Immucillins in Infectious Diseases - ACS Publications - American

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Immucillins in Infectious Diseases Gary B. Evans, Peter C. Tyler, and Vern L. Schramm ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00172 • Publication Date (Web): 19 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

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Immucillins in Infectious Diseases Gary B. Evans,1 Peter C. Tyler1 and Vern L. Schramm2* 1

Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Road, Gracefield, Lower Hutt, New Zealand, 5010 2

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 *Corresponding author. Telephone 718-430-2813. Email [email protected]

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Abstract: The Immucillins are chemically stable analogues that mimic the ribocation and leaving-group features of N-ribosyltransferase transition states. Infectious disease agents often rely on ribosyltransferase chemistry in pathways involving precursor synthesis for nucleic acids, salvage of nucleic acid precursors or synthetic pathways with nucleoside intermediates. Here we review three infectious agents and the use of the Immucillins to taget the disease agent. First: DADMe-Immucillin-G is a purine nucleoside phosphorylase (PNP) inhibitor that blocks purine salvage and shows clinical potential for treatment for the malaria parasite Plasmodium falciparum. Plasmodium falciparum is a purine auxotroph requiring hypoxanthine for purine nucleotide synthesis. Inhibition of the PNPs in the host and in parasite cells leads to apurinic starvation and death. Second: Helicobacter pylori, a causative agent of human ulcers, synthesizes menaquinone, an essential electron transfer agent, in a pathway requiring aminofutalosine nucleoside hydrolysis. Inhibitors of the H. pylori methylthioadenosine nucleosidase (MTAN) are powerful antibiotics for this organism. Synthesis of menaquinone by the aminofutalosine pathway does not occur in most bacteria populating the human gut microbiome. Thus, MTAN inhibitors provide high-specificity antibiotics for H. pylori, and are not expected to disrupt the normal gut bacterial flora. Third: Immucillin-A was designed as a transition state analogue of the atypical PNP from Trichomonas vaginalis. In antiviral screens, Immucillin-A was shown to act as a prodrug. It is active against filoviruses and flaviviruses. In virus-infected cells, Immucillin-A is converted to the triphosphate, incorporated into the viral transcript and functions as an atypical chain-terminator for RNA-dependent RNA polymerases. Immucillin-A has entered clinical trials for use as an antiviral. We also summarize other Immucillins that have been characterized in successful clinical trials for T-cell lymphoma and gout, supporting the potential development of the Immucillins reviewed here in infectious diseases.

Key Words: malaria antibiotics, antivirals, species specific antibiotics, futalosine pathway, purine-less death, Immucillin-A, DADMe-Immucillin-G, RNA chain termination, purine nucleoside phosphorylase, methylthioadenosine phosphorylase, RNA-dependent RNA polymerase

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Introduction and background: Immucillins are chemically stable inhibitor molecules with features similar to the transition states of N-ribosyltransferases. They have been used to target disease-related enzymes in humans, protozoan parasites, bacteria and viruses (Figure 1). Several Immucillins have entered clinical trials. Examples are provided by Immucillin-H (aka BCX1777, Forodesine and Mundesine®) which is approved for resistant and relapsed peripheral T-cell lymphoma (PTCL) in Japan; Immucillin-A (aka BCX4430 and Galidesivir), which is currently in phase I trials as an antiviral; and DADMe-Immucillin-H (aka BCX4208 and Ulodesine), which has completed phase II trials for gout.1-8 Other Immucillins are in preclinical studies for malaria, solid tumors and antibacterial indications (Figure 1).

Figure 1. Clinical and preclinical applications for the Immucillin family of transition state analogues. Immucillin-H is 1 2 approved in Japan. Immucillin-A (BCX4430; Galidesivir) is a prodrug antiviral in phase I trials. DADMe3 Immucillin-H (Ulodesine) has completed phase II trials for gout. DADMe-Immucillin-G is an effective antimalarial in 4 a primate model for malaria. MT-DADMe-Immucillin-A is effective in mouse xenograft models of human 5,6 7,8 cancers. BT-DADMe-Immucillin-A and related analogues are powerful antibiotics for Helicobacter pylori.

