N2 - Orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, the last two enzymes in the pathway for de novo pyrimidine biosynthesis, exist as a multi-enzyme complex in mammalian cells. Because the two enzymes are not separable, assays for the phosphoribosyltransferase are normally dependent on the decarboxylase, and this has in the past produced misinterpretations in evaluating the effects of inhibitors of either enzyme activity. We have developed a procedure for calculating each enzyme activity independently of the other activity in the complex. Instead of 2 separate assays, one reaction vessel is used, (starting with orotate and phosphoribosyl pyrophosphate). The two products (i.e. orotidylate (OMP), the product of the transferase, and UMP, the product of the decarboxylase) are recovered separately, and the amounts recovered were used to calculate both enzyme activities. Purine or pyrimidine bases did not inhibit the decarboxylase activity, but the transferase activity is inhibited by uracil and uracil analogs, barbiturate, and most effectively by 5-F orotate. Both purine and pyrimidine nucleotides inhibit the decarboxylase activity, although di- and tri-phos-phonucleosides were much less effective than monophosphonucleosides. XMP, UMP, and AMP were the most effective natural inhibitors of the decarboxylase, but the most potent inhibitors were 6-AzaUMP and 1-ribosylallopurinol-5'-phosphate. High concentrations of nucleotides inhibit both enzyme activities.
AB - Orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, the last two enzymes in the pathway for de novo pyrimidine biosynthesis, exist as a multi-enzyme complex in mammalian cells. Because the two enzymes are not separable, assays for the phosphoribosyltransferase are normally dependent on the decarboxylase, and this has in the past produced misinterpretations in evaluating the effects of inhibitors of either enzyme activity. We have developed a procedure for calculating each enzyme activity independently of the other activity in the complex. Instead of 2 separate assays, one reaction vessel is used, (starting with orotate and phosphoribosyl pyrophosphate). The two products (i.e. orotidylate (OMP), the product of the transferase, and UMP, the product of the decarboxylase) are recovered separately, and the amounts recovered were used to calculate both enzyme activities. Purine or pyrimidine bases did not inhibit the decarboxylase activity, but the transferase activity is inhibited by uracil and uracil analogs, barbiturate, and most effectively by 5-F orotate. Both purine and pyrimidine nucleotides inhibit the decarboxylase activity, although di- and tri-phos-phonucleosides were much less effective than monophosphonucleosides. XMP, UMP, and AMP were the most effective natural inhibitors of the decarboxylase, but the most potent inhibitors were 6-AzaUMP and 1-ribosylallopurinol-5'-phosphate. High concentrations of nucleotides inhibit both enzyme activities.
Our therapeutic arsenal to treat viral diseases is extremely limited, and there is a critical need for molecules that could be used against multiple viruses. Among possible strategies, there is a growing interest for molecules stimulating cellular defense mechanisms. We recently developed a functional assay to identify stimulators of antiviral genes, and selected compound DD264 from a chemical library using this approach. While searching for its mode of action, we identified this molecule as an inhibitor of pyrimidine biosynthesis, a metabolic pathway that fuels the cell with pyrimidine nucleobases for both DNA and RNA synthesis. Interestingly, it was recently shown that inhibitors of this metabolic pathway prevent the replication of RNA viruses. Here, we established a functional link between pyrimidine biosynthesis pathway and the induction of antiviral genes, and demonstrated that pyrimidine biosynthesis inhibitors like DD264 or brequinar critically rely on cellular immune response to inhibit virus growth. Thus, pyrimidine deprivation is not directly responsible for the antiviral activity of pyrimidine biosynthesis inhibitors, which rather involves the induction of a metabolic stress and subsequent triggering of cellular defense mechanisms.
