Design of inhibitors of ODCase - Future Science

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Design of inhibitors of ODCase ODCase is a highly proficient enzyme responsible for the decarboxylation of orotidine monophosphate to generate uridine monophosphate. ODCase has attracted early attention due to its interesting mechanism of catalysis. In order to exploit therapeutic advantages due to the inhibition of ODCase, one must have selective inhibitors of this enzyme from the pathogen, or a dysregulated molecular mechanism involving ODCase. ODCase inhibitors have potential applications as anticancer agents, antiviral agents, antimalarial agents and potentially act against other parasitic diseases. A variety of C6-substituted uridine monophosphate derivatives have shown excellent inhibition of ODCase. 6-iodouridine is a potent inhibitor of the malaria parasite, and its monophosphate form covalently inhibits ODCase. A variety of inhibitors of ODCase with potential applications as therapeutic agents are discussed in this review. ODCase (EC 4.1.1.23), an enzyme discovered almost 50 years ago, was initially viewed only in the context of nucleotide biosynthesis, where it catalyzes the final reaction in the de novo biosynthesis of uridine monophosphate (UMP) (Figure 1) [1]. In the mid-1990s, however, it was discovered that ODCase is able to catalyze the decarboxylation of orotidine monophosphate (OMP) at extremely high rates compared with the conversion in its absence, popularizing the enzyme and leading to an increased interest in this enzyme [2,3]. ODCase is one of the most proficient enzymes known today [4]. The enzyme enhances the catalytic decarboxylation of OMP (1) to UMP (2) over 17 orders of magnitude compared with the conversion seen in water at neutral pH and ambient temperature, and is able to do so with no assistance from cofactors or metal ions [1,5]. In bacteria and parasites, ODCase is a monofunctional enzyme [6,7], while in humans it is part of the bifunctional enzyme, UMP synthase. ODCase’s sophisticatedly evolved mechanism of catalysis for decarboxylation has the enzyme a highly investigated subject [8,9]. The catalytic decarboxylation of OMP to UMP is a critical process within a cell. The triphosphate form of UMP is a precursor for the synthesis of RNA, as well as a precursor for the synthesis of other pyrimidine nucleotides. Pyrimidine nucleotides are also important building blocks for the synthesis of DNA. Thus, in most species, including humans, these pyrimidine nucleotides are obtained by two pathways, the de novo pathway and the salvage pathway. ODCase has a central role in the synthesis of pyrimidines as part of the de novo pathway and is therefore present in most species including

bacteria, parasites and higher level species, but not in viruses. Viruses depend on their host cells to supply them with nucleotides [10]. However, certain parasitic organisms, such as Plasmodia, are dependent solely on the de novo synthesis to obtain pyrimidine nucleotides because their cells are not equipped to utilize the salvage pathway [11]. When there is a high demand for pyrimidine nucleotides, such as during cell replication and abnormal cell growth, the de novo synthesis is upregulated [12,13]. Using ODCase inhibitors to limit or inhibit pyrimidine production in the cell could potentially elicit a variety of desirable biological activities including antiv iral, anti plasmodial and anticancer activities [14]. A handful of investigations in the past focused on developing ODCase inhibitors against cancer because of the important cellular roles of activated intermediates such as UDP-glucose and CDP-choline in biosynthetic pathways. Preclinical studies suggested that the therapeutic effects of 5-fluorouracil can be enhanced by pretreatment with agents such as phosphonacetyl-l-aspartic acid (PALA), an inhibitor of aspartate transcarbamylase. The objective of treatment with PALA was to increase the activation of 5-fluorouracil by inhibiting the normal pathway of de novo pyrimidine biosynthesis. However, this combination therapy led to mild to moderate myelosuppression, mucositis, diarrhea, nausea and vomiting [15]. Therefore, de novo pyrimidine biosynthesis, and ODCase in particular, is a potential and challenging target for developing novel therapeutics. One must note that ODCase requires that the nucleoside ligands carry a monophosphate group at the 5´-position, which is responsible for a significant amount of binding energy for the initial binding

10.4155/FMC.13.198 © 2014 Future Science Ltd

Future Med. Chem. (2014) 6(2), 165–177

Sourabh Mundra1,2 & Lakshmi P Kotra*1,3 Center for Molecular Design & Preformulations, & Toronto General Research Institute, University Health Network, Toronto, Ontario, M5G 1L7, Canada 2 Birla Institute of Technology & Science, Pilani, Rajasthan, 333031, India 3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Ontario, M5S 3M2, Canada *Author for correspondence: Tel.: +1 416 581 7601 E-mail: [email protected] 1

ISSN 1756-8919

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Review | Mundra & Kotra O O

O-

HN

O

PPi

PRPP

HN H3N

O-

L-aspartate

O

O O3PO

-2

+

O

N H Orotate

O

N

O

COOOrotate phosphoribosyl transferase

HN COO-

OH OH OMP (1)

H+

CO2 O3PO

-2

Orotidine monophosphate decarboxylase

N

O O

OH OH UMP (2)

