PEG Linker Length Strongly Affects Tumor Cell Killing by PEGylated Carbonic Anhydrase Inhibitors in Hypoxic Carcinomas Expressing Carbonic Anhydrase IX

Int J Mol Sci. 2021 Jan 23;22(3):1120. doi: 10.3390/ijms22031120.

Abstract

Hypoxic tumors overexpress membrane-bound isozymes of carbonic anhydrase (CA) CA IX and CA XII, which play key roles in tumor pH homeostasis under hypoxia. Selective inhibition of these CA isozymes has the potential to generate pH imbalances that can lead to tumor cell death. Since these isozymes are dimeric, we designed a series of bifunctional PEGylated CA inhibitors (CAIs) through the attachment of our preoptimized CAI warhead 1,3,4-thiadiazole-2-sulfonamide to polyethylene glycol (PEG) backbones with lengths ranging from 1 KDa to 20 KDa via a succinyl linker. A detailed structure-thermal properties and structure-biological activity relationship study was conducted via differential scanning calorimetry (DSC) and via viability testing in 2D and 3D (tumor spheroids) cancer cell models, either CA IX positive (HT-29 colon cancer, MDA-MB 231 breast cancer, and SKOV-3 ovarian cancer) or CA IX negative (NCI-H23 lung cancer). We identified PEGylated CAIs DTP1K 28, DTP2K 23, and DTP3.4K 29, bearing short and medium PEG backbones, as the most efficient conjugates under both normoxic and hypoxic conditions, and in the tumor spheroid models. PEGylated CAIs did not affect the cell viability of CA IX-negative NCI-H23 tumor spheroids, thus confirming a CA IX-mediated cell killing for these potential anticancer agents.

Keywords: PEG; carbonic anhydrase; hypoxia; inhibitor; polymer conjugate; sulfonamide; tumor.

MeSH terms

  • Antigens, Neoplasm / metabolism*
  • Antineoplastic Agents / chemical synthesis
  • Antineoplastic Agents / chemistry*
  • Antineoplastic Agents / pharmacology*
  • Calorimetry, Differential Scanning
  • Carbonic Anhydrase IX / antagonists & inhibitors
  • Carbonic Anhydrase IX / metabolism*
  • Carbonic Anhydrase Inhibitors / chemical synthesis
  • Carbonic Anhydrase Inhibitors / chemistry*
  • Carbonic Anhydrase Inhibitors / pharmacology*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Drug Screening Assays, Antitumor
  • Female
  • Humans
  • Neoplasms / drug therapy
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Polyethylene Glycols / chemistry
  • Structure-Activity Relationship
  • Tumor Hypoxia / drug effects

Substances

  • Antigens, Neoplasm
  • Antineoplastic Agents
  • Carbonic Anhydrase Inhibitors
  • Polyethylene Glycols
  • CA9 protein, human
  • Carbonic Anhydrase IX