This review emphasizes the Immucillins as emerging candidates for drug therapy in three classes of infectious disease organisms. Thus, DADMe-Immucillin-G inhibits purine nucleoside phosphorylase (PNP) both in Plasmodium falciparum and in its host erythrocytes to prevent hypoxanthine formation, an essential step in the purine salvage pathway for the parasite. This treatment causes parasite death in culture and in animal models.4 DADMe-Immucilin-G is orally available and clears P. falciparum infections in Aotus monkeys, a primate model for human disease. No toxicity has been observed at effective pharmacological doses. 3 ACS Paragon Plus Environment

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Most enteric gut bacteria, including Escherichia coli, synthesize menaquinone from chorismate in a well-established pathway of seven steps catalyzed by the MenA-G proteins.9 More recently, a few bacterial species including Helicobacter pylori, Campylobacter jejuni, Acidothermus cellulolyticus and Streptomyces coelicolor have been found to lack the genes for this pathway. Instead, enzymes encoded by the Mqn genes convert chorismate into menaquinone using 6-amino-6-deoxyfutalosine as a pathway intermediate.10-12 Elucidation of the pathway enzymes indicated the requirement for an Nribosyltransferase to convert 6-amino-6-deoxyfutalosine into a ribosyl-linked precursor of futalosine. Discovery of this reaction suggested the possibility of species-specific antibiotics. Thus, enzyme-specific inhibitors for the the 6-amino-6-deoxyfutalosine pathway would kill the species mentioned above, while sparing bacteria with the MenA-G pathway. Species specificity has been achieved in vitro with Immucillin transition state analogues against the 6-amino-6-deoxyfutalosine hydrolase from H. pylori.7,8 Inhibitors of the 6-amino-6-deoxyfutalosine hydrolase are described with IC90 values in the ng/mL range, orders of magnitude more effective than antibiotics currently approved for clinical treatment of gastric ulcers of H. pylori origin.13 Our third example of Immucillin application to infectious disease is the use of Immucillin-A as an antiviral for RNA-dependent RNA polymerase viruses, including the filoviruses.2 Immucillin-A was originally developed as a potential antibiotic against Trichomonas vaginalis.14 T. vaginalis is a purine auxotrophic protozoan and the cause of trichomoniasis, the most common curable sexually transmitted infection in the United States. The PNP expressed in Trichomonas vaginalis includes adenosine in its substrate specificity, different from the human PNP. Synthesis of Immucillin-A was proposed as a parasite-specific analogue. Immucillin-A is a picomolar inhibitor, but did not kill cultured parasites. T. vaginalis expresses redundant purine salvage enzymes, making PNP non-essential. However, a screen of virus susceptibility to Immucillin-A at the cellular level found antiviral activity against a broad range of viruses, including filovirus. The mechanism of action has been reported to be chain-termination of the product of viral RNA-dependent RNA polymerase. Antiviral activity includes the Ebola and Marburg filoviruses as well as the Yellow Fever and Zika flaviviruses. Human phase I safety trials are underway.2 Nucleoside analogues used historically as antibiotics frequently act as prodrugs for conversion to active nucleoside triphosphates.15,16 The triphosphates are inhibitors of DNA or RNA polymerases, preferably with specificity for the targeted organism. Nucleoside analogues missing the ribosyl 3'hydroxyl group are chain terminators of nucleic acid synthesis.17-19 Many natural product nucleoside antibiotics have been discovered by cell inhibitor screening, or more recently by genomic interpretation, and can inhibit cell wall biosynthesis, protein translation or enzymes of nucleic acid synthesis.20,21 The Immucillins differ from the usual ribosyl or base modifications of nucleoside antibotics, as they are produced by directed chemical synthesis to be transition state analogues against specific targets.22,23 The inhibition potential of transition state analogues is enhanced by the picomolar affinity commonly attained with transition state analogues, as demonstrated by the examples of inducing purine starvation in P. falciparum and inhibition of the menaquinone pathway in H. pylori. Protozoan purine nucleoside phosphorylase and malaria Metabolism—P. falciparum live in a metabolite-rich environments in human cells and are purine auxotrophs.24 Purine precursors for nucleic acid synthesis are obtained through salvage pathways using 4 ACS Paragon Plus Environment

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Figure 2. Purine salvage pathways in Plasmodium falciparum in the environment of the human erythrocyte. Human (h) and P. falciparum (Pf) enzymes are adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT), IMP dehydrogenase (IMPDH), adenylosuccinate synthetase (AdSS), AMP deaminase (AMPDA), adenylosuccinate lyase (AdSL), adenylate kinase (AK), adenine phosphoribosyl transferase (APRT), and GMP synthetase (GMPS). 5’-Methylthioadenosine (MTA) is a product of the polyamine pathway and 5’-methylthioinosine (MTI) is formed in a pathway unique to the parasite. Membrane transport proteins are indicated by shaded ovals. Transport of AMP into P. falciparum 34 occurs only upon adenosine loading to elevate AMP beyond physiological concentrations. Reprinted from reference 4, PLoS One. 6 (11), e26916 (2011), under open access license CC-BY.