Figure 1. De novo biosynthesis of uridine monophosphate from the amino acid, l-aspartate. Orotate phosphoribosyl transferase and OMP decarboxylase enzymatic functions are a part of one single enzyme, UMP synthase, in some species such as human, and others have these two enzymes as monofunctional enzymes. OMP: Orotidine monophosphate; UMP: Uridine monophosphate.

of the ligand to the catalytic site. However, such phosphorylated nucleosides, carrying a highly polar monophosphate group, would not be able to enter cells. Thus, for any biological activity in cell-based assays, one must either mask the polar groups or use the corresponding nucleoside forms and expect that cellular kinases would phosphorylate these nucleosides once they enter the cell. Due to this requirement, most of the cell assays have been carried out using the nucleoside forms of the inhibitors of ODCase, as is the typical practice for nucleoside drugs. This review will first discuss the mechanism of catalysis and the structure of the ODCase enzyme, leading into a discussion on multiple approaches to ODCase drug discovery, emphasizing the varied strategies that are likely to provide important new drugs in the future.

Key Terms Bifunctional enzyme:

Contiguous protein molecule carrying two catalytic sites with ability to conduct two distinct biochemical reactions.

De novo pathway: Synthesis of complex molecules from simple molecules in a cell.

Salvage pathway: Synthesis

of nucleotides from intermediates in the degradative pathway for nucleotides.

Direct decarboxylation:

Carboxylation of orotidine-5´monophosphate without involving a covalent intermediate, but induced by stress and a variety of other factors.

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Catalytic mechanism of ODCase Many investigative studies were undertaken based on chemical and biochemical rationale, x-ray crystal structures and computer simulations, in an attempt to elucidate the mechanism of catalysis of ODCase. Here, we catalog the main mechanisms that were proposed over the past few decades, although since 2000, there has been an intense research into confirming the most possible mechanism of decarboxylation by ODCase based on the x-ray structures of ODCase, mutants of ODCase, and through probe molecules. Beak and Siegel, proposed the ‘nitrogen ylide’ mechanism as a result of examining the non-enzymatic decarboxylation of 1,3-dimethylorotic acid and related compounds. They proposed that the reaction is initiated by substrate (OMP) protonation at O4 to yield a betaine structure with a positive charge localized at N-1 (Figure 2A) [16]. The positive charge then, by inductively stabilizing the adjacent vinylic Future Med. Chem. (2014) 6(2)

carbanion of the intermediate state, facilitates the decarboxylation reaction yielding UMP. A second mechanism, proposed by Silverman and Groziak, involves a Michael addition at the C5 position of the pyrimidine ring followed by decarboxylation (Figure 2B) [17]. Nucleophilic attack by the active site occurs at the C5 position of OMP followed by enzyme-mediated proton donation. This causes a change in the geometry at C5 from a trigonal (sp2) to a tetrahedral (sp3) center, which facilitates the decarboxylation/elimination of the trans intermediate to release UMP as the product. The basis for this mechanism was the observation that orotic acid analogs are susceptible to nucleophilic attack at C5. This proposal was later ruled out when Acheson and co-workers demonstrated a lack of any secondary deuterium isotope effect with the C5 hydrogen [18]. A mechanism involving direct decarboxylation of OMP was proposed after the x-ray structure of ODCase was resolved (Figure 2C) [9], based on x-ray structure analysis of various ODCase inhibitor complexes [19–22]. According to this mechanism, the decarboxylation is initiated by charge repulsion experienced by the carboxyl group on OMP and the Asp residues in the catalytic site of ODCase, followed by proton transfer from Lys73 to the C6 position of the pyrimidine moiety. The addition of the proton is able to destabilize the ground state and stabilize the transition state, which facilitates the decarboxylation of OMP. Computational models investigated by Warshel et al. suggest the ground-state destabilization further supporting this mechanism [23]. Gao and co-workers proposed that the proximity of the aspartate residue to OMP carboxylate, in the active site, produces a ‘ground-state destabilization’ effect that facilitates direct decarboxylation [24]. Recently, there has been more evidence future science group

Design of inhibitors of ODCase towards this mechanism, involving stress onto the carboxyl group at the C6 position of OMP and elimination of the CO2 molecule or direct decarboxylation facilitated through the stress [25]. Lee and Houk proposed a mechanism with a carbene intermediate for the enzymatic decarboxylation [26]. They based their mechanism on quantum mechanical calculations for the ground state and the energies of potential intermediates during OMP decarboxylation. They proposed that protonation at O4 gave a further stabilized intermediate that has both a zwitterionic and a neutral carbene resonance structure. In contrast to Beak and Siegel, this proposal predicts that a positive charge at N1 in OMP would not be important in the enzymatic reaction. This proposal could not be supported by any experimental means and is not widely accepted. Another proposed catalytic mechanism (F igure 2D) by Kollman assumes that Asp70 – one of the two Asp residues in the catalytic tetrad – is protonated [27], and predicts that the decarboxylation is initiated by an enamine protonation at the C5 position by Lys72 (according to the Methanobacterium thermoautotrophicum (Mt) ODCase numbering). Hur and Bruice, studying molecular dynamics simulations of the ground state and intermediate/transition states of the ODCase/OMP structure, provided additional support for the idea of stabilization of the O

