Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase

Abstract

The enzyme aminoimidazole ribonucleotide (AIR) carboxylase catalyzes the synthesis of the purine intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Previously, we have shown that the compound 4-nitro-5-aminoimidazole ribonucleotide (NAIR) is a slow, tight binding inhibitor of the enzyme with a K i of 0.34 nM. The structural attributes and the slow, tight binding characteristics of NAIR implicated this compound as a transition state or reactive intermediate analog. However, it is unclear what molecular features of NAIR contribute to the mimetic properties for either of the two proposed mechanisms of AIR carboxylase. In order to gain additional information regarding the mechanism for the potent inhibition of AIR carboxylase by NAIR, a series of heterocyclic analogs were prepared and evaluated. We find that all compounds are weaker inhibitors than NAIR and that CAIR analogs are not alternative substrates for the enzyme. Surprisingly, rather subtle changes in the structure of NAIR can lead to profound changes in binding affinity. Computational investigations of enzyme intermediates and these inhibitors reveal that NAIR displays an electrostatic potential surface similar to a proposed reaction intermediate. The result indicates that AIR carboxylase is likely sensitive to the electrostatic surface of reaction intermediates and thus compounds which mimic these surfaces should possess tight binding characteristics. Given the evolutionary relationship between AIR carboxylase and N 5 -CAIR mutase, we believe that this concept extends to the mutase enzyme as well. The implications of this hypothesis for the design of selective inhibitors of the N 5 -CAIR mutase are discussed.

The synthesis, antiviral, cytostatic and cytotoxic evaluation of a new series of acyclonucleotide analogues with a 1,2,3-triazole linker

“…In drug discovery replacement of canonical nucleobases [5] by substituted five-membered heterocyclic rings such as imidazole or triazole has been particularly successful. Ribavirin [6e12], AICA [13,14], Bredinin [15,16] and TSAO analogues [17,18] are the best known examples among these analogues (Fig. 2).…”

Synthesis of 13C‐labeled 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐[13C5] ribofuranosyl 5′‐monophosphate

“…16 The resulting inosine acetonide 2′ (not shown) was chromatographed to obtain 2′ free of buffer salts and enzyme in 69% yield over two steps. In the carbon-13 labeled case, commercially available 1 was subjected to adenosine deaminase enzyme to cleanly form [ 13 C 5 -ribose] inosine 2.…”

“…[12][13][14][15] An acetonide protecting group was then installed as previous reports have indicated that phosphorylation of a similar ribose 5′-hydroxyl in the presence of unblocked 2′-and 3′-hydroxyls is not completely selective. 16 The resulting inosine acetonide 2′ (not shown) was chromatographed to obtain 2′ free of buffer salts and enzyme in 69% yield over two steps. 17,18 Subsequent acetylation afforded compound 3 in 94% yield that was used without further purification.…”

Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity

“…3 The architecture of the active sites of bacterial PurE and the PurE domain in hPur6 are nearly superimposable, and yet despite these conserved features, biochemical studies have shown that both enzymes are highly specific. 4 Subsequent studies have shown that bacterial PurE has the potential to be a target for new antibiotic development. 5 In this study, a fluorescence-based 6 thermal shift assay 7,8 was applied to B. anthracis PurE (BaPurE) for high-throughput screening (HTS) to identify molecules (hits) that bind to BaPurE.…”