enzymes that are similar to those used by their mammalian hosts, with a few exceptions. The purine nucleoside phosphorylases (PNPs) are purine salvage enzymes common to protozoan parasites with homologs also found in the human host. PNPs catalyze the phosphorolysis of 6-oxypurine nucleosides and 2'-deoxynucleosides to yield the nucleobase and ribose or 2-deoxyribose 1-phosphates. The purine base is available for salvage by phosphoribosyltransferases, both in the host and the parasite.25-27 The purine phosphoribosyltransferase from P. falciparum accepts hypoxanthine, guanine and xanthine, in decreasing order of physiological significance.28,29 Their product is purine nucleoside monophosphates and these serve as precursors for conversion to all purines as RNA and DNA precursors (Fig. 2). In P. falciparum, hypoxanthine is the central metabolite, as blocking the production of hypoxanthine formation in vitro or in vivo causes death of the parasites by purine starvation.4,30 The relative fluxes through purine salvage pathways dictate their essential contributions to cell survival. In some protozoa, including T. vaginalis, alternative purine salvage pathways with specificity overlapping that of PNP make it an ineffective drug target.31-33 5 ACS Paragon Plus Environment

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PNP Enzymology—Knowledge of the purine salvage pathways in P. falciparum led to the hypothesis that blocking PNP would prevent formation of hypoxanthine. Hypoxanthine is the most important source of purines in this purine auxotroph and is essential for formation of cofactors and nucleic acids.35 However, there are two sources of hypoxanthine in infected erythrocytes since both human (hPNP) and parasite (PfPNP) PNPs contribute to the hypoxanthine pool. Both erythrocytes and parasites have transport proteins for hypoxanthine.36 The anti-malaria design goal was to produce an inhibitor that is effective against both PNP targets. The hPNP and PfPNP differ considerably in structure and function (Figure 3). The human enzyme is a homotrimer, the PfPNP is a hexamer, and the catalytic rates (kcat values) are different, 56 s-1 and 0.34 s-1 for the human and PfPNPs, respectively.37

B

-1

kcat = 56 s

kcat = 0.34 s

-1

Figure 3. Human and P. falciparum PNP enzyme subunit structures and catalytic efficiencies. Inhibition of the homotrimeric human PNP (A) occurs when inhibitor binds at the first catalytic site, rendering the remaining sites inactive. The six catalytic sites of PfPNP (B) act independently. Both enzymes convert inosine and phosphate to hypoxanthine and ribose 1-phosphate. PNP structures are from PDB files 3K8Q (human) and 1NW4 (P. falciparum).

Transition state structure studies revealed the hPNP and PfPNP enzymes to have similar fullydissociated (SN1-like) ribocation character.38 Knowledge of the transition states led to the synthesis of ribocation-like transition state mimics. Synthetic efforts directed toward mimics of these transition states gave analogues with similarity to the transition state and consequently, tight binding to both enzymes (Figure 4). A feature of transition state analogues is their ability to convert the enzyme potential for catalysis into binding energy. Thus, the more catalytically efficient the enzyme, the more 6 ACS Paragon Plus Environment

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tightly transition state analogues are expected to bind.39 This principle for transition state analogue affinity is apparent in the inhibitors for human and PfPNPs. Without exception, binding is tighter to hPNP, despite their catalysis of the same reaction and the similarity of their transition states (Figure 4).40 Among these inhibitors, several are highly suitable as potential anti-malaria agents by virtue of their 7H 5 N

HO

5'

H N

4' 3'

O NH

8 1' 2'