O

N

+H + N

HO

O +H

HN O

N

Nu

O

O

-CO2 O

HN

+H N R I O

O

HN O

N R II

N R IV

N

O HN

+H

O

O

R III

O

R II

I O

HN

HN O

N

O

+ N

O

HN

O

O

HO

O

N

-CO2

HN

R II O

-CO2

O

R III

H

N

O

HN

R

Nu

HN

O

O

O

R II O

O

R I

O

HN

+H O

R I O

O

carboanionic intermediate by a nearby lysine and reorganization of the 203–218 loop [28]. In addition, they found that the intermediate and the transition state have similar associated energies, thus, the stabilization of the former reduces the free energy of the latter. Shostack and Jones proposed the Schiff base formation mechanism in which the active site lysine residue plays an important role [29]. However, the lack of 18O exchange of O4 with water seems to rule this out as a possible mechanism. Appleby and co-workers proposed a concerted mechanism for the decarboxylation. In this mechanism, C6 of OMP is protonated while the carboxylate bond is broken [30]. Accordingly, C6 would start forming a bond with hydrogen on the catalytic Lys residue in the active site, as the p-orbital of C6 turned to the e-ammonium group of the same catalytic lysine, creating the transition state. The carboxylate group is still loosely linked to the C6 while a gradual elongation of the C6–C7 bond occurs, facilitating the decarboxylation. This could explain the experimental results reported by Radzicka and Wolfenden [3]. However, recent studies have confirmed that concerted mechanism for decarboxylation can be ruled out, in favor of direct decarboxylation (vide infra) [31,32]. Jordan and Patel conducted a comparative study of three types of decarboxylases in the

O

HN

| Review

N R III

H

O H

-CO2

H

HN O

N R

O H

HN O

III

N R

IV

Figure 2. Proposed mechanisms of decarboxylation by ODCase. (A) Nitrogen-ylide mechanism, (B) nucleophilic addition mechanism, (C) direct decarboxylation, (D) C5-protonation mechanism.

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Review | Mundra & Kotra

Ligand binding site

3.2 Å

Asp141

3.4 Å 3.3 Å

2.9 Å Lys138

Lys102 Asp136

Figure 3. 3D structure of ODCase from Plasmodium falciparum (PDB code: 2ZA2). Catalytic tetrad is shown in a capped stick representation and the binding site is shown as a Connolly surface. Secondary structure of the ODCase backbone is shown in a cartoon representation. Hydrogen bonds between the functional groups in the catalytic tetrad are shown in broken lines in orange. Residue numbering is as per the P. falciparum ODCase sequence.

context of their catalytic mechanisms of decarboxylation, including ODCase, and highlighted the challenges in explaining the unusual catalytic mechanism of ODCase. While there are common features between decarboxylases such as pyridoxal phosphate-dependent decarboxylases and ODCase, there are some differential features with respect to substrate binding [5]. Using computational approaches, that is, hybrid quantum-molecular mechanics simulations, decarboxylation of OMP and a close analog, 5-fluoro-OMP were conducted. An integral role for Lys72, one of the two lysine residues in the catalytic tetrad, in stabilizing the transition state during the decarboxylation of OMP and the direct decarboxylation mechanism were confirmed [33]. Fujihashi et al. have recently determined a 1 Å-resolution structure of ODCase, including a Lys72 mutant, in complex with the product UMP. Their results strongly support the notion that electrostatic stress and substrate distortion contribute to the direct decarboxylation of OMP [25]. A relatively non-polar microenvironment within the active site in turn may also enhance the strength of electrostatic bonds. One must note that there are other effects such as 168

Future Med. Chem. (2014) 6(2)

strain on the pyrimidine ring, indirect effect of the role of 5´-monophosphate for the binding of the ligand and so on, which ultimately lead to the disruption of the electronic conjugation of the pyrimidine ring. Thus, the contributions of a number of these factors collectively explain the very high catalytic efficiency of this enzyme, which cannot be explained by any single factor. In a separate set of experiments, Gerlt, Richard and co-workers conducted ODCase-catalyzed deuterium exchange reactions between solvent D2O and 5-fluoro-UMP, UMP and the truncated fluorinated substrate FEU [34–37]. These experiments provide strong support for direct loss of CO2 molecule through a vinyl carbanion reaction intermediate. Structures of ODCase The function and enzymatic activity are intrinsically linked to the structure of the ODCase enzyme. To date, 152 ODCase x-ray crystal structures from both prokaryotic and eukaryotic organisms have been solved and appear in the Protein Data Bank [201]. This large collection of ODCase structures along with various mechanistic studies have led to a slow but steady increase of our understanding of the underlying biochemical mechanism for its catalytic activity. As described above, there have been many mechanisms proposed for the decarboxylation of OMP to UMP, and some uncertainty may still exist on the catalytic mechanism. x-ray crystallography of ODCase in complex with its various ligands helps in understanding the interaction between the ligands with the amino acids of the active site and as a result, the active site of this enzyme has been well mapped [22,38,39]. The catalytic site of ODCase consists of two aspartate residues (Asp136 and Asp141B, the latter contributed by the second subunit of the dimeric ODCase; Plasmodium falciparum (Pf ) ODCase numbering) and two lysine residues (Lys102 and Lys138) that are held in place by a strong network of hydrogen bonds (Figure 3) [22]. Analyses of several co-crystal structures of ODCase with a variety of ligands confirm that these residues are in close proximity to substitutions at the C6 position of the pyrimidine in various nucleotide ligands, including the product UMP [22]. The ribosyl moiety and the 5´-monophosphate are involved in an extensive hydrogen bonding network and the resulting binding energy between the enzyme and the ligands is the driving force to place the pyrimidine base into the active site pocket [22,25,39–41]. A recent study has shown that future science group

