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

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.

Folate-Targeted Monodisperse PEG-Based Conjugates Made by Chemo-Enzymatic Methods for Cancer Diagnosis and Treatment

“…Long-term cytotoxicity study showed ( Figure 2 c) that after treatment with FA 2 -dPEG-DOX 2 at 100 μM concentration (regarding DOX), viability values of the treated MDA-MB-231 cells were similar to the untreated control until 48 h. However, treatment with FA 2 -dPEG-DOX 2 reduced cell viability significantly after 48 h (compared to the control) and sustained at an approximately constant level for 168 h. The results show slow DOX release in the cell culture medium investigated. Similar studies often do not investigate simulated drug release profiles because they may not be predictive for in vivo conditions [ 15 , 16 , 17 , 18 , 19 , 20 ].…”

“…PDCs. Research carried out with various polymers, including PEG, showed promise due to increased water solubility and circulation time in the body, and multivalent attachment to receptors FR [ 15 , 16 , 17 , 18 , 19 , 20 ]. Despite the progress achieved in the field, several obstacles currently impede the clinical translation of PDC-based therapeutics [ 21 ].…”

Exploring the potency of diazo‐coumarin containing hybrid molecules: Selective inhibition of tumor‐associated carbonic anhydrase isoforms IX and XII

“…Each inhibitor concentration was tested in triplicate, and the reported values represent the mean of these results. The inhibition constants were calculated using nonlinear least‐squares methods, employing the Cheng‐Prusoff equation, as previously reported, and are the average of at least three separate determinations [43–53] …”

“…The inhibition constants were calculated using nonlinear least-squares methods, employing the Cheng-Prusoff equation, as previously reported, and are the average of at least three separate determinations. [43][44][45][46][47][48][49][50][51][52][53] . All CA isozymes used in this study were recombinant proteins previously obtained by our research group.…”

“…All CA isozymes used in this study were recombinant proteins previously obtained by our research group. [43][44][45][46][47][48][49][50][51][52][53]…”