1506568
Discovery of novel inhibitors of human S-adenosylmethionine decarboxylase based on in silico high-throughput screening and a non-radioactive enzymatic assay
Natural polyamines are small polycationic molecules essential for cell growth and development, and elevated level of polyamines is positively correlated with various cancers. As a rate-limiting enzyme of the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (AdoMetDC) has been an attractive drug target. In this report, we present the discovery of novel human AdoMetDC (hAdoMetDC) inhibitors by coupling computational and experimental tools. We constructed a reasonable computational structure model of hAdoMetDC that is compatible with general protocols for high-throughput drug screening, and used this model in in silico screening of hAdoMetDC inhibitors against a large compound library using a battery of computational tools. We also established and validated a simple, economic, and non-radioactive enzymatic assay, which can be adapted for experimental high-throughput screening of hAdoMetDC inhibitors. Finally, we obtained an hAdoMetDC inhibitor lead with a novel scaffold. This study provides both new tools and a new lead for the developing of novel hAdoMetDC inhibitors.
5313851
Toxicity of polyamines and their metabolic products.
Polyamines are ubiquitous and essential components of mammalian cells. They have multiple functions including critical roles in nucleic acid and protein synthesis, gene expression, protein function, protection from oxidative damage, the regulation of ion channels, and maintenance of the structure of cellular macromolecules. It is essential to maintain a correct level of polyamines, and this amount is tightly regulated at the levels of transport, synthesis, and degradation. Catabolic pathways generate reactive aldehydes including acrolein and hydrogen peroxide via a number of oxidases. These metabolites, particularly those from spermine, can cause significant toxicity with damage to proteins, DNA, and other cellular components. Their production can be increased as a result of infection or cell damage that releases free polyamines and activates the oxidative catabolic pathways. Since polyamines also have an important physiological role in protection from oxidative damage, the reduction in polyamine content may exacerbate the toxic potential of these agents. Increases in polyamine catabolism have been implicated in the development of diseases including stroke, other neurological diseases, renal failure, liver disease, and cancer. These results provide new opportunities for the early diagnosis, prevention, and treatment of disease.
572638
Polyamines in aging and disease
Polyamines are polycations that interact with negatively charged molecules such as DNA, RNA and proteins. They play multiple roles in cell growth, survival and proliferation. Changes in polyamine levels have been associated with aging and diseases. Their levels decline continuously with age and polyamine (spermidine or high-polyamine diet) supplementation increases life span in model organisms. Polyamines have also been involved in stress resistance. On the other hand, polyamines are increased in cancer cells and are a target for potential chemotherapeutic agents. In this review, we bring together these various results and draw a picture of the state of our knowledge on the roles of polyamines in aging, stress and diseases.
7498261
Identification and assay of allosteric regulators of S-adenosylmethionine decarboxylase.
Polyamine biosynthesis is extensively regulated in cells by multiple mechanisms, including regulation of enzyme activity posttranslationally. The identified regulatory factors include both small molecules and regulatory proteins, and the mechanisms vary in different species across the evolutionary tree. Based on this diversity of mechanism, it is likely that regulatory factors of the pathway remain unidentified in many species. This article focuses on methods for identifying novel regulatory factors of polyamine biosynthesis as illustrated by the discovery of a novel protein activator of the key biosynthetic enzyme S-adenosylmethionine decarboxylase in the protozoan trypanosomatid parasites.