Design of inhibitors of ODCase purine nucleotides, in addition to pyrimidines, also inhibit ODCase, such as xanthosine monophosphate (XMP; 28), GMP, AMP and IMP [22,42]. Interestingly ODCases from diverse species exhibit remarkable differences in their affinity to various ligands. For example, XMP binds approximately 150-fold tighter to the plasmodial ODCase than to the human enzyme [39,42]. ODCase continues to attract attention due to its unusual catalytic efficiency, the discovery of a variety of ligands its active site can accommodate and the new twists it imparts onto the ligands. Along these lines, two cytidine-based analogs undergo quite an interesting rearrangement facilitated by ODCase within the catalytic site, providing ideas for novel inhibitors design [43]. Comparison of known structures of ODCase from various sources, namely bacteria, the malaria parasite and humans will illuminate the conserved residues in catalysis of this most proficient enzyme. Considering the vast repertoire of this structural information, this could be the subject of a separate article. However, this is important knowledge to explore the design of inhibitors targeting ODCase when a structure-based approach is undertaken for drug design. Nucleoside inhibitors of ODCase as anticancer agents Nucleosides and compounds bearing nucleosidelike features interfering with the pyrimidine biosynthesis pathway have shown interesting anticancer activities [44]. 5-fluorouracil and methotrexate are two such examples of drugs that are able to impair nucleotide synthesis in cancer cells, and thereby functionally reduce the uncontrolled proliferation of these cells [45]. During uncontrolled and fast replication of cells, the de novo pathway must also contribute toward supplying nucleotides despite the existence of the salvage pathway because of the higher demand for nucleotides in a short period of time [46]. 6-aza-uridine (17) and pyrazofurin (3) exhibited good anticancer activities against a number of clinical tumor models [47,48]. Pyrazofurin (3) is a C-nucleoside antibiotic that also blocks de novo pyrimidine synthesis by inhibiting ODCase. In vivo, it is converted to its 5´-phosphate derivative (4) and then engages in competitive inhibition of the ODCase enzyme (UMP synthase). The 5´-monophosphate derivative of pyrazofurin (4) was originally isolated from the broth filtrates of Streptomyces candidus [101] and is a potent inhibitor of ODCase with inhibition constants in the low nanomolar range future science group

| Review

(Figure 4) [49].

It has a breadth of clinically relevant biological activities and subsequently has been the focus of numerous investigations as an anticancer agent [50]. Pyrazofurin was shown to inhibit the growth of several solid animal tumors but has no effect on most transplantable carcinomas [51]. Pyrazofurin was considered a breakthrough molecule in late 1970s and early 1980s, and was considered for clinical development. However, pyrazofurin (3) had a limited antitumor effect with toxicity mostly to the oral mucosa in Phase I trials and was not pursued any further [52,53]. An x-ray crystal structure of the complex of pyrazofurin-5´-monophosphate (4) and human ODCase indicated a very strong network of hydrogen bonds between the ligand and the enzyme [10]. Interestingly, 4 is a competitive inhibitor of Mt ODCase (K i = 6.2 ± 0.6 µM), O H2NOC

RO

HN

NH N

HO O

HO

O

R

HO

O

N NH

H2NOC

OH

8 R = COOMe 9 R = CONH2

HN N

NH

O

HO

CONH2

CONH2

O

OH OH

OH OH

N N H

7

5 R=H 6 R = PO3 2-

CONH2

OH

OH OH

OH OH

3 R=H 4 R = PO32-

HO

I

O

OH OH

S

N

O RO

OH

10

OH

11

O NH

N O HO

N

N

NH2

O Br

OH

N H

OH

12

H N N

N

O

NH2

O

HO

CONH2

14

13

NO2 NO2 COOMe

MeOOC N H

15 (Nifedipine)

O

O

O

O

OMe

N H 16 (Nimodipine)

Figure 4. Structures of nucleoside-like inhibitors of ODCase and non-nucleoside ODCase inhibitors. (A) Nucleoside-like inhibitors of ODCase, (B) non-nucleoside ODCase inhibitors.