To identify chemical compounds stimulating this pathway, several strategies have been developed. Molecules that engage TLRs or IFN-α/β receptors have been identified using various combinations of functional screens, in silico molecular docking and binding assays , . Phenotypic screens have also been used to identify stimulators of the antiviral gene cluster , , , , , , . Several groups have recently described similar assays based on cells transfected with a reporter gene under control of IFN-stimulated response elements (ISRE) , , . This led to the identification of small molecules showing some antiviral activity in vitro. In the current report, we used a similar strategy to screen a compound collection of 41,353 molecules, and identified compound DD264 as a molecule stimulating the expression of antiviral genes in treated cells and exhibiting a potent antiviral activity in vitro. While searching for its mode of action, we found that compound DD264 targets the de novo pyrimidine biosynthesis pathway. This allowed us to establish for the first time a link between inhibition of pyrimidine biosynthesis, amplification of antiviral gene expression, and inhibition of RNA virus infections.
Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.
Sources of Drugs: Biological, marine, mineral and plant tissue cultures as sources of drugs;
Classification of Drugs: Morphological, taxonomical, chemical and pharmacological classification of drugs; Study of medicinally important plants belonging to the families with special reference to: Apocynacae, Solanaceae, Rutacease, Umbelliferae, Leguminosae, Rubiaceae, Liliaceae, Graminae, Labiatae, Cruciferae, Papaveraceae; Cultivation, Collection, Processing and Storage of Crude Drugs: Factors influencing cultivation of medicinal plants, Types of soils and fertilizers of common use. Pest management and natural pest control agents, Plant hormones and their applications, Polyploidy, mutation and hybridization with reference to medicinal plants. Quality Control of Crude Drugs: Adulteration of crude drugs and their detection by organoleptic, microscopic, physical, chemical and biological methods and properties. Introduction to Active Constituents of Drugs: Their isolation, classification and properties.
Systematic pharmacognostic study of the followings:
CARBOHYDRATES and derived products: agar, guar gum acacia, Honey, Isabagol, pectin, Starch, sterculia and Tragacanth; Lipids: Bees wax, Castor oil, Cocoa butter, Codliver oil, Hydnocarpus oil, Kokum butter, Lard, Linseed oil, Rice, Bran oil, Shark liver oil and Wool fat; RESINS: Study of Drugs Containing Resins and Resin Combinations like Colophony, podophyllum, jalap, cannabis, capsicum, myrrh, asafoetida, balsam of Tolu, balsam of Peru, benzoin, turmeric, ginger;
TANNINS: Study of tannins and tannin containing drugs like Gambier, black catechu, gall and myrobalan;
VOLATILE OILS: General methods of obtaining volatile oils from plants, Study of volatile oils of Mentha, Coriander, Cinnamon, Cassia, Lemon peel, Orange peel, Lemon grass, Citronella, Caraway, Dill, Spearmint, Clove, Fennel, Nutmeg, Eucalyptus, Chenopodium, Cardamom, Valerian, Musk, Palmarosa, Gaultheria, Sandal wood; Phytochemical Screening: Preparation of extracts, Screening of alkaloids, saponins, cardenolides and bufadienolides, flavonoids and leucoanthocyanidins, tannins and polyphenols, anthraquinones, cynogenetic glycosides, amino acids in plant extracts; FIBERS: Study of fibers used in pharmacy such as cotton, silk, wool, nylon, glass-wool, polyester and asbestos.
Study of the biological sources, cultivation, collection, commercial varieties, chemical constituents, substitutes, adulterants, uses, diagnostic macroscopic and microscopic features and specific chemical tests of following groups of drugs:
GLYCOSIDE CONTAINING DRUGS: Saponins : Liquorice, ginseng, dioscorea, sarsaparilla, and senega. Cardioactive glycosides: Digitalis, squill, strophanthus and thevetia, Anthraquinone cathartics: Aloe, senna, rhubarb and cascara, Others: Psoralea, Ammi majus, Ammi visnaga, gentian, saffron, chirata, quassia.
