Johan Vande Voorde

Phosphoramidate ProTides of the Anticancer Agent FUDR Successfully Deliver the Preformed Bioactive Monophosphate in Cells and Confer Advantage over the Parent Nucleoside

The fluorinated pyrimidine family of nucleosides continues to represent major current chemotherapeutic agents for treating solid tumors. We herein report their phosphate prodrugs, ProTides, as promising new derivatives, which partially bypass the dependence of the current drugs on active transport and nucleoside kinase-mediated activation. They are also resistant to metabolic deactivation by phosphorolytic enzymes. We report 39 ProTides of the fluorinated pyrimidine FUDR with variation in the aryl, ester, and amino acid regions. Notably, only certain ProTide motifs are successful in delivering the nucleoside monophosphate into intact cells. We also find that the ProTides retain activity in mycoplasma infected cells, unlike FUDR. Data suggest these compounds to be worthy of further progression.

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (favipiravir)

6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5′-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5′-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Kmapp values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.

Background: Gemcitabine is used to treat solid tumors. Some mycoplasmas preferentially colonize tumors in patients. Results: Mycoplasma-encoded cytidine deaminase and pyrimidine nucleoside phosphorylase compromise the cytostatic/antitumor activity of gemcitabine in mycoplasma-infected tumor cell cultures and xenografts in mice. Conclusion: Tumor-associated mycoplasmas may decrease the therapeutic efficiency of gemcitabine. Significance: Current treatment of mycoplasma-infected tumors with gemcitabine may be suboptimal. The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2′,2′-difluoro-2′-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2′-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 μM) and deoxyuridine (Km=578 μM), it prefers uridine (Km=92 μM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.

Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics

Significance The polymerization of nucleotides by DNA polymerases occurs through a common mechanism based on similar highly conserved amino acid motifs and the universal role of the coordination of Mg2+ by three dNTP phosphate oxygens. Based on these universal principles, we aimed at designing a dNTP mimic that could interact with a broad variety of DNA polymerases and should consist of three major indispensable entities: a nucleobase for Watson–Crick base-pairing, an enzymatically and chemically stable triphosphate replacement that can efficiently coordinate the Mg2+ cation, and a variable linker moiety between the nucleobase and the modified phosphate. The resulting α-carboxy nucleoside phosphonates (α-CNPs) were structurally, kinetically, and biochemically investigated, and the novel dNTP mimics were successfully validated in several DNA polymerase models. Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg2+ cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg2+, mimicking the chelation by the β- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg2+ ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg2+-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.

Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase

8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy.

Mycoplasma hyorhinis-encoded purine nucleoside phosphorylase: kinetic properties and its effect on the cytostatic potential of purine-based anticancer drugs

A mycoplasma-encoded purine nucleoside phosphorylase (designated PNPHyor) has been cloned and characterized for the first time. Efficient phosphorolysis of natural 6-oxopurine and 6-aminopurine nucleosides was observed, with adenosine the preferred natural substrate (Km = 61 µM). Several cytostatic purine nucleoside analogs proved to be susceptible to PNPHyor-mediated phosphorolysis, and a markedly decreased or increased cytostatic activity was observed in Mycoplasma hyorhinis–infected human breast carcinoma MCF-7 cell cultures (MCF-7.Hyor), depending on the properties of the released purine base. We demonstrated an ∼10-fold loss of cytostatic activity of cladribine in MCF-7.Hyor cells and observed a rapid and complete phosphorolysis of this drug when it was exposed to the supernatant of mycoplasma-infected cells. This conversion (inactivation) could be prevented by a specific PNP inhibitor. These findings correlated well with the high efficiency of PNPHyor-catalyzed phosphorolysis of cladribine to its less toxic base 2-chloroadenine (Km = 80 µM). In contrast, the cytostatic activity of nucleoside analogs carrying a highly toxic purine base and being a substrate for PNPHyor, but not human PNP, was substantially increased in MCF-7.Hyor cells (∼130-fold for fludarabine and ∼45-fold for 6-methylpurine-2′-deoxyriboside). Elimination of the mycoplasma from the tumor cell cultures or selective inhibition of PNPHyor by a PNP inhibitor restored the cytostatic activity of the purine-based nucleoside drugs. Since several studies suggest a high and preferential colonization or association of tumor tissue in cancer patients with different prokaryotes (including mycoplasmas), the data presented here may be of relevance for the optimization of purine nucleoside–based anticancer drug treatment.

Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs

Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased inMycoplasma hyorhinis‐infected tumor cell cultures compared with non‐infected tumor cell cultures. This mycoplasma‐driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. TheM. hyorhinis‐encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine‐based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency.