9

4

HO

2

N 3

NH2

N

HO

HO

HO Ki* = 140 ± 10 pM Ki* = 2200 ± 100 pM Hu 16-fold

Ki* = 42 ± 6 pM Ki* = 900 ± 200 pM Hu 21-fold

O

H N

NH HO

N

N

O

H N

NH NH2

N

N HO

O NH

N

HO N

H N

N 8-azaDADMe-ImmH

Ki* = 2000 ± 100 pM Ki* = 5500 ± 200 pM Hu 3-fold

MeS N HO

NH

5'-d-5'-MeDADMe-ImmH

HO

Ki* = 7 ± 1 pM Ki* = 890 ± 60 pM Hu 130-fold

Ki* = 16 ± 1 pM Ki* = 500 ± 40 pM Hu 31-fold

O

N

N

DADMe-ImmG

DADMe-ImmH HO

H N

N 2'-d-ImmH

HO OH

H N

NH

H N

ImmG

Ki* = 56 ± 15 pM Ki* = 860 ± 80 pM Hu 15-fold

O

H N

NH

H N

ImmH

HO OH

HO

O

H N

6 1

Ki* = 360 ± 30 pM Ki* = 1600 ± 400 pM Hu 4-fold

O

H N

NH S

N

N

5'-MTDADMe-ImmH

Ki* = 71 ± 1 pM Ki* = 900 ± 60 pM Hu 13-fold

HO

O NH N

5'-PrTDADMe-ImmH

Ki* = 41 ± 5 pM Ki* = 160000 ± 40 pM Hu 3900-fold

Figure 4. Examples of transition state analog inhibitors binding to human and PfPNPs. The top row are Immucillins, and represent the first generation of PNP transition state analogues. Rows two and three are the DADMe-Immucillins, secondgeneration inhibitors, also active on both PNPs. Dissociation constants (individual Ki or Ki * values) are shown for human (in blue) or PfPNP (in red). The dissociation constant Ki is from classical competitive inhibition and Ki* is a dissociation constant following slow-onset tight-binding inhibition. The bottom line (in blue) indicates the extent of tighter binding to hPNP than to PfPNP as the ratio of dissociation constants. Adapted with permission from reference 42, Journal of Biological Chemistry 277 (5), 3219-3225 (2002). Copyright 2002, American Society for Biochemistry and Molecular Biology.

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sub-nanomolar inhibitory potential for both enzymes.41,42 Immucillin-H (ImmH) and DADMe-ImmG were selected for additional studies as sub-nanomolar inhibitors of both human and PfPNPs (Figure 4).

P. falciparum cultured in human erythrocytes and grown in the presence of minimal hypoxanthine were killed by the presence of ImmH with IC50 values of between 16 and 148 nM, depending on the erythrocyte content of the assay.30 This variation resulted from the relatively high PNP content of human erythrocytes and the need to completely inhibit PNP in both the parasites and erythrocytes to prevent formation of hypoxanthine. Rescue from the cell killing induced by ImmH was accomplished by the addition of hypoxanthine but not by inosine. This rescue experiment demonstrated the metabolic block at PNP for both the erythrocyte and the parasite PNPs and also established the lack of a catalytic pathway to convert inosine to inosine monophosphate (IMP) or other nucleic acid precursors in infected human erythrocytes.

Figure 5. The effect of DADMe-ImmG on P. falciparum growth (A), incorporation of inosine (B) and hypoxanthine (C) into purine metabolite pools. Reprinted from reference 4, PLoS One. 6 (11), e26916 (2011), under open access license CC-BY.

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The more potent DADMe-ImmG (also, BCX4945) was selected for metabolic studies in infected human erythrocytes and in Aotus primates as an animal model (Figures 5, 6).4 BCX4945 blocked the growth of P. falciparum strains, including those resistant to other antimalarials, in human erythrocytes cultured with 10 µM hypoxanthine (sufficient to permit growth; Figure 5A). Addition of isotopically labeled inosine to infected erythrocytes caused robust incorporation into the cellular nucleotide pool. The presence of BCX4945 completely blocked inosine incorporation, demonstrating that PNP is an essential step to convert inosine to hypoxanthine as the purine nucleotide precursor (Figure 5B). In contrast, isotopically labeled hypoxanthine is beyond the PNP step and was incorporated into the

Figure 6. DADMe-ImmG clears P. falciparum growth in Aotus primates (A), inhibits whole blood PNP, where uric acid is detected from the coupled assay of PNP and xanthine oxidase (B) and causes the accumulation of inosine and depletion of hypoxanthine (C) in Aotus blood. Reprinted from reference 4, PLoS One. 6 (11), e26916 (2011), under open access license CC-BY.”