14863747
Polyamines and transglutaminases: biological, clinical, and biotechnological perspectives
The history of polyamines dates back to the fifteenth century when spermine was discovered by Antoni van Leeuwenhoek [born in Delft, Holland (1632–1723)], and yet it took many years before serious attention was given to understanding the role of spermine or other polyamines in the biology of living cells. It is now clear that regulation of polyamine homeostasis is complex and has excited polyamine researchers who have continued to focus on this productive area of research. Therefore, enough new research findings have prompted to organize conferences and congresses worldwide to disseminate the new knowledge generated. New research in the areas of biosynthesis and action of the ubiquitous polyamines, with particular focus on the biology of normal and cancerous eukaryotic cells, and plants, was the driving force to hold a 6-day International Congress on ‘‘Polyamines Biological and Clinical Perspectives’’ in Turkey at Istanbul Kultur University, in the beautiful city of Istanbul, from 2 September to 7 September, 2012. This conference was organized by Professor Narcin Palavan Unsal (Istanbul Kultur University) and Professor Enzo Agostinelli (SAPIENZA University of Rome, Rome, Italy). Istanbul is a largest marvelous city in Turkey. Located on both sides of the Bosporus, Istanbul bridges Asia and Europe. The ancient name of Istanbul was Bisantium, a city founded by Greeks in 659 B.C. on the banks of the Bosporus. Bisantium was renamed Constantinopolis in honor of the Roman emperor Constantine I, becoming a center of Greek culture and Christianity. Throughout its long history, Istanbul (the old Constantinopolis) was the capital of three important empires: Roman, Byzantine, and Ottoman. Today, Istanbul as one of the largest cities in the world is also one of the European capitals of culture while its historic areas are part of the UNESCO list of World Cultural Heritage. This Special Issue of amino acids brings together 28 peer-reviewed manuscripts that provide the essence of the lectures and posters presented at the above-mentioned conference on the biological and physiological roles of Polyamines and Transglutaminases (TGases), as well as a few manuscripts authored by internationally recognized experts who could not attend the meeting. All the articles deal with high-class research data obtained until the mid2013. This issue also provides short overviews of some important concepts and notions in the subject matter, which represent important tools for those new investigators who may embark on investigating this intriguing field. The papers project the role of polyamines in cell growth and differentiation, cell cycle regulation, gene expression and signal transduction in animals, plants and microorganisms as well as in several pathophysiological processes including carcinogenesis and other diseases. Recently, the significance of polyamines in cell proliferation and carcinogenesis has stimulated further research interest in their biochemical and metabolic regulation at genomic level (Zhu et al. 2012; Cervelli et al. 2013; Murray-Stewart E. Agostinelli (&) Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy e-mail: enzo.agostinelli@uniroma1.it
206207609
Polyamines and cancer: implications for chemotherapy and chemoprevention
Polyamines are small organic cations that are essential for normal cell growth and development in eukaryotes. Under normal physiological conditions, intracellular polyamine concentrations are tightly regulated through a dynamic network of biosynthetic and catabolic enzymes, and a poorly characterised transport system. This precise regulation ensures that the intracellular concentration of polyamines is maintained within strictly controlled limits. It has frequently been observed that the metabolism of, and the requirement for, polyamines in tumours is frequently dysregulated. Elevated levels of polyamines have been associated with breast, colon, lung, prostate and skin cancers, and altered levels of rate-limiting enzymes in both biosynthesis and catabolism have been observed. Based on these observations and the absolute requirement for polyamines in tumour growth, the polyamine pathway is a rational target for chemoprevention and chemotherapeutics. Here we describe the recent advances made in the polyamine field and focus on the roles of polyamines and polyamine metabolism in neoplasia through a discussion of the current animal models for the polyamine pathway, chemotherapeutic strategies that target the polyamine pathway, chemotherapeutic clinical trials for polyamine pathway-specific drugs and ongoing clinical trials targeting polyamine biosynthesis.
21412838
Identification of a pyruvoyl residue in S-adenosylmethionine decarboxylase from Saccharomyces cerevisiae.
S-Adenosylmethionine decarboxylase from Saccharomyces cerevisiae has been purified to homogeneity. Acid hydrolysis of NaB3H4-reduced enzyme released 2.2 mol of tritiated lactate per mol of dimeric enzyme, indicating that a pyruvate moiety is present. Inhibition of enzymatic activity by NaBH4 reduction and by carbonyl-binding reagents indicates that this pyruvoyl residue is required for the activity of the enzyme. This is the first example reported of a eukaryotic enzyme containing a covalently linked pyruvoyl residue.
5820242
On the role of S-adenosyl-L-methionine in the biosynthesis of spermidine by rat prostate.
