Crystal structure of human carbonic anhydrase II at 1.95 Å resolution in complex with 667-coumate, a novel anti-cancer agent

Abstract

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 A (1 A=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 A through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.

In Vitro Metabolism of Irosustat, a Novel Steroid Sulfatase Inhibitor: Interspecies Comparison, Metabolite Identification, and Metabolic Enzyme Identification

“…1). Desulfamoylation in aqueous solution probably occurs via the E1cB elimination reaction, assisted by the extended conjugation present in the coumarin motif (Lloyd et al, 2005). However, the results of the present work showed how incubations of irosustat with microsomes in the absence of cofactor produced increased amounts of 667-coumarin compared with incubations of irosustat in buffer alone ( Table 2), demonstrating that 667-coumarin can be also formed by non-NADPH-dependent enzymatic hydrolysis.…”

“…1A). The presence of the sulfamoyl ester group is indispensable for its STS inhibitory activity and, in addition, confers to the irosustat molecule the ability to bind and to reversibly inhibit carbonic anhydrase II (Ho et al, 2003;Lloyd et al, 2005), an enzyme that is highly expressed in mammalian erythrocytes. Irosustat undergoes spontaneous desulfamoylation in aqueous solutions at nearly physiological pH (Ireson et al, 2003) leading to the formation of its major degradation derivative, 667-coumarin (structure shown in Fig.…”

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

“…S2). These alternative loop conformations have been observed with HCA II:inhibitor structures solved in the orthorhombic space group as well as for other HCA II structures with mutations in or near the opening of the active site (Ippolito et al, 1995;Lloyd et al, 2005). These displaced surface loops are most likely a direct consequence of the crystal packing forces.…”

“…It is not obvious why Lys225 occupies this unique conformation in TS5 compared with TS1 -4. In addition, previous studies of HCA II in an orthorhombic space group report a Zn coordinated to His4 located near the opening of the active site cavity (Lloyd et al, 2005; PDB: 2X7S). Interestingly, in our …”

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

“…Crystal structure of hCA II in complex with compound 3 was determined at 1.80 Å resolution, revealing a clear electron density for the inhibitor molecule in the enzyme active site (Figure 2 [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] , compound 1 interacts directly with the zinc ion of the active site, with its sulphamate nitrogen atom N1 (for atom numbering see Figure 1) displacing the water molecule/hydroxide ion, which in the not-inhibited enzyme occupies the fourth coordination position. Additional hydrogen bonds between the sulphamate moiety and residues within the enzyme active site contribute to stabilise the binding.…”