nucleotide pool with or without the PNP inhibitor (Figure 5C). The ability of the Immucillins to induce ‘purine-less death’ in cultured parasites led to trials in the Aotus primate animal model for antimalarials.4,43 Aotus are highly susceptible to human strains of P. falciparum causing lethal infections without anti-parasite intervention. The experimental animal protocol for Aotus requires animals bred in captivity and pharmacological intervention to prevent overt illness or death in any animals.4 Infected Aotus showed robust growth of parasites in blood samples, increasing to 104 parasites/µL of blood within three days of infection (Figure 6A). Oral treatment of once a day with 50 mg/kg of DADMe-ImmG was provided for 7 days. Parasitemia decreased each day of therapy to clear the blood of parasites by day 6 (< 10-1 parasites/µL). Despite the robust clearance, residual parasites regrew within days post-therapy (Figure 6A). Treatment with DADMe-ImmG completely inhibited the PNP catalytic activity and eliminated hypoxanthine from Aotus blood (Figure 5B, C). Conversely, the inosine concentration during treatment reached 50 – 100 µM, increasing by approximately an order of magnitude compared to untreated animals (Figure 5C). No toxicity could be detected for this therapy, indicating a wide margin of safety. The hypoxanthine levels found in Aotus 9 ACS Paragon Plus Environment

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blood is remarkable. Humans are reported to have 1 to 3 µM hypoxanthine, providing a small precursor pool for purine synthesis in the infecting P. falciparium parasites. In contrast, Aotus have 20 to 50 µM hypoxanthine, making the PNP blockade of hypoxanthine formation a more stringent test of the drug designed to decrease hypoxanthine concentration. No human trials have been initiated with DADMe-ImmG as an antimalarial. However, DADMeImmH (Figure 4) has similar inhibitory potential for both the human and parasite PNPs. It has completed several phase 2 clinical trials for gout, as PNP inhibition blocks uric acid formation in humans.3 Clinical studies have shown DADMe-ImmH to be orally available, safe and to have a long lifetime on the PNP target. In human trials, single oral doses of DADMe-ImmH as low as 0.5 mg/kg cause complete inhibition of human erythrocyte PNP. More remarkably, the tight binding kinetics between DADMe-ImmH and human erythrocyte PNP caused complete inhibition of the enzyme for the 120 day lifetime of erythrocytes.44 Both DADMe-ImmG and DADMe-ImmH appear to have significant clinical potential as oral agents for testing against malaria. Bacterial menaquinone synthesis—the futalosine pathway Menaquinone is synthesized in bacteria and its prenylated product acts as an essential electron transfer agent. The well-defined pathway in Escherichia coli converts chorismate into menaquinone by the actions of the menA to menG genes (MenA to MenG enzymes).9 Genomic analysis indicates that most bacterial species, including those of the human gut microbiome, rely on this pathway. Reports in 2008 indicated that several bacterial species do not posess homologues of menB to menF and thus must encode a distinct pathway for menaquinone biosynthesis.11,12 Subsequent genetic and biochemical analysis indicated an alternative menaquinone synthetic pathway involving the mqn genes with deoxyfutalosine or 6-amino-6-deoxyfutalosine as intermediates (Figure 7). These intermediates are processed by a purine nucleoside hydrolase with homology to other bacterial 5'-methylthioadenosine nucleosidases (MTANs).11,45 Tansition state analogue design work on pathways of quorum sensing in Gram-negative bacteria had already targeted MTAN to produce powerful inhibitors with dissociation constants as low as 47 fM.46-48 These were immediate candidates for the H. pylori MTAN.

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Figure 7. Inhibition strategy for the menaquinone pathway proposed for H. pylori. The pathway (A) involves hydrolysis of adenine from 6-amino-6-deoxyfutalosine by nucleoside hydrolase annotated as an MTAN based on bacterial sequence homology. The inhibition strategy (B) involves a transition state analog with a hydrophobic 5'-substituent to mimic the substituent in 6-amino-6-deoxyfutalosine. The R’-group in the catalytic site is a catalytic site base accepting the proton from the attacking water nucleophile. The inhibitor BuT-DADMe-ImmA (C) is a 36 pM transition state analog inhibitor of the H. pylori MTAN. Adapted from reference 7, Biochemistry 51 (35), 6892-6894 (2012). Copyright 2012 by the American Chemical Society.