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Review | Mundra & Kotra Table 1. C6 substituted and C5 fluorinated uridine monophosphates that exhibit potent ODCase inhibition and anticancer activities. Compound number

Compound

18

6-aza-uridine-5´-Omonophosphate 5-fluoro-6-azidouridine-5´-Omonophosphate 5-fluoro-6-aminouridine-5´-Omonophosphate 5-fluoro-6-ethyluridine-5´-Omonophosphate 6-azidouridine -5’-O-monophosphate 6-aminouridine -5’-O-monophosphate

19 20 21 23 24

Mt ODCase Ki (µM) Mt ODCase

Hs ODCase

11

NA

NA

0.36 ± 0.03

16.60 ± 0.70

11.40 ± 0.60

0.35 ± 0.01

29.00 ± 0.30

NA

0.20 ± 0.10

NA

0.84 ± 0.02

Enzyme kinetics conducted in different laboratories may have used distinct experimental conditions, and comparisons must be made with care. Hs: Homo sapiens; Mt: Methanobacterium thermoautotrophicum; NA: Not available. Reproduced with permission from [47] © American Chemical Society (2009).

but inhibits human ODCase, as a slow tightbinding inhibitor with nanomolar potency (estimated K i = 17 nM) [10]. 6-aza-uridine (17) is also converted into its mononucleotide form in vivo, 6-aza-uridine-5´-O-monophosphate (18), and the resulting mononucleotide inhibits ODCase, impairing the de novo production of pyrimidine nucleotides [54]. This compound inhibits yeast and Mt ODCases with the inhibition constants (K i) of 64 nM and 11 µM, respectively (Table 1) [55,56]. Kotra and co-workers have developed a variety of C6 substituted 5-fluoro-UMPs derivatives that exhibit potent ODCase inhibition and evaluated their anticancer activities (Table 1) [46]. These compounds carried small substitutions at the C6 position of the 5-fluoropyrimidinyl backbone, including cyano, azido, amino, iodo, N-methylamino, N,N-dimethylamino, aldehyde, ethyl ester and hydroxyl amino moieties. The inhibition of Mt ODCase by various inhibitors in Table 1 were evaluated in a competitive inhibition assay and the reversible inhibition of human ODCase was studied at 37°C using isothermal titration calorimetry, a novel method developed by Kotra and co-workers [56]. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine5´-monophosphate. The presence of the fluorine moiety at C5 of uridine or UMP was expected to offer a stabilizing effect to the C6 substitutions as well as generate an electrophilic center at C6 due to the electronegativity of the fluorine moiety [46]. These mononucleotide derivatives were evaluated for ODCase inhibitory activities, 170


while the corresponding nucleoside derivatives were evaluated in the cellular assays. The parent compound, 5-fluoro-UMP was a very weak inhibitor of ODCase, but 6-azido-5-fluoro (19) and 5-fluoro-6-iodo derivatives were covalent inhibitors of ODCase, akin to their structural analogs without the fluorine moiety. The active site residue Lys145 (human ODCase) covalently binds to the ligand after the elimination of the 6-substitution, which is clearly seen in the cocrystal structure of the ligand–enzyme complex, supporting the enzyme kinetics [46]. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro, 6-amino-5-fluoro and 6-carbaldehyde-5-fluoro (22) derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. Specifically, 6-azido, 6-amino, 6-ethyl and 6-aldehyde derivatives of 5-fluorouridine exhibited potent anticancer activities in cell lines of hematopoietic origin, including leukemias (acute myelogenous leukemia lines OCIAML-1 and OCI-AML-2, and T-cell leukemia line, SKW3), lymphomas (non-Hodgkin’s B cell lymphoma-OCI-Ly-7), and multiple myeloma (OCI-My-2), with IC50 values in the range of 0.3–2.1 µM. Adherent cancer cell lines, including breast cancer lines MCF7 and MDA468, were generally less sensitive to these derivatives, with IC50 values ranging between 1 and 31 µM [46]. There appears to be some selectivity towards cancer lines when compared with the cytotoxicity of these compounds to concanavalin A-activated healthy human PBMC cells, offering the possibility that ODCase inhibitors could have potential as anticancer agents. ODCase (i.e., UMP synthase in humans) as an anticancer target will need to be further explored, but there is a prospect to design inhibitors to this enzyme to inhibit cancer cells due to its importance in pyrimidine biosynthesis. Potent inhibitors of de novo pyrimidine biosynthesis may have limited value as anticancer drugs because of the important cellular roles of activated intermediates such as UDP-glucose and CDP-choline. Consistent with this, PALA and pyrazofurin are clinically too toxic to be used as anticancer drugs. Nucleoside inhibitors of ODCase as antivirals Viral infections are unique since the invading virus becomes a component of the host cell, integrating with and utilizing the host cell’s biosynthetic machinery. In this aspect, the chemical modification of nucleosides has become one future science group