Abstract The isolation from the rat ventral prostate of a soluble enzyme preparation producing spermidine, CO2, and methylthioadenosine from putrescine and S-adenosyl-l-methionine is described. This preparation catalyzes limited decarboxylation of S-adenosylmethionine in the absence of any additional reactants but maximal rates of CO2 release from S-adenosylmethionine require the presence of putrescine. When putrescine is present, spermidine and methylthioadenosine are produced in amounts stoichiometric with the CO2 released. The decarboxylation of S-adenosylmethionine in the presence of putrescine is inhibited by isonicotinic acid hydrazide and by 4-bromo-3-hydroxybenzyloxyamine. This finding suggests that pyridoxal phosphate is a cofactor of the reaction. The prostatic enzyme preparation also catalyzes the formation of spermidine from putrescine and exogenous decarboxylated S-adenosylmethionine (prepared by the action of Escherichia coli S-adenosylmethionine decarboxylase). The affinities for S-adenosylmethionine, decarboxylated S-adenosylmethionine, and putrescine were measured. The prostatic preparation could not be separated into two enzymic fractions, one catalyzing the decarboxylation of S-adenosylmethionine, and the other catalyzing spermidine synthesis, as is the case with the spermidine-synthesizing system of E. coli. The differences between the prostatic and the bacterial spermidine-synthesizing systems are discussed. Spermine is synthesized by the preparation from the rat ventral prostate in the presence of S-adenosylmethionine and spermidine, but at a considerably slower rate than spermidine synthesis from putrescine and S-adenosylmethionine.
35179512
Polyamines in cancer.
Polyamines are organic cations shown to control gene expression at the transcriptional, posttranscriptional, and translational levels. Multiple cellular oncogenic pathways are involved in regulation of transcription and translation of polyamine-metabolizing enzymes. As a consequence of genetic alterations, expression levels and activities of polyamine-metabolizing enzymes change rapidly during tumorigenesis resulting in high levels of polyamines in many human epithelial tumors. This review summarizes the mechanisms of polyamine regulation by canonical tumor suppressor genes and oncogenes, as well as the role of eukaryotic initiation factor 5A (EIF5A) in cancer. The importance of research utilizing pharmaceutical inhibitors and cancer chemopreventive strategies targeting the polyamine pathway is also discussed.
12662606
Novel S-adenosyl-L-methionine decarboxylase inhibitors as potent antiproliferative agents against intraerythrocytic Plasmodium falciparum parasites☆
Graphical abstract
32896579
Mammalian polyamine metabolism and function
Polyamines are ubiquitous small basic molecules that play multiple essential roles in mammalian physiology. Their cellular content is highly regulated and there is convincing evidence that altered metabolism is involvement in many disease states. Drugs altering polyamine levels may therefore have a variety of important targets. This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism. © 2009 IUBMB IUBMB Life 61(9): 880–894, 2009
20537846
Polyamine-Blocking Therapy Reverses Immunosuppression in the Tumor Microenvironment
Hayes and colleagues report that polyamine elevation in cancer contributes significantly to tumor immunosuppression; they developed a novel polyamine-depletion strategy combining inhibitors of the polyamine transport system and ornithine decarboxylase to prevent tumor immune escape and promote antitumor immunity. Correcting T-cell immunosuppression may unleash powerful antitumor responses; however, knowledge about the mechanisms and modifiers that may be targeted to improve therapy remains incomplete. Here, we report that polyamine elevation in cancer, a common metabolic aberration in aggressive lesions, contributes significantly to tumor immunosuppression and that a polyamine depletion strategy can exert antitumor effects that may also promote immunity. A polyamine-blocking therapy (PBT) that combines the well-characterized ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) with AMXT 1501, a novel inhibitor of the polyamine transport system, blocked tumor growth in immunocompetent mice but not in athymic nude mice lacking T cells. PBT had little effect on the proliferation of epithelial tumor cells, but it increased the number of apoptotic cells. Analysis of CD45+ tumor immune infiltrates revealed that PBT decreased levels of Gr-1+CD11b+ myeloid suppressor cells and increased CD3+ T cells. Strikingly, in a model of neoadjuvant therapy, mice administered with PBT one week before surgical resection of engrafted mammary tumors exhibited resistance to subsequent tumor rechallenge. Collectively, our results indicate that therapies targeting polyamine metabolism do not act exclusively as antiproliferative agents, but also act strongly to prevent immune escape by the tumor. PBT may offer a general approach to heighten immune responses in cancer. Cancer Immunol Res; 2(3); 274–85. ©2013 AACR.