Expression, isolation and characterization of MTAN from H. pylori established its action on 6amino-6-deoxyfutalosine and demonstrated that BuT-DADMe-ImmA was an inhibitor with a dissociation constant of 36 pM.10 Evidence that BuT-DADMe-ImmA acts as a transition state analogue for the MTAN from H. pylori is based on kinetic, crystal structure and mass spectrometry studies of the complex. The inhibitor is a slow-onset, tight-binding inhibitor with the equilibrium dissociation constant of 36 pM. The Km value for 6-amino-6-deoxyfutalosine is 0.8 µM, to give a Km/Kd ratio of 22,200. In the catalytic site, the adenine group is held by favorable hydrogen bonds to Asp198 and Val154 while the hydrophobic butylthio group is surrounded by a hydrophobic channel large enough to also accommodate the 5'group of 6-amino-6-deoxyfutalosine (Figure 8). The nucleophilic water molecule (Figure 7B, 8) is 2.6 Å from the cationic hydroxypyrrolidine nitrogen, a position expected from transition state properties of closely related MTANs. Glu13 and Arg194 are both H-bonded to the water nucleophile based on neutron diffraction studies, and play the role of orienting the lone pair of the water oxygen nucleophile toward the developing ribocation at the transition state.49 The water nucleophile is unique, as mass spec analysis of the MTAN·BuT-DADMe-ImmA complex demonstrates a mass that includes the inhibitor and

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one water molecule remaining stable in the gas phase. Thus, the nucleophilic water can be assumed to be stabilized as an integral part of the complex with the transition state analogue.50

Figure 8. Active site of HpMTAN in complex with BuT-DADMe-ImmA. (A) Crystal structure of the active site of HpMTAN with bound BuT-DADMe-ImmA. The figure shows a 2Fo – Fc map around the inhibitor (blue grid) and catalytic water molecule (red grid) contoured at 1.5σ. (B) Protein interactions to BuT-DADMe-ImmA (blue), a water molecule (red sphere), and residues of HpMTAN. Green residues come from the neighboring monomer of the HpMTAN dimer. Dashed lines represent hydrogen bonds (≤3 Å) except for water—3′-OH, 3.1 Å; —Glu175, 3.3 Å; —Glu13, 3.2 Å). Reprinted from reference 7, Biochemistry 51 (35), 6892-6894 (2012). Copyright 2012 by the American Chemical Society.

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Powerful enzyme inhibitors may not be effective antibiotics because of the diffusion barrier and relative impermeability of the cell wall structure in Gram-negative bacteria. The antibiotic action of BuTDADMe-ImmA on H. pylori was tested with cultured organisms and compared directly with amoxicillin, metronidazole and tetracycline, antibiotics often used to treat H. pylori infections in cases of human gastic ulcers.51

Figure 9. Inhibition of H. pylori growth by BuT-DADMe-ImmA. In (A), the indicated concentrations of BuT-DADMe-ImmA were incorporated into horse blood agar culture and spread with media containing H. pylori. The IC90 values are compared with other antibiotics for H. pylori (values from reference 42). Note the unit scales, BuT-DADMe-ImmA (BUTDIA) is 1000 times more effective than clarithromycin. In (B), paper disks containing up to 20 ng of antibiotic were placed on plates pre-innoculated with H. pylori for direct comparison. Only BuT-DADMe-ImmA gave significant zones of clearance. Part of the figure is adapted from reference 7, Biochemistry 51 (35), 6892-6894 (2012). Copyright 2012 by the American Chemical Society.”

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NH2

H N

N

R

Kd

IC90

(pM)

(ng/mL)

S

89

9

S

120

16

S

7

10

S

36

7

S

110

9

S

5

6

S

4

8

S

40

16

S

170

11

6

8

N

N

R group

HO

OH

N N

S 8

Figure 10. Transition state analogues of H. pylori MTAN and IC90 values for inhibition of cell growth. On the left, substituents of the 3-hydroxypyrrolidine group were explored. On the right, substituents of an acyclic amino propanol were also found to be active.

