Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC-APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Šresolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA's high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate-binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.

Regulation of polyamine homeostasis through an antizyme citrullination pathway.

This study reveals an uncovered mechanism for the regulation of polyamine homeostasis through protein arginyl citrullination of antizyme (AZ), a natural inhibitor of ornithine decarboxylase (ODC). ODC is critical for the cellular production of polyamines. AZ binds to ODC dimers and promotes the degradation of ODC via the 26S proteasome. This study demonstrates the protein citrullination of AZ catalyzed by peptidylarginine deiminase type 4 (PAD4) both in vitro and in cells. Upon PAD4 activation, the AZ protein was citrullinated and accumulated, leading to higher levels of ODC proteins in the cell. In the PAD4-overexpressing and activating cells, the levels of ODC enzyme activity and the product putrescine increased with the level of citrullinated AZ proteins and PAD4 activity. Suppressing cellular PAD4 activity reduces the cellular levels of ODC and downregulates cellular polyamines. Furthermore, citrullination of AZ in the C-terminus attenuates AZ function in the inhibition, binding, and degradation of ODC. This paper provides evidence to illustrate that PAD4-mediated AZ citrullination upregulates cellular ODC and polyamines by retarding ODC degradation, thus interfering with the homeostasis of cellular polyamines, which may be an important pathway regulating AZ functions that is relevant to cancer biology.

The rational discovery of multipurpose inhibitors of the ornithine decarboxylase.

Metabolic reprograming is a hallmark of cancer, and the polyamine metabolic network is dysregulated in many cancers. Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis in the polyamine metabolic network. In many cancer cells, ODC is over-expressed, so this enzyme has been an attracting anti-cancer drug target. In the catalysis axis (pathway), ODC converts ornithine to putrescine. Meanwhile, ODC's activity is regulated by protein-protein interactions (PPIs), including the ODC-OAZ1-AZIN1 PPI axis and its monomer-dimer equilibrium. Previous studies showed that when ODC's activity is inhibited, the PPIs might counteract the inhibition efficiency. Therefore, we proposed that multipurpose inhibitors that can simultaneously inhibit ODC's activity and perturb the PPIs would be very valuable as drug candidates and molecular tools. To discover multipurpose ODC inhibitors, we established a computational pipeline by combining positive screening and negative screening. We used this pipeline for the forward screening of multipurpose ligands that might inhibit ODC's activity, block ODC-OAZ1 interaction and enhance ODC non-functional dimerization. With a combination of different experimental assays, we identified three multipurpose ODC inhibitors. At last, we showed that one of these inhibitors is a promising drug candidate. This work demonstrated that our computational pipeline is useful for discovering multipurpose ODC inhibitors, and multipurpose inhibitors would be very valuable. Similar with ODC, there are a lot of proteins in human proteome that act as both enzymes and PPI components. Therefore, this work is not only presenting new molecular tools for polyamine study, but also providing potential insights and protocols for discovering multipurpose inhibitors to target more important protein targets.

Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ95-228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the α1 helix, ß5 and ß6 strands, and connecting loop between ß6 and α2 (residues 142-178), which is the posterior part of AZ95-228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ; amino acid residues within the anterior part of AZ95-228 (residues 120-145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

The antizyme family for regulating polyamines.

The polyamines spermidine, spermine, and their precursor putrescine are organic polycations involved in various cellular processes and are absolutely essential for cellular proliferation. Because of their crucial function in the cell, their intracellular concentration must be maintained at optimal levels. To a large extent, this regulation is achieved through the activity of an autoregulatory loop that involves two proteins, antizyme (Az) and antizyme inhibitor (AzI), that regulate the first enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), and polyamine uptake activity in response to intracellular polyamine levels. In this Minireview, I will discuss what has been learned about the mechanism of Az expression and its physical interaction with both ODC and AzI in the regulation of polyamines.

L-arginine Attenuates Hypobaric Hypoxia-Induced Increase in Ornithine Decarboxylase 1.

BACKGROUND: Chronic hypoxia-induced pulmonary hypertension and vascular remodeling have been shown to be associated with ornithine decarboxylase 1 (ODC1). However, few animal studies have investigated the role of ODC1 in acute hypoxia. OBJECTIVES: We investigated ODC1 gene expression, morphologic and functional changes, and the effect of L-arginine as an attenuator in lung tissues of rats exposed to acute hypobaric hypoxia at a simulated altitude of 6000 m. METHODS: Sprague-Dawley rats exposed to simulated hypobaric hypoxia (6000 m) for 24, 48, or 72 hours were treated with L-arginine (L-arginine group, 20 mg/100 g intraperitoneal; n=15) or untreated (non-L-arginine group, n=15). Control rats (n=5) were maintained at 2260 m in a normal environment for the same amount of time but were treated without L-arginine. The mean pulmonary artery pressure was measured by PowerLab system. The morphologic and immunohistochemical changes in lung tissue were observed under a microscope. The mRNA and protein levels of ODC1 were measured by real-time polymerase chain reaction and Western-blot, respectively. RESULTS: Hypobaric hypoxia induced pulmonary interstitial hyperemia and capillary expansion in the lungs of rats exposed to acute hypoxia at 6000 m. The mean pulmonary artery pressure and the mRNA and protein levels of ODC1 were significantly increased, which could be attenuated by treatment with L-arginine. CONCLUSIONS: L-arginine attenuates acute hypobaric hypoxia-induced increase in mean pulmonary artery pressure and ODC1 gene expression in lung tissues of rats. ODC1 gene contributes to the development of hypoxic pulmonary hypertension.