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

Utpal K Mondal,Claudiu T Supuran,Md Raqibul Alam,Ahmed M Shabana,Rachel Adelberg,Kate Doroba,Marc A Ilies

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.

Highlights

Genetic and epigenetic mutations can transform normal human cells into malignant ones
The series allowed us to test whether the polyethylene glycol (PEG) linker separating the two individual warheads elicit significant effects on the biological properties of this type of Carbonic anhydrases (CAs) inhibitors (CAIs)
Differential scanning calorimetry (DSC) data allowed us to conclude that, starting from a 3.4 KDa linker length, the two warheads are spaced enough to have minimum influence on each other and on the PEG backbone. This linker length was found relevant for the ability of CAIs to kill cancer cells in 2D and 3D that express CA IX

Summary

Introduction

Genetic and epigenetic mutations can transform normal human cells into malignant ones. The malignant cells lose their normal physiologic functions and contact inhibition and gain characteristics that facilitate their survival, growth, and metastasis [1,2]. Cancer cells grow aggressively, forming tumors in different parts of the body. Often, their aggressive growth outpaces the growth of supporting vasculature, so they become hypoxic [1,2,3,4]. Their aggressive growth outpaces the growth of supporting vasculature, so they become hypoxic [1,2,3,4] Many aggressive tumors such as breast, colorectal, ovarian, pancreatic, bladder, and other carcinomas are associated with hypoxia and the upregulation and stabilization of hypoxia-inducible factor

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Results

Conclusion