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The efficacy of BuT-DADMe-ImmA against H. pylori led to a synthetic chemistry program to explore and expand the transition state analogues as potential antibiotics.52 Derivatives of two distinct chemical scaffolds gave fourteen compounds with IC90 values at or below 16 ng/mL for H. pylori growth (Figure 10). Blocking growth of the bacteria requires entry into the cell and inhibition of the H. pylori MTAN. All of the inhibitors have sub-namomolar Kd values, but these do not directly correspond to the efficiency of cell growth inhibition. The Kd values ranged from 4 to 170 pM, with IC90 values in a more narrow range from 6 to 16 ng/mL. The lack of correlation indicates different efficiency for diffusion, uptake or transport into the bacterial cells. The promise for these compounds as anti-ulcer antibiotics has not yet been developed into animal models or human clinical trials. Inhibition of viral RNA-dependent RNA polymerases Immucillin-A (ImmA) is currently in clinical trials as an antiviral, but it was originally synthesized as an anti-protozoan agent. The success in blocking purine metabolism with PNP inhibitors in P. falciparium, led to the proposal that Immucillin transition state analogues might be effective to block purine salvage in the protozoan parasite Trichomonas vaginalis, the causative agent of trichomonia. T. vaginalis expresses an unusual PNP (TvPNP) with a preference for adenosine as the substrate.14 In contrast, human and P. falciparum PNPs prefer inosine or guanosine (6-oxypurin nucleosides), and the human enzyme is inactive with adenosine. ImmA and DADMe-Immucillin-A (DADMe-ImmA) were tested for their ability to inhibit the TvPNP and found to be an 87 and 30 pM slow-onset-tight binding inhibitors (Figure 11). In contrast, DADMe-ImmG (see above), the analogue successful for blocking hPNP and PfPNPs, was orders of magnitude weaker for TvPNP with a Kd of 18 nM.14 Finally, ImmA is virtually inactive against hPNP as it did not inhibit even at 10 µM. Subsequent studies to determine if ImmA or DADMe-ImmA are effective as an antibiotic against T. vaginalis showed them to be inactive (unpublished results, Ching C. Wang, UCSF).

Figure 11. Transition state analogues of Trichomonas vaginalis PNP bound to TvPNP and their slow-onset inhibition constants. Amino acids H4* and R43* are contributed by the neighboring subunit. TvPNP is a hexameric enzyme similar to that of PfPNP shown in Figure 3B. Reprinted with permission from reference 14, Biochemistry 46 (3), 659-668 (2007). Copyright 2007 by the American Chemical Society.

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An antiviral screen of ImmA (BCX4430 in the original trials) against a panel of human viruses revealed significant activity against filoviruses, bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. The most potent activity was against the filoviruses with EC50 values from 3.4 to 11.8 µM.53 At present, there is no small-molecule approach for treatment of most of these viruses. Flavivirus infections alone cause an estimated 200 million clinical cases worldwide.54 Subsequent studies demonstrated broad-specificity against both mosquito-borne and TBEV serocomplex flaviviruses with low or no toxicity against the mammalian host cells.55 No incorporation into human RNA or DNA was detected. The mechanism of inhibition for BCX4430 is reported to be as a prodrug. Human nucleoside kinases (presumably adenosine kinase) converts the prodrug to the 5'-monophosphate followed by conversion to the triphosphate by other kinases. The triphosphate is a substrate for viral RNA-dependent RNA polymerase and permits the addition of a few additional encoded bases at which point the BCX4430-bearing RNA cannot be elongated. (Figure 12). ImmA (tested as BCX4430 in the antiviral studies) is converted to the mono- di- and triphosphates in virus- infected human Huh-7 cells. Treatment of cells with BCX4430 inhibited the replication of an ebola virus minigenome RNA replicon and inhibited HCV NS5B RNA polymerase activity. In infected HeLa cells, addition of BCX4430 blocked the expression of Ebola virus and Marburg virus glycoprotein.53 Mice infected with RAVV were tested for response to twice daily i.m. dosing of 150 mg/kg BCX4430. The drug was given for 9 days at timed intervals beginning 4 h before infection, and 24, 48, 72, 96 and 120 h post infection. Mice treated with vehicle only expired by day 8. Groups treated within 96 h post-infection had 80 to 100% survival rates, while those treated at 120 h post-infection gave a 25% survival rate. The encouraging studies with rodents led to a study of Marburg virus infection in cynomolgus macaques, conducted at the biosafety level 4 containment facilities at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID).53 The study was modeled after the mouse experiments with macaques being challenged with Marburg virus by subcutaneous injection. Treatment with (15 mg/kg i.m.) BCX4430 was initiated 1, 24 and 48 hr post infection. Control animals treated only with vehicle expired within 11 days post infection. In contrast, treatment with BCX4430 rescued all but one of the eighteen animals in the treatment group (Figure 12).

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Figure 12. Challenge of Marburg virus infections in cynomolgus macaques by BCX4430 (ImmA; Tx). Six animals in each group were infected with virus on day 0. BCX4430 (15 mg/kg) was given twice a day by i.m. injection starting at 1 h, 24 h or 48 h after exposure to the virus. Vehicle indicates no BCX4430 was given. Reprinted with permission from reference 53, Nature 508 (7496), 402-405 (2014). Copyright 2014 by Nature Publishing Group.