19702536
Clinical Efficacy, Tolerability, and Safety of SAM486A, a Novel Polyamine Biosynthesis Inhibitor, in Patients with Relapsed or Refractory Non-Hodgkin’s Lymphoma
Purpose: SAM486A is a new inhibitor of S-adenosyl-methionine-decarboxylase, a key enzyme for polyamine biosynthesis. It is more potent than the first generation S-adenosyl-methionine-decarboxylase inhibitor methylglyoxal bis-guanylhydrazone. This Phase IIa study reports the findings of SAM486A monotherapy in patients with refractory or relapsed non-Hodgkin’s lymphoma (NHL). Patients and Methods: Forty-one previously treated patients with either diffuse large cell, follicular, or peripheral T-cell NHL were treated i.v. with 100 mg/m2 SAM486A as a daily 1-h infusion for 5 days repeated every 3 weeks. Treatment was continued for a total of eight cycles or until disease progression. Results: Two patients, both with large B-cell lymphoma, showed a complete response at cycle 3 that was maintained for ≥13 and ≥28 months. Five patients had a partial response, and 3 had stable disease at last follow-up. The overall response rate (complete response plus partial response) was 18.9% for evaluable patients (7 patients). Anemia was the primary hematological toxicity and observed in 7 (17.1%) patients. Five patients experienced grade 3/4 anemia. Four patients (9.8%) experienced grade 3/4 febrile neutropenia and grade 3/4 thrombocytopenia, respectively. Nonhematological toxicities were mild to moderate in intensity. The most frequent side effects were nausea (39%), vomiting (22%), diarrhea (19.5%), asthenia (17.1%), abdominal pain (14.6%), and flushing (9.8%). Conclusion: SAM486A has a promising clinical activity in patients with poor prognosis NHL and manageable safety profile. To further define the role of SAM486A, in the treatment of NHL, additional studies are warranted.
35936199
Drug repositioning by structure-based virtual screening.
Approved drugs have favourable or validated pharmacokinetic properties and toxicological profiles, and the repositioning of existing drugs for new indications can potentially avoid expensive costs associated with early-stage testing of the hit compounds. In recent years, technological advances in virtual screening methodologies have allowed medicinal chemists to rapidly screen drug libraries for therapeutic activity against new biomolecular targets in a cost-effective manner. This review article outlines the basic principles and recent advances in structure-based virtual screening and highlights the powerful synergy of in silico techniques in drug repositioning as demonstrated in several recent reports.
21311409
Rational drug design: the search for Ras protein hydrolysis intermediate conformation inhibitors with both affinity and specificity.
Computer-aided drug design (CADD) plays significant roles in all stages of today's drug discovery. Many CADD technologies and methods were employed in finding promising hits against different targets during the past several decades. In this review, the most common molecular modeling methods applied to computer-aided drug design are discussed. However, how to effectively integrate these computational methods and then combine them with experiments to improve the hit rate is still a challenge. In addition, the present study reviews the ISR (intrinsic specificity ratio) as a novel concept and quantitative criterion for binding specificity to be applied as a complement in addition to binding affinity for finding new leads. Using Ras protein as a case study, Molecular modeling calculations and the subsequent biological testings for the hits are performed, the specificity of these hits is also studies against the normal and cancer cells aiming at discovering the novel chemical compounds with minimal side effects. Herein, the case study also includes the evaluations for tumor-specific cytotoxicity on different cell lines. The current results suggest that ISR is useful for quantitative assessment of specificity of small molecules.
8588838
Structure and Ligand Based Drug Design Strategies in the Development of Novel 5-LOX Inhibitors
Lipoxygenases (LOXs) are non-heme iron containing dioxygenases involved in the oxygenation of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA). Depending on the position of insertion of oxygen, LOXs are classified into 5-, 8-, 9-, 12- and 15-LOX. Among these, 5-LOX is the most predominant isoform associated with the formation of 5-hydroperoxyeicosatetraenoic acid (5-HpETE), the precursor of non-peptido (LTB4) and peptido (LTC4, LTD4, and LTE4) leukotrienes. LTs are involved in inflammatory and allergic diseases like asthma, ulcerative colitis, rhinitis and also in cancer. Consequently 5-LOX has become target for the development of therapeutic molecules for treatment of various inflammatory disorders. Zileuton is one such inhibitor of 5-LOX approved for the treatment of asthma. In the recent times, computer aided drug design (CADD) strategies have been applied successfully in drug development processes. A comprehensive review on structure based drug design strategies in the development of novel 5-LOX inhibitors is presented in this article. Since the crystal structure of 5-LOX has been recently solved, efforts to develop 5-LOX inhibitors have mostly relied on ligand based rational approaches. The present review provides a comprehensive survey on these strategies in the development of 5-LOX inhibitors.
