Research Progress in Competitive Purine Antagonists.

Purine, one of the nucleotides, is an important substance for the metabolism and regulation of the body. Purine plays a key role not only in the composition of coenzymes but also in the supply of energy. Since purine was artificially synthesized, it has always been an important scaffold for respiratory diseases, cardiovascular diseases, and anti- tumor and anti-viral drugs. In addition to being widely used as competitive antagonists in the treatment of diseases, purines can be used in combination with other drugs and as precursors to benefit human life. Unfortunately, few new discoveries have been made in recent years. In this article, purine drugs in the market have been classified according to their different targets. In addition, their mechanism of action and structure-activity relationship have also been introduced. This paper provides details of the signaling pathways through which purine drugs can bind to the respective receptors on the surface of cells and cause consequent reactions within the cell, which finally affect the targeted diseases. The various receptors and biological reactions involved in the signaling for respective disease targets within the cells are discussed in detail.

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors.

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.

The nucleotide ppGpp is a highly conserved regulatory molecule in bacteria that helps tune growth rate to nutrient availability. Despite decades of study, how ppGpp regulates growth remains poorly understood. Here, we developed and validated a capture-compound mass spectrometry approach that identified >50 putative ppGpp targets in Escherichia coli. These targets control many key cellular processes and include 13 enzymes required for nucleotide synthesis. We demonstrated that ppGpp inhibits the de novo synthesis of all purine nucleotides by directly targeting the enzyme PurF. By solving a structure of PurF bound to ppGpp, we designed a mutation that ablates ppGpp-based regulation, leading to dysregulation of purine-nucleotide synthesis following ppGpp accumulation. Collectively, our results provide new insights into ppGpp-based growth control and a nearly comprehensive set of targets for future exploration. The capture compounds developed should also enable the rapid identification of ppGpp targets in any species, including pathogens.

Genome-wide analysis of differentially expressed genes and the modulation of PEDV infection in Vero E6 cells.

PEDV remains one of the most important swine diseases that infects pigs of all ages. It causes devastating viral enteric disease in piglets with a high mortality rate, leading to significant threats and huge economic loss to the pork industry. In this study, a transcriptomic shotgun sequencing (RNA-Seq) procedure was used to study gene responses against PEDV infection. Genome-wide analysis of differentially expressed genes (DEGs) was performed in Vero E6 cells post-PEDV infection. mTOR signaling pathway activator-MHY1485, and inhibitor-PP242 were used to study the antiviral function. Results revealed that the IRF3 was significantly up-regulated post-PEDV infection. Although most of the IFN-regulatory and -related genes evaluated in this study were either down-regulated or remained unchanged, IL11 behaved significantly up-regulated, with the peak at 16 hpi. Nearly 90% of PEDV infections were suppressed in the PP242 pretreated cells whereas the reverse effect was observed in the MYH1485 pretreated cells. Results indicated that the mTOR signaling pathway played a vital role in the PEDV antiviral regulation in the Vero E6 cells. Future studies will contribute to better understand the cellular antiviral mechanism against PEDV.

Loss of nitric oxide-mediated inhibition of purine neurotransmitter release in the colon in the absence of interstitial cells of Cajal.

Regulation of colonic motility depends on the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor-α-positive (PDGFRα+) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in KitW/KitW-v (W/Wv ) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We used small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD+)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP), and metabolites from the tunica muscularis of human, monkey, and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/Wv, Nos1-/- , and Prkg1-/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/Wv mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/Wv mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provide novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.NEW & NOTEWORTHY This is the first study investigating the role of nitric oxide (NO) and intramuscular interstitial cells of Cajal (ICC-IM) in modulating neural release of purines in colon. We found that NO inhibited release of purines in human, monkey, and murine colons and that colons from KitW/KitW-v (W/Wv ) mice, which present with partial loss of ICC-IM, demonstrated augmented neural release of purines. Interactions between nitrergic and purinergic neurotransmission may affect motility in disease conditions with ICC-IM deficiencies.

TRPA1 mediates trigeminal neuropathic pain in mice downstream of monocytes/macrophages and oxidative stress.

