ABSTRACT
The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.
Subject(s)
Adenine Phosphoribosyltransferase/metabolism , Adenine/chemistry , Adenine/metabolism , Adenine Phosphoribosyltransferase/chemistry , Biocatalysis , Crystallography, X-Ray , Humans , Kinetics , Models, Molecular , Protein Structure, Tertiary , Quantum Theory , Substrate SpecificityABSTRACT
In the course of a programme aimed at identifying Nurr1/NOT agonists for potential treatment of Parkinson's disease, a few hits from high throughput screening were identified and characterized. A combined optimization pointed to a very narrow and stringent structure activity relationship. A comprehensive program of optimization led to a potent and safe candidate drug displaying neuroprotective and anti-inflammatory activity in several in vitro and in vivo models.
Subject(s)
Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/pharmacology , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Parkinson Disease/drug therapy , Animals , Cell Line , Cricetinae , Drug Discovery , Gene Expression Regulation/drug effects , Homeodomain Proteins/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Mice , Microglia/drug effects , Molecular Structure , Neurons/drug effects , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Rats , Retinoid X Receptors/genetics , Retinoid X Receptors/metabolismABSTRACT
Phosphoribosyltransferases catalyze the displacement of a PRPP α-1'-pyrophosphate to a nitrogen-containing nucleobase. How they control the balance of substrates/products binding and activities is poorly understood. Here, we investigated the human adenine phosphoribosyltransferase (hAPRT) that produces AMP in the purine salvage pathway. We show that a single oxygen atom from the Tyr105 side chain is responsible for selecting the active conformation of the 12 amino acid long catalytic loop. Using in vitro, cellular, and in crystallo approaches, we demonstrated that Tyr105 is key for the fine-tuning of the kinetic activity efficiencies of the forward and reverse reactions. Together, our results reveal an evolutionary pressure on the strictly conserved Tyr105 and on the dynamic motion of the flexible loop in phosphoribosyltransferases that is essential for purine biosynthesis in cells. These data also provide the framework for designing novel adenine derivatives that could modulate, through hAPRT, diseases-involved cellular pathways.
Subject(s)
Adenine Phosphoribosyltransferase/metabolism , Adenine Phosphoribosyltransferase/chemistry , Adenine Phosphoribosyltransferase/isolation & purification , Crystallography, X-Ray , Humans , Models, Molecular , Protein ConformationABSTRACT
From potent and selective inhibitors of GSK3beta displaying CYP1A2 inhibition and poor PK properties, mostly linked to metabolic instability and in vivo hydrolysis of the amide bond, we were able to obtain safe and orally available inhibitors with good half lives.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacokinetics , Animals , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1A2 Inhibitors , Glycogen Synthase Kinase 3 beta , Humans , Mice , Models, Molecular , Protein Kinase Inhibitors/chemistryABSTRACT
From an HTS hit, a series of potent and selective inhibitors of GSK3beta have been designed based on a Cdk2-homology model and with the help of several crystal structures of the compounds within Cdk2.
