate the clearance of these therapeutics upon repeat injection, resulting in toxicity and hindered therapeutic efficacy. We observed this phenomenon in our polymer platform, virus-inspired polymer for endosomal release (VIPER), which employs pH-sensitive triggered display of a lytic peptide, melittin, to facilitate endosomal escape. While the polymer-peptide conjugate was well tolerated after a single injection, we observed unexpected mortality upon repeat injection. Thus, the goal of this work was to enhance the safety and tolerability of VIPER for frequent dosing. Based on previous reports on anti-PEG antibodies and the adjuvant activity of melittin, we characterized the antibody response to polymer, peptide, and polymer-peptide conjugates after repeat-dosing and measured high IgM titers that bound PEG. By substituting the L-amino acid peptide for its D-amino acid enantiomer, we significantly attenuated the anti-PEG antibody generation and toxicity, permitting repeat-injections. We attempted to rescue mice from L-melittin induced toxicity by prophylactic injection of platelet activating factor (PAF) antagonist CV-6209, but observed minimal effect, suggesting that PAF is not the primary mediator of the observed hypersensitivity response. Overall, we demonstrated that the D-amino acid polymer-peptide conjugates, unlike L-amino acid polymer-peptide conjugates, exhibit good tolerability in vivo, even upon repeat administration, and do not elicit the generation of anti-PEG antibodies.

Discovery of specific inhibitors for intestinal E. coli ß-glucuronidase through in silico virtual screening.

Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli ß-glucuronidase (eßG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eßG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eßG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eßG but not human ßG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eßG activity. Compound 4041 (IC50 = 2.8 µM) shows a higher inhibiting ability than compound 7145 (IC50 = 31.6 µM) against eßG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413) at active site of eßG via hydrogen-bonding interactions. These novel NYBS-based eßG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eßG to reduce eßG-based carcinogenesis and intestinal injury.