28025930
Computer-aided drug design: lead discovery and optimization.
Over the past decade, there have been remarkable advances in the area of computer-aided drug design (CADD), which has been applied at almost all stages in the drug discovery pipeline. The generation of initial lead compounds and the subsequent optimization aimed at improving potency and pharmacological properties are the core activities among all. The development in these aspects over the past years will be the focus of this review.
5204683
Integration of in silico and in vitro tools for scaffold optimization during drug discovery: predicting P-glycoprotein efflux.
In silico tools are regularly utilized for designing and prioritizing compounds to address challenges related to drug metabolism and pharmacokinetics (DMPK) during the process of drug discovery. P-Glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) transporters with broad substrate specificity that plays a significant role in absorption and distribution of drugs that are P-gp substrates. As a result, screening for P-gp transport has now become routine in the drug discovery process. Typically, bidirectional permeability assays are employed to assess in vitro P-gp efflux. In this article, we use P-gp as an example to illustrate a well-validated methodology to effectively integrate in silico and in vitro tools to identify and resolve key barriers during the early stages of drug discovery. A detailed account of development and application of in silico tools such as simple guidelines based on physicochemical properties and more complex quantitative structure-activity relationship (QSAR) models is provided. The tools were developed based on structurally diverse data for more than 2000 compounds generated using a robust P-gp substrate assay over the past several years. Analysis of physicochemical properties revealed a significantly lower proportion (<10%) of P-gp substrates among the compounds with topological polar surface area (TPSA) <60 Å(2) and the most basic cpKa <8. In contrast, this proportion of substrates was greater than 75% for compounds with TPSA >60 Å(2) and the most basic cpKa >8. Among the various QSAR models evaluated to predict P-gp efflux, the Bagging model provided optimum prediction performance for prospective validation based on chronological test sets. Four sequential versions of the model were built with increasing numbers of compounds to train the models as new data became available. Except for the first version with the smallest training set, the QSAR models exhibited robust prediction profiles with positive prediction values (PPV) and negative prediction values (NPV) exceeding 80%. The QSAR model demonstrated better concordance with the manual P-gp substrate assay than an automated P-gp substrate screen. The in silico and the in vitro tools have been effectively integrated during early stages of drug discovery to resolve P-gp-related challenges exemplified by several case studies. Key learning based on our experience with P-gp can be widely applicable across other DMPK-related challenges.
7682651
The holistic integration of virtual screening in drug discovery.
During the past decade, virtual screening (VS) has come of age. In this review, we document the evolution and maturation of VS from a rather exotic, stand-alone method toward a versatile hit and lead identification technology. VS campaigns have become fully integrated into drug discovery campaigns, evenly matched and complementary to high-throughput screening (HTS) methods. Here, we propose a novel classification of VS applications to help to monitor the advances in VS and to support future improvement of computational hit and lead identification methods. Several relevant VS studies from recent publications, in both academic and industrial settings, were selected to demonstrate the progress in this area. Furthermore, we identify challenges that lie ahead for the development of integrated VS campaigns.
8625573
Complementarity between in Silico and Biophysical Screening Approaches in Fragment-Based Lead Discovery against the A2A Adenosine Receptor
Fragment-based lead discovery (FBLD) is becoming an increasingly important method in drug development. We have explored the potential to complement NMR-based biophysical screening of chemical libraries with molecular docking in FBLD against the A(2A) adenosine receptor (A(2A)AR), a drug target for inflammation and Parkinson's disease. Prior to an NMR-based screen of a fragment library against the A(2A)AR, molecular docking against a crystal structure was used to rank the same set of molecules by their predicted affinities. Molecular docking was able to predict four out of the five orthosteric ligands discovered by NMR among the top 5% of the ranked library, suggesting that structure-based methods could be used to prioritize among primary hits from biophysical screens. In addition, three fragments that were top-ranked by molecular docking, but had not been picked up by the NMR-based method, were demonstrated to be A(2A)AR ligands. While biophysical approaches for fragment screening are typically limited to a few thousand compounds, the docking screen was extended to include 328,000 commercially available fragments. Twenty-two top-ranked compounds were tested in radioligand binding assays, and 14 of these were A(2A)AR ligands with K(i) values ranging from 2 to 240 μM. Optimization of fragments was guided by molecular dynamics simulations and free energy calculations. The results illuminate strengths and weaknesses of molecular docking and demonstrate that this method can serve as a valuable complementary tool to biophysical screening in FBLD.