Design of inhibitors of ODCase of the most important chemical strategies to design antiviral agents. However some of these agents have targeted host enzymes rather than viral-specific enzymes. ODCase thus has been a potential target for drugs directed against RNA viruses such as pox and flaviviruses, and potentially for antibacterial drugs [57–59]. Pyrazofurin (3) was evaluated as an antiviral agent for respiratory syncytial virus, influenza, vaccinia, West Nile virus and other RNA viruses [60–63]. Pyrazofurin (3) and its a-epimer, pyrazofurin B (7) exhibit a broad spectrum of antiviral and antitumor activities at low concentrations, but also show significant toxicity [59–66]. Reduced toxicity was observed by replacing the pyrazole ring in pyrazofurin with a thiophene ring [64] in such compounds as methyl-5-carboxamido4-hydroxy-3-(b-d-ribofuranosyl)-2-thiophene carboxylate (8) and its corresponding 2,5-thiophene dicarboxamide derivative (9), two new analogs of pyrazofurin (Figure 4). Certain nucleoside analogs have been found to exhibit an ability to stimulate the immune system, which is important for fighting viruses. For example, 8-substituted (8-bromo and 8-mercapto) guanosines and phenyl-substituted pyrimidinones possessing a guanine-like functionality can activate B cells and natural killer cells, induce interferons and exhibit antiviral activity in vivo [67–74]. Another related compound, 7-methyl-8-oxoguanosine (12), is also reported to be a B-cell activator [75]. In these series of compounds, the important molecular configurations for this activity appear to reside in the pyrimidine portion of the molecule (resembling guanosine) and in the substitutions at the 7 and 8 positions on the guanine moiety. Wicker et al. have suggested the role of a biochemical pathway common to several immune cell types in immune cell activation [68]. Interestingly, adenosine- or inosine-like structures do not appear to be immuno potentiators. 2-amino-5-bromo6-phenyl-4-pyrimidinone or bropirimine (13) is one such compound that is extensively studied and exhibits antiviral activity. The pyrazolebased nucleoside 3-(2-hydroxyethoxymethyl] pyrazole-5-carboxamide (14), which is an analog of 4-deoxypyrazofurin-3-(b-d-ribofuranosyl)pyrazole-4-carboxamide (10), also possesses antiviral activity (Figure 4) [71]. Nucleoside inhibitors of ODCase as antimalarials Malaria is caused by Plasmodia parasites, specifically Pf, Plasmodium vivax and Plasmodium future science group

| Review

ovalae, and increasingly Plasmodium malariae and Plasmodium knowlesi as the causative agents for human malaria cases [76,77]. The de novo pathway for the biosynthesis of pyrimidine nucleotides in Plasmodia provides essential precursors for the synthesis of DNA and RNA. Unlike the cells of its human host, Plasmodia are unable to salvage preformed pyrimidines from the extracellular environment and therefore, are restricted to using the de novo pathway for pyrimidine nucleotide synthesis, the only one operational inside the parasite cell [78,79]. Since mature human red blood cells lack the de novo pyrimidine pathway [80], inhibiting this pathway could selectively kill the parasites that infect these red blood cells. The de novo pathway includes six enzyme-catalyzed reactions, the last of these being the ODCase catalyzed decarboxylation to produce UMP. ODCase in fact is a very attractive target for designing antimalarial agents due to the essential nature of this enzyme for Plasmodia [81–83]. A number of C6 substituted UMPs were synthesized using the principles of bioisosterism (Table 2). A structure–activity relationship study with strategically placed groups at the C6 position of uridine and UMP, such as 6-iodo, 6-cyano, 6-azido, 6-amino, 6-methyl, 6-N-methyl and 6-N,N-dimethyl groups revealed interesting findings about the binding of these nucleotides at the Table 2. Inhibition kinetics of C6-substitued uridine monophosphate derivatives and xanthosine monophosphate against Plasmodium falciparum ODCase. Compound number Compound

Pf ODCase Ki (µM)

2

UMP 6-aza-UMP Allopurinol-MP Pyrazofurin-5´-O monophosphate

210 ± 10 0.012 ± 0.003 0.24 ± 0.02 0.0036 ± 0.0007

[44]

6-azido-UMP 6-amino-UMP 6-methyl-UMP 6-cyano-UMP Xanthosine 5´-O monophosphate 6-iodo-UMP CMP-N3-oxide 6-NHMe-uridine -5’-O-monophosphate 6-N(Me)2-uridine†

2.0 ± 0.1 2.1 34.1 ± 0.4 26 ± 2 0.0044 ± 0.0007

[87]

18 25 4 23 24 26 27 28 6 29 30 31

Ref. [39] [39] [39]

[87] [87] [44,87] [39] [39]

6.2 ± 0.7 22.1 ± 3.2 28.5 ± 8.9

[44]

31.7 ± 10.0

[87]

[87]

Enzyme kinetics conducted in different laboratories may have used distinct experimental conditions, and comparisons must be made with care. † This molecule is tested in its 5’-O-monophosphate form against Pf ODCase. Pf: Plasmodium falciparum; UMP: Uridine monophosphate.


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Review | Mundra & Kotra Key Term Promiscuous: Ability of

ODCase (or any other enzyme) to facilitate biochemical reactions other than its native decarboxylation, and also its ability to bind to molecules with a variety of structures as ligands.

