Biophysical, Biochemical, and Cell Based Approaches Used to Decipher the Role of Carbonic Anhydrases in Cancer and to Evaluate the Potency of Targeted Inhibitors

Abstract

Carbonic anhydrases (CAs) are thought to be important for regulating pH in the tumor microenvironment. A few of the CA isoforms are upregulated in cancer cells, with only limited expression in normal cells. For these reasons, there is interest in developing inhibitors that target these tumor-associated CA isoforms, with increased efficacy but limited nonspecific cytotoxicity. Here we present some of the biophysical, biochemical, and cell based techniques and approaches that can be used to evaluate the potency of CA targeted inhibitors and decipher the role of CAs in tumorigenesis, cancer progression, and metastatic processes. These techniques include esterase activity assays, stop flow kinetics, and mass inlet mass spectroscopy (MIMS), all of which measure enzymatic activity of purified protein, in the presence or absence of inhibitors. Also discussed is the application of X-ray crystallography and Cryo-EM as well as other structure-based techniques and thermal shift assays to the studies of CA structure and function. Further, large-scale genomic and proteomic analytical methods, as well as cell based techniques like those that measure cell growth, apoptosis, clonogenicity, and cell migration and invasion, are discussed. We conclude by reviewing approaches that test the metastatic potential of CAs and how the aforementioned techniques have contributed to the field of CA cancer research.

Figure 1

Biophysical and biochemical methods used to investigate the potency of CA targeted inhibitors, CA structure determination and drug development.

Figure 2

Large-scale analysis and cell based assays currently used to determine the role of CAs in cancer and the anticancer properties of CA targeted inhibitors.

Figure 3

(a) CA IX and CA II and (b) CA XII activities were assessed using MIMS, under normoxic or hypoxic conditions for 16 h in the presence or absence of the impermeant sulfonamide inhibitor N-3500. DSF was used to determine the binding affinity of N-3500 to purified (c) CA II and (d) CA IX.

Figure 4

X-ray crystallography structures showing sulfonamide based inhibitors bound in the active site of CA II (top) and CA IX (bottom). Catalytic zinc (magenta sphere), hydrophilic (blue), and hydrophobic (orange) residues are as shown. Red double-headed arrows indicate isoform specificity relative to residue 131 (labeled in white). These arrows also show flexibility in tail conformations seen in CA II and CA IX. The K_i values of each compound bound to purified CA II and CA IX are also given. Figure was designed using PyMol.

Figure 5

(a) Heatmap comparing microarray data of differentially expressed genes between UFH-001 (UFH, representative of triple negative breast cancer) and MCF10A (MCF, normal breast cells) cells (top 200 differentially expressed genes are shown). (b) Cell growth curves of MCF10A, UFH-001, and T47D (representative of luminal breast cancer) cells were determined using MTT assays. (c) Migration capacity of cell lines and (d) Invasion capacity of cell lines as determined using transwell Boyden chamber assays.