32831353
Simple and rapid enzymatic assay of ornithine decarboxylase activity.
Since the induction of putrescine synthesis by ornithine decarboxylase (ODC) is observed in many pathological and physiological processes, a useful and simple method to assay this enzyme activity should be an interesting tool to quantify the biological importance of its induction. An enzymatic method to assay ODC is reported here. This method is based on the reaction between putrescine and soya diamine oxidase. The reaction releases H(2)O(2), which is measured by a colorimetric method. The validation of this method showed good accuracy (98+/-5% of recovery). High precision and reproducibility were obtained. A linearity with a correlation coefficient of 0.999 in the range of 2.5-25 nmol was obtained. This method is also rugged and specific. The application of the assay of ODC activity showed that it is useful as a rapid and simple tool for assaying ODC activity in vitro. Comparison with the HPLC determination of ODC activity shows strong correlation along with the high accuracy of the two methods.
1924360
Spectrophotometric assay for ornithine decarboxylase.
A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.
5149435
Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes.
Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).
19366193
On the purification of L-ornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme.
Abstract The activity and stability of l-ornithine decarboxylase from rat ventral prostate is markedly increased by certain thiol compounds, notably dithiothreitol. The enzyme was purified about 300-fold. Many properties of the purified enzyme are described. In the absence of added thiol compounds, the purified ornithine decarboxylase apparently undergoes polymerization, as evidenced by its behavior on sucrose density gradients and in molecular sieving experiments. The larger forms of the enzyme appear to be catalytically inert, but can be reactivated by a number of sulfhydryl compounds, certain dithiols being superior to a number of monothiols in this respect.
17410238
Design of polyamine-based therapeutic agents: new targets and new directions.
Enzymes in the biosynthetic and catabolic polyamine pathway have long been considered targets for drug development, and early drug discovery efforts in the polyamine area focused on the design and development of specific inhibitors of the biosynthetic pathway, or polyamine analogues that specifically bind DNA. More recently, it has become clear that the natural polyamines are involved in numerous known and unknown cellular processes, and disruption of polyamine functions at their effector sites can potentially produce beneficial therapeutic effects. As new targets for polyamine drug discovery continue to evolve, the rational design of polyamine analogues will result in more structurally diverse agents. In addition, the physical linkage of polyamine-like structures to putative drug molecules can have beneficial effects resulting from increases in DNA affinity and selective cellular uptake. The present chapter will summarize recent advances in the development of alkylpolyamine analogues as antitumour agents, and describe subsequent advances that have resulted from incorporating polyamine character into more diverse drug molecules. Specifically, new polyamine analogues, and the role of polyamine fragments in the design of antiparasitic agents, antitumour metal complexes, histone deacetylase inhibitors and lysine-specific demethylase 1 inhibitors, will be described.
9396787
Minimal Antizyme Peptide Fully Functioning in the Binding and Inhibition of Ornithine Decarboxylase and Antizyme Inhibitor
Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC50: 0.20 µM) similar to that of AZ-95-228 (IC50: 0.16 µM), even though a large segment spanning residues 177–228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC50 values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC50 values comparable to that of AZ_WT and formed AZ-ODC complexes with K d,AZ-ODC values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.
32633744
Critical Factors Determining Dimerization of Human Antizyme Inhibitor*
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer Kd, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (Kd value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a Kd value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.