catalytic site of ODCase, and the antimalarial activities of the corresponding nucleoside analogs in Pf cultures [84,85,201]. 6-iodo-UMP (6) and 6-azido-UMP (23) covalently inhibit ODCase, while 6-cyano-UMP (27) and 6-amino UMP (24) are competitive inhibitors [42,86,87]. 6-aza-UMP (18) is a moderate inhibitor of Mt ODCase with a K i of 12.4 ± 0.7 µM, but it inhibits Pf ODCase with higher potency (K i = 1.1 ± 0.03 µM) (F igure 5) . 6-cyano-UMP (27), an apparent competitive, non-covalent inhibitor, inactivates Pf ODCase with a K i of 29 ± 2 µM (Mt ODCase, 26 ± 2 µM), but against P. vivax ODCase it exhibits a weaker inhibition with a K i of 62 ± 5 µM [57,85,88]. However, in time-dependent studies, upon incubation of 6-cyano-UMP (27) with Pf ODCase for several hours, barbiturate-5’-monophosphate (BMP) is generated and this transformation also occurs with Mt ODCase [86,88]. The inhibition constant (Ki) for 6-methyl-UMP (26) is O

O

HN N

RO

N

O3PO

OH

O

17 R = H 18 R = -PO32-

O3PO

N

OH

OH

O3PO

2-

O

2-

O3PO

27

28

OH OH 25 O

NH N H

O

NH2 N

O

O3PO

2-

N

O OH

OH

29

O

HN

HN N

NHCH3

O HO

O OH

O OH OH

OH OH

O

O3PO

N

CN

N

O

O N

O

26

O

O3PO

24

N

N

N 2-

OH OH

OH

O

NH2

O

HN N

HO N

O

OH OH

2-

O3PO

23 HN

O OH OH 21

N

O 2-

O

O3PO

N3

O

22

2-

OH

C2H5

N

HN

O 2-

O

O

O3PO

2-

O

HN

CHO

NH2

O

O

N

O

N

OH

F

HN

20

F

HN

OH

O3PO

19

O

HO

O 2-

OH OH

OH

O

N3

N

O

F

HN

O 2-

O

O

F

HN

O

a competitive inhibitor of ODCases from Pf and P. vivax with moderate potency (K i = 34.1 ± 0.4 and 22.4 ± 0.7 µM, respectively). 6-amino-UMP (24) inhibited Mt ODCase competitively with a K i of 840 ± 25 nM, but showed slightly less potency against Pf ODCase (K i ~2.1 ± 0.0 µM) [85]. When evaluated as a noncovalent inhibitor, 6-azido-UMP (23) is a good inhibitor of Mt and Pf ODCases (K i = 0.19 ± 0.01 and 2.0 ± 0.1 µM, respectively). However, in time-dependent assays 6-azido-UMP (23) inactivated Mt and Pf ODCases with first-order rates of inactivation (kinact) of 61.2 and 0.35 h-1, respectively. By comparison, 6-iodo-UMP (6) inhibited Pf ODCase with a second-order rate constant (k obs/[I]) of 680,000 M-1sec-1; 6-azido-UMP (23) inhibited this enzyme with the second-order rate constant (kinact/K I) of 13.2 M-1sec-1, clearly indicating the superior inactivation of ODCase by 6-iodo-UMP (6) [85,88]. In certain cases, kinact /K I could not be

OH

N

N(CH3)2

O OH

30

OH

31

Figure 5. Various nucleoside inhibitors of ODCase with antimalarial and anticancer activities.

172



Design of inhibitors of ODCase derived and the second-order rate constant is estimated to be kobs /[I], and were used for comparative purposes only. Derivation of these parameters is pertinent to the type of irreversible inactivation that a compound exhibits. kobs /[I] (slope of a linear fit) indicates one-step inactivation without the reversible enzyme–inhibitor complex formation, where as kinact /K I is derived from a non-linear (hyperbolic) profile indicating the presence of reversible complex formation before the irreversible covalent bond is formed. This discussion is elaborately included in the reference [46] in the context of ODCase inhibitors, and a general discussion of the enzymology can be gleaned from reference [88]. Several 6-substituted uridine derivatives were evaluated in cell-based assays as potential antimalarial agents. 6-N-methylamino uridine and 6-N,N-dimethylamino uridine (31) were moderate inhibitors of Pf with IC50 values of 28 ± 9 and 32 ± 10 µM, respectively. 6-methyluridine exhibited very weak activity against the Pf 3D7 isolate (IC50 of 530.5 ± 0.1 µM) although its 5´-monosphosphate derivative (26) is a potent inactivator of ODCase. 6-methyluridine did not show toxicity to CHO cells (IC50 > 1.2 mM). 6-N-methylamino uridine and 6-N,N-dimethylamino uridine (31) inhibited CHO cells with IC50 values in the range of 68–72 µM (Figure 5). Interestingly, 6-iodouridine (5), the most potent inhibitor of Pf ODCase, other than BMP, exhibited good antimalarial activity against several strains of Pf ranging from 1.2 ± 0.2 to 56.5 ± 14.0 µM including mefloquine-resistant and chloroquine-resistant isolates [84,90]. 6-iodouridine (5) inhibited drug-resistant isolates ItG, and KC98 at low micromolar concentrations, while multidrug resistant isolates such as MB and SB were less susceptible to 6-iodouridine (5). Interestingly, the human parasite Pf exhibited much higher sensitivity, almost 25-fold higher, than the rodent parasites Plasmodium berghei and Plasmodium chabaudi chabaudi, to 6-iodouridine (5). This compound did not generate rapid drugresistant populations of the parasite in vitro [89]. 6-iodouridine (5) was evaluated in combination with artemisinin and azithromycin, in malaria mouse models, and showed good efficacy in vivo with artemisinin and azithromycin. 6-iodouridine exhibited an additive effect in combination with artemisinin in vitro, and in an in vivo study; a drop in parasitemia was observed during treatment but a sharp ‘rebound’ in the parasitemia was observed when the drug treatment was stopped. Overall, however, the combination of 6-iodouridine and artemisinin showed a beneficial outcome in this future science group