Despite intense investigation, the mechanisms of the different forms of trigeminal neuropathic pain remain substantially unidentified. The transient receptor potential ankyrin 1 channel (encoded by TRPA1) has been reported to contribute to allodynia or hyperalgesia in some neuropathic pain models, including those produced by sciatic nerve constriction. However, the role of TRPA1 and the processes that cause trigeminal pain-like behaviours from nerve insult are poorly understood. The role of TRPA1, monocytes and macrophages, and oxidative stress in pain-like behaviour evoked by the constriction of the infraorbital nerve in mice were explored. C57BL/6 and wild-type (Trpa1(+/+)) mice that underwent constriction of the infraorbital nerve exhibited prolonged (20 days) non-evoked nociceptive behaviour and mechanical, cold and chemical hypersensitivity in comparison to sham-operated mice (P < 0.05-P < 0.001). Both genetic deletion of Trpa1 (Trpa1(-/-)) and pharmacological blockade (HC-030031 and A-967079) abrogated pain-like behaviours (both P < 0.001), which were abated by the antioxidant, α-lipoic acid, and the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin (both P < 0.001). Nociception and hypersensitivity evoked by constriction of the infraorbital nerve was associated with intra- and perineural monocytic and macrophagic invasion and increased levels of oxidative stress by-products (hydrogen peroxide and 4-hydroxynonenal). Attenuation of monocyte/macrophage increase by systemic treatment with an antibody against the monocyte chemoattractant chemokine (C-C motif) ligand 2 (CCL2) or the macrophage-depleting agent, clodronate (both P < 0.05), was associated with reduced hydrogen peroxide and 4-hydroxynonenal perineural levels and pain-like behaviours (all P < 0.01), which were abated by perineural administration of HC-030031, α-lipoic acid or the anti-CCL2 antibody (all P < 0.001). The present findings propose that, in the constriction of the infraorbital nerve model of trigeminal neuropathic pain, pain-like behaviours are entirely mediated by the TRPA1 channel, targeted by increased oxidative stress by-products released from monocytes and macrophages clumping at the site of nerve injury.

Distinct TRPV1- and TRPA1-based mechanisms underlying enhancement of oral ulcerative mucositis-induced pain by 5-fluorouracil.

In many patients with cancer, chemotherapy-induced severe oral ulcerative mucositis causes intractable pain, leading to delays and interruptions in therapy. However, the pain mechanism in oral ulcerative mucositis after chemotherapy has not been extensively studied. In this study, we investigated spontaneous pain and mechanical allodynia in a preclinical model of oral ulcerative mucositis after systemic administration of the chemotherapy drug 5-fluorouracil, using our proprietary pain assay system for conscious rats. 5-Fluorouracil caused leukopenia but did not induce pain-related behaviors. After 5-fluorouracil administration, oral ulcers were developed with topical acetic acid treatment. Compared with saline-treated rats, 5-fluorouracil-exposed rats showed more severe mucositis with excessive bacterial loading due to a lack of leukocyte infiltration, as well as enhancements of spontaneous pain and mechanical allodynia. Antibacterial drugs, the lipid A inhibitor polymyxin B and the TRPV1/TRPA1 channel pore-passing anesthetic QX-314, suppressed both the spontaneous pain and the mechanical allodynia. The cyclooxygenase inhibitor indomethacin and the TRPV1 antagonist SB-366791 inhibited the spontaneous pain, but not the mechanical allodynia. In contrast, the TRPA1 antagonist HC-030031 and the N-formylmethionine receptor FPR1 antagonist Boc MLF primarily suppressed the mechanical allodynia. These results suggest that 5-fluorouracil-associated leukopenia allows excessive oral bacterial infection in the oral ulcerative region, resulting in the enhancement of spontaneous pain through continuous TRPV1 activation and cyclooxygenase pathway, and mechanical allodynia through mechanical sensitization of TRPA1 caused by neuronal effects of bacterial toxins. These distinct pain mechanisms explain the difficulties encountered with general treatments for oral ulcerative mucositis-induced pain in patients with cancer and suggest more effective approaches.

Systemic use of non-biologics in orofacial diseases: 2. Purine synthesis inhibitors.

Systemic non-biological agents (NBAs) have been extensively used for immunosuppression in clinical medicine, often with considerable efficacy, although sometimes accompanied with adverse effects as with all medicines. With the advent of biological agents, all of which currently are restricted to systemic use, there is a rising need to identify which agents have the better therapeutic ratio. The NBAs include a range of agents, most especially the corticosteroids (corticosteroids). This article reviews the purine synthesis inhibitors (azathioprine and mycophenolate), which are currently the most commonly used systemically immunosuppressive agents in the management of orofacial mucocutaneous diseases. Subsequent articles discuss other corticosteroid-sparing agents used in the management of orofacial disease, such as calcineurin inhibitors, and the cytotoxic and other immunomodulatory agents.

Thymidylate synthase expression determines pemetrexed targets and resistance development in tumour cells.

Although treatment options for cancer patients are increasing every year, the drug resistance problem remains very present. It is very difficult to find a drug that acts equally on tumours of the same histology as the individual's genetic characteristics often determine the response to treatment. Furthermore, tumours that initially respond to anti-tumour therapy are able to adapt and develop resistance to the drug, while others do not. In addition, this usually implies resistance development to agents to which the cells have not been exposed, a phenomenon called cross-resistance or multidrug resistance. Given this situation, it has been suggested that the most appropriate treatment would be able to act in parallel on multiple pathways constitutively altered in tumour cells. Pemetrexed is a multitargeted antifolate that exerts its activity against folate-dependent enzymes involved in de novo pyrimidine and purine synthesis. It is currently in use in combination with cisplatin against malignant pleural mesothelioma and non-squamous non-small cell lung cancer with favourable results. By real-time RT-PCR gene expression assays and restoration viability assays we demonstrated that Pemetrexed targets folate-dependent enzymes involved in de novo biosynthesis of purines differently depending on the intrinsic genetic characteristics of the tumour. These differences did not, however, interfere either with the initial response to the drug or with the activation of apoptotic pathways. In addition, these genetic fingerprints can differentiate two groups of tumours: those capable of developing resistance to antifolate, and not capable. These results may be useful to employ targets gene expression as resistance markers, a valuable tool for identifying patients likely to receive combination therapy to prevent the development of resistance.