20495156
X-ray Structure Determination of Trypanosoma brucei Ornithine Decarboxylase Bound to d-Ornithine and to G418
Ornithine decarboxylase (ODC) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the rate-determining step in the biosynthesis of polyamines. ODC is a proven drug target to treat African sleeping sickness. The x-ray crystal structure of Trypanosoma brucei ODC in complex with d-ornithine (d-Orn), a substrate analog, and G418 (Geneticin), a weak non-competitive inhibitor, was determined to 2.5-Å resolution. d-Orn forms a Schiff base with PLP, and the side chain is in a similar position to that observed for putrescine and α-difluoromethylornithine in previous T. brucei ODC structures. The d-Orn carboxylate is positioned on the solvent-exposed side of the active site (si face of PLP), and Gly-199, Gly-362, and His-197 are the only residues within 4.2 Å of this moiety. This structure confirms predictions that the carboxylate of d-Orn binds on the si face of PLP, and it supports a model in which the carboxyl group of the substrate l-Orn would be buried on the re face of the cofactor in a pocket that includes Phe-397, Tyr-389, Lys-69 (methylene carbons), and Asp-361. Electron density for G418 was observed at the boundary between the two domains within each ODC monomer. A ten-amino acid loop region (392–401) near the 2-fold axis of the dimer interface, which contributes several residues that form the active site, is disordered in this structure. The disordering of residues in the active site provides a potential mechanism for inhibition by G418 and suggests that allosteric inhibition from this site is feasible.
254374
Formation of functional cross-species heterodimers of ornithine decarboxylase.
The two active sites in ornithine decarboxylase (ODC) are formed at the dimer interface with Lys-69 and Cys-360 contributing to each active site from opposite monomers [Tobias, K. E., & Kahana, C. (1993) Biochemistry 32, 5842-5847]. To gain insight into the organization of the substrate binding site and the nature of the dimer interface, analysis of ornithine decarboxylase from two parasitic protozoa, Trypanosoma brucei and Leishmania donovani, and from mouse was undertaken. Though T. brucei and mouse ornithine decarboxylase share only 60% sequence identity, the cross-species heterodimers form spontaneously, as measured by the restoration of enzyme activity upon mixing inactive K69A and C360A mutant enzymes. Thus, the amino acid composition of the dimer interface is apparently highly conserved between the T. brucei and mouse enzymes. Cross-species heterodimers were not formed between either T. brucei or mouse ODC and L. donovani ODC. Unlike the mouse and T. brucei ODC, the subunits of L. donovani ODC are not in rapid equilibrium, and incubation with a denaturant is required to induce reassociation. Kinetic analysis of the wild-type mouse and parasite ODCs revealed differences in the substrate binding sites between the three enzymes. The substrate binding properties of the restored active site in the T. brucei:mouse cross-species heterodimer mimic the characteristics of the wild-type enzyme from the species which contributes the subunit with a functional Lys-69.
6563107
Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog
African sleeping sickness is a fatal disease that is caused by the protozoan parasite Trypanosoma brucei. Polyamine biosynthesis is an essential pathway in the parasite and is a validated drug target for treatment of the disease. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in polyamine biosynthesis. Here, we show that trypanosomatids uniquely contain both a functional AdoMetDC and a paralog designated prozyme that has lost catalytic activity. The T. brucei prozyme forms a high-affinity heterodimer with AdoMetDC that stimulates its activity by 1,200-fold. Both genes are expressed in T. brucei, and analysis of AdoMetDC activity in T. brucei extracts supports the finding that the heterodimer is the functional enzyme in vivo. Thus, prozyme has evolved to be a catalytically dead but allosterically active subunit of AdoMetDC, providing an example of how regulators of multimeric enzymes can evolve through gene duplication and mutational drift. These data identify a distinct mechanism for regulating AdoMetDC in the parasite that suggests new strategies for the development of parasite-specific inhibitors of the polyamine biosynthetic pathway.
1037842
Structural biology of S-adenosylmethionine decarboxylase
S-adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and a subject of many structural and biochemical investigations for anti-cancer and anti-parasitic therapy. The enzyme undergoes an internal serinolysis reaction as a post-translational modification to generate the active site pyruvoyl group for the decarboxylation process. The crystal structures of AdoMetDC from Homo sapiens, Solanum tuberosum, Thermotoga maritima, and Aquifex aeolicus have been determined. Numerous crystal structures of human AdoMetDC and mutants have provided insights into the mechanism of autoprocessing, putrescine activation, substrate specificity, and inhibitor design to the enzyme. The comparison of the human and potato enzyme with the T. maritima and A. aeolicus enzymes supports the hypothesis that the eukaryotic enzymes evolved by gene duplication and fusion. The residues implicated in processing and activity are structurally conserved in all forms of the enzyme, suggesting a divergent evolution of AdoMetDC.
12809662
Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.
1952227
Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli. Determination of sites for putrescine activation of activity and processing.
Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.