ABSTRACT
AIMS: Arginine depleting enzymes are found effective to treat arginine-auxotrophic cancers and therapy-resistant malignancies, alone or in combination with cytotoxic agents or immune checkpoint inhibitors. We aim to select and validate a long-lasting, safe and effective PEGylated and cobalt-chelated arginase conjugated at the selective cysteine residue as a therapeutic agent against cancers. MAIN METHODS: Exploring pharmacokinetic and pharmacodynamic properties of the three arginase conjugates with different PEG modality (20 kDa linear as A20L, 20 kDa branched as A20Y, and 40 kDa branched as A40Y) by cell-based and animal studies. KEY FINDINGS: Arginase conjugates showed comparable systemic half-lives, about 20 h in rats and mice. The extended half-life of PEGylated arginase was concurrent with the integrity of conjugates of which PEG and protein moieties remain attached in bloodstream for 72 h after drug administration. Arginase modified with a linear 20 kDa PEG (A20L) was chosen as the lead candidate (PT01). In vitro assays confirmed the very potent cytotoxicity of PT01 against cancer cell lines of breast, prostate, and pancreas origin. In MIA PaCa-2 pancreatic and PC-3 prostate tumor xenograft models, weekly infusion of the PT01 at 5 and 10 mg/kg induced significant tumor growth inhibition of 44-67%. All mice experienced dose-dependent but rapidly reversible weight loss following each weekly dose, suggesting tolerable toxicity. SIGNIFICANCE: These non-clinical data support PT01 as the lead candidate for clinical development that may benefit cancer patients by providing an alternative cytotoxic mechanism.
Subject(s)
Antineoplastic Agents/chemical synthesis , Arginase/chemical synthesis , Arginine/deficiency , Chemical Engineering/methods , Drug Design , Polyethylene Glycols/chemical synthesis , Animals , Antineoplastic Agents/administration & dosage , Arginase/administration & dosage , Arginine/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Isoenzymes/administration & dosage , Isoenzymes/chemical synthesis , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Polyethylene Glycols/administration & dosage , Protein Structure, Secondary , Rats , Rats, Sprague-Dawley , Treatment Outcome , Xenograft Model Antitumor Assays/methodsABSTRACT
Carbapenems are one of the last lines of defense for Gram-negative pathogens, such as members of the Enterobacteriaceae. Despite the fact that most carbapenems are resistant to extended-spectrum ß-lactamase (ESBL), emerging metallo-ß-lactamases (MBLs), including New Delhi metallo-ß-lactamase 1 (NDM-1), that can hydrolyze carbapenems have become prevalent and are frequently associated with the so-called "superbugs," for which treatments are extremely limited. Crystallographic study sheds light on the modes of antibiotic binding to NDM-1, yet the mechanisms governing substrate recognition and specificity are largely unclear. This study provides a connection between crystallographic study and the functional significance of NDM-1, with an emphasis on the substrate specificity and catalysis of various ß-lactams. L1 loop residues L59, V67, and W87 were important for the activity of NDM-1, most likely through maintaining the partial folding of the L1 loop or active site conformation through hydrophobic interaction with the R groups of ß-lactams or the ß-lactam ring. Substitution of alanine for L59 showed greater reduction of MICs to ampicillin and selected cephalosporins, whereas substitutions of alanine for V67 had more impact on the MICs of carbapenems. K224 and N233 on the L3 loop played important roles in the recognition of substrate and contributed to substrate hydrolysis. These data together with the structure comparison of the B1 and B2 subclasses of MBLs revealed that the broad substrate specificity of NDM-1 could be due to the ability of its wide active site cavity to accommodate a wide range of ß-lactams. This study provides insights into the development of efficient inhibitors for NDM-1 and offers an efficient tactic with which to study the substrate specificities of other ß-lactamases.