The DPP-4 inhibitor linagliptin restores ß-cell function and survival in human isolated islets through GLP-1 stabilization.

CONTEXT: Inhibition of dipeptidyl peptidase-4 (DPP-4) is a potent strategy to increase glucose-dependent insulinotropic polypeptide and glucagon like peptide 1 (GLP-1) induced insulin secretion in diabetes. It is important to know whether new drugs approved for the treatment of type 2 diabetes have direct effects on the ß-cell. OBJECTIVE: Herein we investigated the effect of linagliptin, a novel DPP-4 inhibitor, on ß-cell function and survival. DESIGN: Human islets were exposed to a diabetic milieu (11.1-33.3 mM glucose, 0.5 mM palmitate, the mixture of 2 ng/mL IL-1ß+1000 U/mL interferon-γ, or 50 µM H2O2) with or without 500 ng/mL IL-1 receptor antagonist (IL-1Ra) or 30-50 nM linagliptin. RESULTS: Linagliptin restored ß-cell function and turnover, which was impaired when islets were exposed to elevated glucose, palmitate, cytokines, or H2O2. Pretreatment with IL-1Ra was similarly effective, except against H2O2 treatment. Nitrotyrosine concentrations in islet lysates, an indicator of oxidative stress, were highly elevated under diabetic conditions but not in islets treated with linagliptin or IL-1Ra. Linagliptin also reduced cytokine secretion and stabilized GLP-1 in islet supernatants. CONCLUSIONS: We show that the novel DPP-4 inhibitor linagliptin protected from gluco-, lipo-, and cytokine-toxicity and stabilized active GLP-1 secreted from human islets. This provides a direct GLP-1 mediated protective effect of linagliptin on ß-cell function and survival.

Genotype frequencies of drug-metabolizing enzymes responsible for purine and pyrimidine antagonists in a healthy Asian-Indian population.

Purine and pyrimidine antimetabolites are used to treat leukemias, autoimmune diseases, and solid tumors. Detection of slow metabolizers before administration of the drugs is necessary to prevent any subsequent drug toxicity. With this aim, we determined the frequencies of normal and slow alleles in our population. Polymorphisms in genes encoding cytidine deaminase (CDA), dihydropyrimidine dehydrogenase (DPYD), and thiopurine-S-methyltransferase (TPMT) were documented in 225 healthy volunteers. The polymorphisms typed included CDA*3, DPYD*2A, TPMT*2A, TPMT*3B, and TPMT*3C. Methods used for genotyping included standard PCR-RFLP and allele-specific PCR reactions. The frequencies were 0.44 % for DPYD*2A, 0.67 % for TPMT*3B, and 0.89 % for TPMT*3C. The CDA*3 and TPMT*2A alleles were not detected. Although these polymorphisms have been demonstrated to be associated with drug toxicity in other populations, they appear to be very rare in the adult Indian population.

Synthesis and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers as inhibitors of de novo purine biosynthesis with selectivity for cellular uptake by high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier.

We previously reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a three- (3a) and four-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells. While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and l-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5, and 6) substitutions on the thienoyl ring and with acetylenic insertions in the four-atom bridge were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.

[Antimetabolites]. / Les antimétabolites.

Antimetabolites are cytotoxic agents, which have been developed for more than 50  years. Which cancer patient did not receive or will not receive 5-fluorouracil or methotrexate during the evolution his or her disease? Antimetabolites are defined as interfering with the synthesis of the DNA constituents; they are structural analogues, either of purine and pyrimidine bases (or the corresponding nucleosides), or of folate cofactors, which are involved at several steps of purine and pyrimidine biosynthesis. Their first mechanism of action is, therefore, to induce depletion in nucleotides inducing in turn an inhibition of DNA replication. However, some of them are able to get inserted fraudulently into nucleic acids, inducing structural abnormalities leading to cell death by other mechanisms, including DNA breaks. We present in this paper, for the three classes of antimetabolites, both ancient and recent molecules as well as molecules still in clinical trials, without exhaustivity.

Potential teratogenic effects of allopurinol: a case report.

We report on a case of a multiple congenital anomalies in a newborn infant whose mother was on allopurinol treatment through the pregnancy. The pattern of congenital anomalies that was noted in our patient was similar to the pattern described in a number of published reports following mycophenolate mofetil [CellCept®] treatment during pregnancy. The anomalies present in our patient include: diaphragmatic hernia, unilateral microtia and absence of external auditory canal, micrognathia, microphthalmia, optic nerve hypoplasia, hypoplasia of the corpus callosum, unilateral renal agenesis, pulmonary agenesis, and cleft lip and palate. Since both allopurinol and mycophenolate mofetil act by disrupting purine biosynthesis and given the similarities in anomalies seen after prenatal exposure, we suggest that allopurinol should also be considered a teratogen.