Success in the animal trials has led to clinical trials of BCX4430 (Galidesivir in human trials). In unreviewed, but publically available studies of BCX4430 efficacy against Ebola virus infections, rhesus monkeys were protected by i.m. injections of 16 or 25 mg/kg twice per day.56 Survival against the otherwise lethal infection was 66% in the low dose group and 100% in the high dose group, and no significant toxicities were encountered. Based on these results, the FDA has approved a phase 1 clinical trial to evaluate the safety, tolerability and pharmacokinetics of BCX4430.57 This double-blind, placebocontrolled, dose-ranging study evaluated the safety, tolerability and pharmacokinetics of BCX4430 administered via intramuscular injection in healthy subjects. The study has been completed but no follow-up studies are evident. Discovery of the significant antiviral activity of ImmA is an important advance as it represents a new chemical scaffold as an antiviral. Additional studies to improve to bioavailability of ImmA are advisable, as nucleoside antivirals can be made more available by synthesis as prodrugs of the monophosphates.58 Summary and Conclusions Immucillins are chemically stable mimics of the transition states of N-ribosyltransferases. Chemical stability comes from replacement of the N-ribosyl bond as a C-C riboside. This change also increases the pKa of N7 in the purine ring to resemble the N7 protonation that occurs at the transition states. Replacement of the ribosyl ring oxygen by nitrogen provides a cationic center to resemble the ribocation transition states. Compounds with dissociation constants in the low picomolar range have advantages in long lifetimes on their drug targets. However, affinity does not predict cellular access. The chemical modifications to convert nucleosides to the Immucillins are sufficiently minor to permit rapid cellular access by nucleoside transporters. Immucillin chemical stability causes them to be stable to human metabolism facilitating excretion of unbound drug while the tight binding keeps the Immucillins on their target enzyme for extended periods. DADMe-ImmG shows these properties in the treatment of P. falciparum in aotus primates. A single dose of DADMe-ImmG inhibits the catalytic activity of PNP in 17 ACS Paragon Plus Environment

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erythrocytes for the life of the cell. However periodic dosing is required to capture the PNP of newly formed erythrocytes and parasites. Inhibitors of the H. pylori MTAN also have dissociation constants in the low picomolar range. The highly effective antibiotic action, as low as 6 ng/mL (20 nM) for cell growth IC90 values is orders of magnitude more powerful than antibiotics now used in the treatment of gastric ulcers. As these molecules are readily water-soluble, it suggests a transport mechanism for the inhibitors although the uptake mechanism has not been studied or resolved. Important features of targeting the futalosine pathway with Immucillins is that this pathway is missing in the bacterial population of a healthy human gut microbiome. Thus, treatment of H. pylori infections to cure ulcers and to reduce the incidence of stomach cancers, is not expected to disrupt the gut microbiome. Broad-spectrum antibiotics disrupt the gut microbiome and encourage infection with Clostridium difficile, a growing health problem as a complication following antibiotic therapy. Although ImmA was designed as a transition state analogue against T. vaginalis PNP, it showed no antibiotic action against this protozoan parasite. However, its conversion to ImmA-triphosphate in human cells generates a unusual nucleotide analogue substrate for the viral RNA polymerase, resulting in termination of viral RNA replication and thus serves as an antiviral. The broad-spectrum antiviral activity of ImmA is impressive. However the mechanism of ImmA uptake and the biochemical metabolism and enzymology to define its exact mechanism of action have lagged behind the impressive antiviral studies. As ImmA was developed from a primary screen of Immucillin analogues against viruses, it is anticipated to be followed by future generations of molecules with a similar mechanism of action but with improved bioavailability and/or potency.

Conflict of Interest: Disclosure of potential conflicts. The authors are named inventors on patents describing the design, synthesis and use of Immucillins as potential drugs. The patents are the joint property of the Albert Einstein College of Medicine and the Victoria University of Wellington. These institutions promote the use of Immucillins in clinical developments. The authors may receive compensation under the policies of these institutions. Acknowledgements: This work has been accomplished through the tireless efforts of postdoctoral fellows, students and the academic and pharmaceutical collaborators named and acknowledged in the references. Support for these research efforts have come from NIH research grants GM04961xxxxx, xxxxxxxx and from the Medical Research Foundation of New Zealand.

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