Novel fluorinated carbonic anhydrase IX inhibitors reduce hypoxia-induced acidification and clonogenic survival of cancer cells

Abstract

Human carbonic anhydrase (CA) IX has emerged as a promising anticancer target and a diagnostic biomarker for solid hypoxic tumors. Novel fluorinated CA IX inhibitors exhibited up to 50 pM affinity towards the recombinant human CA IX, selectivity over other CAs, and direct binding to Zn(II) in the active site of CA IX inducing novel conformational changes as determined by X-ray crystallography. Mass spectrometric gas-analysis confirmed the CA IX-based mechanism of the inhibitors in a CRISPR/Cas9-mediated CA IX knockout in HeLa cells. Hypoxia-induced extracellular acidification was significantly reduced in HeLa, H460, MDA-MB-231, and A549 cells exposed to the compounds, with the IC50 values up to 1.29 nM. A decreased clonogenic survival was observed when hypoxic H460 3D spheroids were incubated with our lead compound. These novel compounds are therefore promising agents for CA IX-specific therapy.

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

“…Interestingly, in the same report a Cys174Ser CA IX variant crystallized with the same packing and dimeric interface but without the disulfide bond (Alterio et al, 2009). Recently, another study reported a CA IX structure, this time determined from protein expressed in yeast, that crystallized in space group H3 (PDB entry 6fe0; unit-cell parameters a = b = 152.9, c = 171.5 Å ; Kazokaitė et al, 2018). In addition to these studies, a crystal structure of CA IX SV was also determined in space group P2 1 2 1 2 1 (PDB entry 5dvx; unit-cell parameters a = 57.9, b = 102.7, c = 108.9 Å ).…”

Inhibitory activity against carbonic anhydrase IX and XII as a candidate selection criterion in the development of new anticancer agents

“…To our delight, however, there is a large number of mechanistically relevant experiments reported in the literature which address the ability of hCA IX/XII inhibitors to block the growth of hypoxic cancer cells overexpressing the target isozymes [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67] . Moreover, a certain cohort of studies revealed compounds possessing both favourable hCA inhibitory profile and significant anticancer activity, as exemplified by structures 5-13 ( Figure 2) [49][50][51][52][53][54][55][56][58][59][60][61][64][65][66][67] . In fact, quite a number of single-digit nanomolar hCA IX/XII inhibitors significantly suppressed cancer cell growth.…”

From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

“…However, the native protein is 459 amino acids (UniProtKB-Q16790) with multiple domains, including a membrane spanning domain [20]. There are two reports on the structure of the catalytic domain of native CA IX, but the unit cell and parameters are unsuitable for neutron studies [21,22]. The most recent is from CA IX prepared by expression in yeast, and the space group was H3 with unit cell parameters: a = b = 152.7 Å, c = 170.7 Å; α = β = 90 • , γ = 120 • (PDB ID 6fe2) [22].…”

“…There are two reports on the structure of the catalytic domain of native CA IX, but the unit cell and parameters are unsuitable for neutron studies [21,22]. The most recent is from CA IX prepared by expression in yeast, and the space group was H3 with unit cell parameters: a = b = 152.7 Å, c = 170.7 Å; α = β = 90 • , γ = 120 • (PDB ID 6fe2) [22]. The first structure that was reported was also for the catalytic domain of CA IX, but produced in insect cells and crystallized in P6 1 with unit cell parameters: a = b = 144.2 Å, c = 208.9 Å; α = β = 90 • , γ = 120 • (PDB ID 3iai) [21].…”

“…While surface mutations are introduced away from the highly conserved active site of the enzyme, the catalytic efficiency of the SV enzyme is decreased in comparison with native protein, as described by Mahon et al [23]. The CA IX SV was previously reported to crystallize in P2 1 2 1 2 1 with unit cell parameters~100 Å on edge using 8% PEG 8000, 0.1 M Tris pH 8.5, and as such is more suitable for neutron studies than working with the native protein [21][22][23]. Despite extensive efforts to reproduce these conditions, we were unable to obtain any crystals.…”