A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides

Abstract

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.

The Enzyme‐Free Release of Nucleotides from Phosphoramidates Depends Strongly on the Amino Acid

“…Thek inetics of hydrolysis suggest that the structural properties of the amino acid residue in nucleosidic phosphoramidates are important for the release of the nucleotide. However,tothe best of our knowledge no crystal structure of an unmodified aminoacidyl-nucleoside phosphoramidate had been published to date.There are crystal structures of methyl esters of L-or D-alanyl or tryptophanyl phosphoramidates of purines complexed to the human Hint1 protein, [33] and as earch of the Cambridge Structural Database gave structures of prodrug forms of phosphoramidates,t hat is,t he phenyl ester form of phosphoramidates (Scheme 1), but no structures of the free aminoacidyl nucleotides.T herefore,we undertook crystallization experiments with aseries of aminoacidyl phosphoramidates,i ncluding Gly-A, Pro-A, bPro-A, Phe-A, and Arg-A. We tested conditions commonly employed for crystallization of nucleosides and nucleotides from aqueous solution, using slow evaporation of solvent and high or saturating concentrations of the target compound, either with or without the addition of precipitants,togradually force the compound out of solution.…”

Histidine triad nucleotide‐binding proteins HINT1 and HINT2 share similar substrate specificities and little affinity for the signaling dinucleotide Ap4A

“…Thus, while the 2′‐methyl group significantly affects the substrate specificity of HINT1 and HINT2, it is one of the few substrates that impacted the catalytic activity of HINT2 more than HINT1. The root of this difference between the enzymes is not clear given that the active sites are nearly identical .…”

“…) . Previously, using steady‐state kinetic and X‐ray crystallographic structural studies, we have established that HINT1 prefers phosphoramidate and acyl nucleotidylate substrates composed of purine nucleobases, ribose sugars, alkyl amines, and d ‐amino acids . Structural analysis of the catalytically dead HINT1 mutant, H112N, bound with these substrates later established that the specificity of the active site results from a large hydrophobic nucleobase binding site, pivotal hydrogen bonds with the ribose 2′ and 3′ hydroxyl groups, and the ability for bulky d ‐amino acids to avoid major steric clashes with the boundaries of the active site .…”

The Enzyme‐Free Release of Nucleotides from Phosphoramidates Depends Strongly on the Amino Acid

“…This attack of an imidazole ring as the main path leading to hydrolysis is interesting, as it is analogous to what has been reported for hydrolysis mediated by Hint enzymes. [33] To confirm that the attack does not require the active site of an enzyme,weincubated Pro-A and Ala-A with the amino acid histidine in buffer devoid of 1-ethylimidazole.I ne ither case, the imidazolides were found in the equilibrium (Figure 5B/ C). ForP ro-A, the formation of this species is much faster than for Ala-A, and its hydrolysis then leads to free AMP, with ar eaction via the Na-amidate as aminor pathway.T his data suggests that imidazolides formed with histidine components of the cell can induce the release of nucleoside 5'monophosphates without the need for aH int enzyme.…”

“…Needless to say, dephosphorylation of a nucleotide metabolite through cleavage of the P−O bond is not desirable for a metabolite of a ProTide prodrug [32] . Rather, a preferred cleavage of the P−N bond, similar to what is found for reactions catalyzed by human hHint1, [33] but without the limitations of substrate specificity and tissue distribution of the enzyme, would be attractive.…”