Drug repositioning to discover novel ornithine decarboxylase inhibitors against visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by Leishmania donovani (Ld), and most cases occur in Brazil, East Africa, and India. The treatment for VL is limited and has many adverse effects. The development of safer and more efficacious drugs is urgently needed. Drug repurposing is one of the best processes to repurpose existing drugs. Ornithine decarboxylase (ODC) is an important target against L. donovani in the polyamine biosynthesis pathway. In this study, we have modeled the 3D structure of ODC and performed high-throughput virtual screening of 8630 ZINC database ligands against Leishmania donovani ornithine decarboxylase (Ld ODC), selecting 45 ligands based on their high binding score. It is further validated through molecular docking simulation and the selection of the top two lead molecules (ceftaroline fosamil and rimegepant) for Molecular Dynamics (MD) simulation, Density functional theory (DFT), and molecular mechanics generalized born surface area (MMGBSA) analysis. The results showed that the binding affinities of ceftaroline fosamil, and rimegepant are, respectively, -10.719 and 10.159 kcal/mol. The docking complexes of the two lead compounds, ceftaroline fosamil, and rimegepant, with the target ODC, were found stable during molecular dynamics simulations. Furthermore, the analysis of MMGBSA revealed that these compounds had a high binding free energy. The DFT analysis showed that the top lead molecules were more reactive than the standard drug (pentamidine). In-silico findings demonstrated that ceftaroline fosamil, and rimegepant might be recognized as potent antagonists against ODC for the treatment of VL.

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.

Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors.

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4-7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10-5 mM-1·s-1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Virtual screening of natural inhibitors targeting ornithine decarboxylase with pharmacophore scaffolding of DFMO and validation by molecular dynamics simulation studies.

Ornithine decarboxylase (ODC) is an enzyme that initiates polyamine synthesis in human. Polyamines play key roles in cell-cell adhesion, cell motility and cell cycle regulation. Higher synthesis of polyamines also occurs in rapidly proliferating cancer cells are mediated by ODC. As per earlier studies, di-flouro-methyl-orninthine (DFMO) is a proven efficient inhibitor ODC targeting the catalytic activity, however, its usage is limited due to side effects. Targeting ODC is considered as a potential therapeutic modality in the treatment of cancer. In this study, it is attempted to use DFMO scaffold to build a ligand-based pharmocophore query using MOE to screen similar active compounds from Universal Natural Products Database with better ADMET properties. The identified compounds were virtually screened against the active cavity of ODC using Glide. Further, potential natural hits targeting ODC were shortlisted based on Molecular Mechanics/Generalized-Born/Surface Area (MM-GBSA) score. Finally, molecular dynamics simulations were performed for the natural molecule hit and DFMO in complex with ODC using Desmond. Among the hits shortlisted, 2-amino-5, 9, 13, 17-tetramethyloctadeca-8, 16-diene-1, 3, 14-triol (UNPD208110) was found to be highly potential, as it showed a higher binding affinity in terms of interactions with key active cavity residues, and also showed better ADMET property, HUMO-LUMO gap energy and more stable complex formation with ODC compared to DFMO. Hence, the proposed molecule (UNPD208110) shall be favourably considered as a potential natural inhibitor targeting ODC-mediated disease conditions.

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.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Ornithine Decarboxylase Inhibition: A Strategy to Combat Various Diseases.

Ornithine decarboxylase is the first enzyme in the polyamine biosynthetic pathway. It is the rate-limiting enzyme which is included during the change of ornithine to putrescine which is the first polyamine. Polyamines (putrescine, spermidine, spermine) are natural and synthetic compounds which contain two or more amino group. Polyamines are highly implicated in cellular functions such as cell growth & multiplication, DNA stabilization, gene transcription and translation, ion-channel activity, etc. Elevated levels of polyamines are found in highly proliferating tumor cells. Hence inhibition of this enzyme was found useful in cancer. α-DL-difluoromethylornithine(DFMO) (Eflornithine) an enzyme- activated irreversible inhibitor was the first of this type. However, its use as an anticancer agent did not continue for long due to various reasons. Polyamines have also been found to play an important role in other infectious microorganisms. Eflornithine is successfully used in diseases such as African sleeping sickness and is being researched against a number of tropical diseases. It is widely used against hirsutism in women. Various other product (putrescine) based analogs and transition state or PLP (cofactor) based analogs are being synthesized against diseases such as Leishmaniasis, Malaria and others discussed in the article.

Molecular Modeling and Virtual Screening Approach to Discover Potential Antileishmanial Inhibitors Against Ornithine Decarboxylase.

BACKGROUND: Visceral leishmaniasis (VL) is a tropical neglected disease, which encounters poorest of poor people living in Asia, Africa and Latin America; causing the mortality of more than 30,000 people worldwide. The armamentarium for the treatment of VL cases is limited and continuously facing decreasing of efficacy for existing drugs. Ornithine decarboxylase (ODC) is one of the interesting drug targets in Leishmania donovani, due to its association with redox metabolism. OBJECTIVE: To search an antileishmanial compound showing the inhibitory effect against ornithine decarboxylase of Leishmania donovani Method: In this study, we have modelled the three dimensional structure of ODC using Phyre2 (Protein Homology/analog Y Recognition Engine V 2.0), followed by validation using VADAR (Volume, Area, Dihedral Angle Reporter), RAMPAGE, ERRAT, Verify3D and ProSA (Protein Structure Analysis). In order to develop potential antileishmanial, we conducted a high throughput virtual screening of ZINC database ligands comprising of 135,966 compounds. Furthermore, QikProp, ADMET predictor and MM-GBSA was performed for ADME (Absorption, Distribution, Metabolism and Elimination), toxicity and binding energy prediction for top ligands, respectively. Finally, molecular dynamics simulation was performed to get potential antileishmanial compounds. RESULT: Screening of zinc database compounds using high throughput virtual screening has given twelve compounds with good inhibition activity against ornithine decarboxylase. Furthermore, the molecular dynamics simulation work reveals that ZINC67909154 could be a potent inhibitor and this compound can be used to combat VL disease Conclusion: This study concludes that ZINC67909154 has the great potential to inhibit L. donovani ODC and would add to the drug discovery process against visceral leishmaniasis.

Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor.

Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant (Kd) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The Kd values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 µM, respectively. Furthermore, the Kd values for CCND1 binding to the AZ N-terminal peptide (AZ34-124) and AZ C-terminal peptide (AZ100-228) were 0.92 and 8.97 µM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The Kd values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 µM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.

Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma.

LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.

N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells.

Many cancer cells have high expression of ornithine decarboxylase (ODC) and there is a concerted effort to seek new inhibitors of this enzyme. The aim of the study was to initially characterize the inhibition properties, then to evaluate the cytotoxicity/antiproliferative cell based activity of N-ω-chloroacetyl-l-ornithine (NCAO) on three human cancer cell lines. Results showed NCAO to be a reversible competitive ODC inhibitor (Ki = 59 µM) with cytotoxic and antiproliferative effects, which were concentration- and time-dependent. The EC50,72h of NCAO was 15.8, 17.5 and 10.1 µM for HeLa, MCF-7 and HepG2 cells, respectively. NCAO at 500 µM completely inhibited growth of all cancer cells at 48 h treatment, with almost no effect on normal cells. Putrescine reversed NCAO effects on MCF-7 and HeLa cells, indicating that this antiproliferative activity is due to ODC inhibition.