Inhibition of pyrimidine and purine nucleoside phosphorylases by a 3,5-dichlorobenzoyl-substituted 2-deoxy-d-ribose-1-phosphate derivative

The 3,5-dichlorobenzoyl-substituted 2-deoxy-D-ribose-1-phosphate derivative, designated Cf2891, was found to inhibit a variety of pyrimidine and purine nucleoside phosphorylases (NPs) with preference for uridine- and inosine-hydrolyzing enzymes [uridine phosphorylase (UP; EC 2.4.2.3), pyrimidine nucleoside phosphorylase (PyNP; EC 2.4.2.2) and purine nucleoside phosphorylase (PNP; EC 2.4.2.1)]. Kinetic analyses revealed that Cf2891 competes with inorganic phosphate (P(i)) for binding to the NPs and, depending on the nature of the enzyme, acts as a competitive or non-competitive inhibitor with regard to the nucleoside binding site. Also, the compound prevents breakdown of pyrimidine analogues used in the treatment of viral infections and cancer. Since NPs are abundantly present in tumor tissue and may be overexpressed due to secondary bacterial infections in immunocompromised patients suffering viral infections, Cf2891 may serve as a lead molecule for the development of inhibitors to be used in nucleoside-based combination therapy.

An emerging understanding of the Janus face of the human microbiome: enhancement versus impairment of cancer therapy

Sir, Commensal bacteria have been shown to modulate myeloidderived cell functions in the tumour microenvironment that are crucial for an optimal response to immunotherapy or platinumbased chemotherapy. Also, the therapeutic efficacy of cyclophosphamide has been proven to be mediated by selected species of Gram-positive bacteria that stimulate a specific anticancer immune response. As a result, a decreased efficiency of cancer therapy was reported in germ-free or antibiotic-treated mice. These results underscore the potential risks of manipulating the microbiome prior to or during cancer treatment. Here, we want to place these findings in a broader perspective by also taking into account the, often opposite, effects microbiota might have on the anticancer activity of nucleoside-based antimetabolite drugs. Nucleoside analogues (NAs) represent 20% of the approved anticancer drugs and have become cornerstones of successful treatment for cancer patients. The clinical use of several potent NAs (e.g. floxuridine and trifluridine) has been limited due to drug inactivation by catabolic enzymes such as nucleoside phosphorylases (NPs), which can be expressed both by mammalian cells and by commensal prokaryotes. Their therapeutic potential may therefore be improved by coadministration of a specific NP inhibitor. Indeed, TAS-102, a combination of trifluridine with the highly potent and specific thymidine phosphorylase (TP) inhibitor TPI, is currently being evaluated in a Phase III clinical trial to treat metastatic colorectal cancer. Non-mammalian enzymes are often characterized by a different substrate specificity or catalytic activity compared with their mammalian counterparts. Therefore, prokaryotic enzymes expressed by commensal bacteria may affect the efficiency of NA treatment or NA-related toxicity. For example, sorivudine, a potent antivaricella zoster virus agent, is catabolized (inactivated) to 5-(2-bromovinyl)uracil (BVU) by prokaryotic but not by mammalian TP upon oral administration. BVU is a potent inhibitor of dihydropyrimidine dehydrogenase, a crucial enzyme in the detoxification of the anticancer drug 5-fluorouracil (5-FU). Simultaneous treatment of cancer patients with sorivudine and fluoropyrimidines may therefore cause acute 5-FU-related toxicity and has in the past resulted in 18 fatalities. More recently, the cytostatic activity of several thymidine-based NAs (e.g. floxuridine and trifluridine) was found to be compromised by 10 –150-fold in mycoplasma-infected tumour cell cultures due to the extensive expression of prokaryote-encoded pyrimidine NP (PyNP). Also, in contrast to mammalian purine NPs (PNPs), prokaryotic PNPs efficiently catalyse the phosphorolysis of (2′-deoxy)adenosine derivatives, such as the anticancer drug cladribine. The activity of cladribine, used to treat lymphoproliferative diseases, was decreased 10-fold in mycoplasma-infected tumour cell cultures due to prokaryote-driven drug catabolism to the inactive base 2-chloroadenine. The antitumour activity of the above-mentioned drugs could be restored by administering a mycoplasmatargeting antibiotic or a specific PyNP or PNP inhibitor. Similarly, the cytostatic and antitumour action of gemcitabine, used in the treatment of solid tumours, and the cytostatic activity of cytarabine, used for acute lymphoblastic and myeloid leukaemia, were heavily compromised due to prokaryote-encoded catabolic enzymes such as cytidine deaminase. Thus, NA-based cancer therapy may benefit from concomitant administration of specific inhibitors of prokaryotic enzymes or antibiotics to prevent drug inactivation by microorganisms. Several studies report prokaryotic colonization of different human tumours, which may be due to aberrant vascularization of the tumour, local immune suppression, increased availability of nutrients or the presence of chemoattractants. When systemically administered, bacteria were shown to replicate specifically within the tumours. Also, a high and preferential colonization of tumour tissue by mycoplasmas compared with control or pre-malignant tissue was observed (additional references in Vande Voorde et al.). These prokaryotes may therefore selectively inactivate NAs at the tumour site and decrease their therapeutic efficiency. However, conversely, other NAs have been shown to benefit from phosphorolysis; fludarabine is selectively metabolized by prokaryotic PNP to a more toxic product (useful in combined suicide gene/ chemotherapy of cancer) and capecitabine requires TP activity to exert anticancer activity. Research letters