Spacer-Based Selectivity in the Binding of “Two-Prong” Ligands to Recombinant Human Carbonic Anhydrase I

Abstract

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu(2+) independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M., Roy, B. C., Jia, X., Haldar, M. K., Mallik, S., and Srivastava, D. K. (2004) J. Am. Chem. Soc. 126, 10875-10883]. By attaching IDA-bound Cu(2+) to benzenesulfonamide via different chain length spacers, we synthesized two "two-prong" ligands, L1 and L2, in which the distances between Cu(2+) and NH(2) group of sulfonamide were 29 and 22 A, respectively. We compared the binding affinities of L1 and L2, vis-a-vis their parent compound, benzenesulfonamide, for recombinant human carbonic anhydrase I (hCA-I) by performing the fluorescence titration and steady-state kinetic experiments. The experimental data revealed that whereas the binding affinity of L1 for hCA-I was similar to that of benzenesulfonamide, the binding affinity of L2 was approximately 2 orders of magnitude higher, making L2 one of the most potent ligands or inhibitors of hCA-I. Since the enhanced binding or inhibitory potency of L2 is diminished (to the level of benzenesulfonamide) either in the presence of EDTA or upon treatment of the enzyme with diethyl pyrocarbonate, it is proposed that Cu(2+) of L2 interacts with one of the surface-exposed histidine residues of the enzyme. A cumulative account of the experimental data leads to the suggestion that the differential binding of L1 versus L2 to hCA-I is encoded in the chain length of the spacer moiety.

How many carbonic anhydrase inhibition mechanisms exist?

“…The His cluster was reported by Briganti et al 23 whereas Zn(II), Cu(II), Co(II) and many other metal complexes of aromatic/heterocyclic sulfonamides incorporating polyaminopolycarboxylic acids (including IDA) in 2002 by Scozzafava et al 48 In all cases in which such metal-containing CAIs were investigated by our group, we observed only binding of the metal ion to His64 and not to the other His residues from the His cluster mentioned above 48 . Compounds sent by Srivastava in our laboratory in 2005-2006, did not show the inhibition pattern published in their paper 158 and the X-ray crystallography performed with some of these compounds (unpublished data from this and De Simone laboratory) showed the normal binding of the sulfonamide to the zinc ion and the Cu(II) always bound to His64. A subsequent crystallographic work from the same group 159 again evidenced binding of the Cu(II) only to His64, but the conclusion of the work was that the two-prong approach was working.…”

“…I have already mentioned in an earlier review 6 that the ''two-prong'' approach 158 (based on our tail approach published several years earlier 81,82 ) is probably fabricated. According to this approach, ideated by Srivastava's group, iminodiacetic (IDA) moieties (which complexate Cu 2+ ) were attached to benzenesulfonamides via different chain length spacers, leading to the so-called ''two-prong'' inhibitors.…”

Synthetic Multivalent Ligands as Probes of Signal Transduction

“…Unlike monovalent ligands, which can only access subsites adjacent to their primary binding pocket, multivalent ligands may gain binding energy from contacting these secondary sites. [180] Either a RE or another component of the scaffold can contact these subsites. In situations in which the multivalent ligand occupies more than 1 binding site --whether it is a subsite or a primary site within an oligomeric receptor --the ligand typically will bind with a high functional affinity.…”

Binding of Uridine 5‘-Diphosphate in the “Basic Patch” of the Zinc Deacetylase LpxC and Implications for Substrate Binding,

“…A similar approach has been adopted in the design of inhibitors of carbonic anhydrases I and II, in which a cupric iminodiacetate moiety is tethered to a sulfonamide group in order to simultaneously bind to the active site Zn 2+ ion and a histidine residue ~8 Å away (39)(40)(41).…”