| Review

study [89]. A combination of 6-iodouridine with azithromycin indicated that the combination is significantly better than either treatment alone and there was no ‘rebound effect’ observed due to the longer half-life of azithromycin than that for artemisinin [89]. Interestingly, supplementation of uridine when treated with 6-iodouridine reduced any drug toxicities to the mouse, thereby facilitating the administration of higher doses of the drug to achieve higher efficacy. This validates the mechanism of these ODCase inhibitors that pyrimidine nucleosides can be salvaged in the host cell but not by the malaria parasite [89], which is an important observation useful for the development of this molecule as a potential antimalarial agent. Investigations into the utility of ODCase inhibitors targeting other parasitic infections are still at an early stage. During a recent study using Trypanosoma brucei ODCase, it was shown that this enzyme may not be the primary target for pyrazofurin-5´-monophosphate (4) and the organism can still survive when UMP synthase is not expressed [90]. However, the parasite virulence is lost when a double-knockout mutant of UMP synthase from T. brucei was generated, and this mutant regained virulence after prolonged culture implying its ability to survive and evolve using the salvage pathway for obtaining UMP through the host cellular machinery [90]. Non-nucleoside inhibitors for ODCase Two non-nucleoside inhibitors, nifedipine (15) and nimodipine (16; Figure 4B), clinically used as calcium channel blockers, have been reported to inhibit ODCase competitively, with inhibition constants (K i) of 105 and 18 µm, respectively. Although ODCase may have evolved to carry out the decarboxylation of OMP with high efficiency, it appears to be an inherently promiscuous enzyme, binding to other nucleosides, both purines and pyrimidines, as well as to non-nucleoside inhibitors [10,91]. Based on the crystal structure of the complexes of various ODCase inhibitors such as BMP, pyrazofurin-5´-monophosphate (4), 6-cyanoUMP (27), 6-aza-UMP (18) as well as non-nucleoside inhibitors, Meza-Avina et al. proposed an empirical pharmacophore describing the characteristics of potent inhibitors to ODCase [10]. According to this model, three separate regional characteristics were identified on the pharmacophore that various compounds should possess as inhibitors of ODCase. These general principles help simplify the complex network of interactions between ODCase and its various ligands. www.future-science.com

173

Review | Mundra & Kotra Future perspective There is strong support that ODCase is a good drug target and a potentially druggable target to design potent inhibitors. With the availability of a large set of x-ray crystal structures, many in complex with ligands, ODCase provides an opportunity for new drug discovery. Due to its essential role in pyrimidine biosynthesis in a variety of organisms’ genomes, such inhibitors could be developed for a variety of clinical indications. ODCase inhibitors have shown promise as potential drug candidates against Plasmodia parasites, although this mechanism could also be applicable in other parasitic diseases and remains to be explored. Development of ODCase inhibitors as anticancer agents may have some merit, especially against fast-growing cancers because cancer cells would depend on the de novo supply of pyrimidines for their fast division. It is important to consider the fundamental nucleotide biosynthesis and the role of inhibition of ODCase in affecting the targeted

disease condition for any clinical development, because nucleotide biosynthesis and its integrity have evolved differently across various species. Acknowledgement The authors are grateful to EF Pai for discussions on the mechanisms of decarboxylation by ODCase. The authors acknowledge W Wei for the preparation of Figure 3.

Financial & competing interests disclosure The authors wish to thank ISTP Canada (LPK; Grant #ICRD08–15) and the Department of Biotechnology, Ministry of Science and Technology, Government of India for financial support. LP Kotra has financial interest in the company that licensed the disclosures in patent number US8067391. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Background ODCase is an important component of the de novo nucleic acid biosynthesis pathway. Catalytic mechanism

There is strong evidence that ODCase decarboxylates orotidine monophosphate via direct decarboxylation imparting stress onto the substrate, that is, the C6 carbanion-based transition-state intermediate appears during the early stage of the reaction, and a variety of stress, including electrostatic, appears to play a role in the process of decarboxylation of the substrate. Nucleoside inhibitors

C6-subsituted derivatives such as 6-azido-uridine monophosphate and 6-iodo-uridine monophosphate are covalent inhibitors of ODCase. Structures of ODCase

A plethora of 3D structures of ODCase and numerous studies on the inhibition mechanisms provide ample opportunity to design novel inhibitors to ODCase. Nucleoside inhibitors as anticancer agents

C6-substituted 5-fluorouridine derivatives and other similar derivatives may possess useful anticancer activities, targeting ODCase. Nucleoside inhibitors of ODCase as antimalarials

A number of C6-substituted uridine monophosphate derivatives with small substitutions exhibit potent antiplasmodial activities. 6-iodouridine is a potential antimalarial agent with a novel mechanism of action.

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