ALKALOID CONTAINING DRUGS: Pyridine-piperidine: Tobacco, areca and lobelia. Tropane: Belladonna, hyoscyamus, datura, duboisia, coca and withania. Quinoline and Isoquinoline: Cinchona, ipecac, opium. Indole: Ergot, rauwolfia, catharanthus, nux-vomica and physostigma. Imidazole: Pilocarpus. Steroidal: Veratrum and kurchi. Alkaloidal Amine: Ephedra and colchicum. Glycoalkaloid: Solanum. Purines: Coffee, tea and cola. Biological sources, preparation, identification tests and uses of the following enzymes: Diastase, papain, pepsin, trypsin, pancreatin. Studies of Traditional Drugs: Common vernacular names, botanical sources, morphology, chemical nature of chief constituents, pharmacology, categories and common uses and marketed formulations of following indigenous drugs: Amla, Kantkari, Satavari, Tylophora, Bhilawa, Kalijiri, Bach, Rasna, Punamava, Chitrack, Apamarg, Gokhru, Shankhapushpi, Brahmi, Adusa, Atjuna, Ashoka, Methi, Lahsun, Palash, Guggal, Gymnema, Shilajit, Nagarmotha and Neem. The holistic concept of drug administration in traditional systems of medicine. Introduction to ayurvedic preparations like Arishtas, Asvas, Gutikas, Tailas, Chumas, Lehyas and Bhasmas.
General Techniques of Biosynthetic Studies and Basic Metabolic Pathways/Biogenesis: Brief introduction to biogenesis of secondary metabolites of pharmaceutical importance. Terpenes: monoterpenes, sesquiterpenes, diterpenes, and triterpenoids. Carotenoids: a-carotenoids, ß-carotenes, vitamin A, Xanthophylls of medicinal importance. Glycosides: Digitoxin, digoxin, hecogenin, sennosides, diosgenin and sarasapogenin. Alkaloids: Atropine and related compounds, Quinine, Reserpine, Morphine, Papaverine, Ephedrine, Ergot and Vinca alkaloids. Lignans, quassanoids and flavonoids. Role of plant-based drugs on National economy: A brief account of plant based industries and institutions involved in work on medicinal and aromatic plants in India. Utilization and production of phyto-constituents such as quinine, calcium sennosides, podophyllotoxin, diosgenin, solasodine, and tropane alkaloids. Utilization of aromatic plants and derived products with special reference to sandalwood oil, mentha oil, lemon grass oil, vetiver oil, geranium oil and eucalyptus oil. World-wide trade in medicinal plants and derived products with special reference to diosgenin (disocorea), taxol (Taxus sps) digitalis, tropane alkaloid containing plants, Papain, cinchona, Ipecac, Liquorice, Ginseng, Aloe, Valerian, Rauwolfia and plants containing laxatives. Plant bitters and sweeteners. Plant Tissue Culture: Historical development of plant tissue culture, types of cultures, nutritional requirements, growth and their maintenance. Applications of plant tissue culture in pharmacognosy. Marine pharmacognosy: Novel medicinal agents from marine sources. Natural allergens and photosensitizing agents and fungal toxins. Herbs as health foods. Herbal cosmetics. Standardization and quality control of herbal drugs, WHO guidelines for the standardization of herbal drugs.
Pathophysiology of common diseases; Basic Principles of Cell Injury and Adaptations: Causes of Cellular injury, pathogenesis, morphology of cell injury, adaptations and cell death. Basic Mechanisms involved in the process of inflammation and repair: Vascular and cellular events of acute inflammation, chemical mediators of inflammation, pathogenesis of chronic inflammation, brief outline of the process of repair. Immunopathophysiology: T and B cells, MHC proteins, antigen presenting cells, immune tolerance, pathogenesis of hypersensitivity reactions, autoimmune diseases, AIDS, Amyloidosis. Pathophysiology of Common Diseases: Asthma, diabetes, rheumatoid arthritis, gout, ulcerative colitis, neoplasia, psychosis, depression, mania, epilepsy, acute and chronic renal failure, hypertension, angina, congestive heart failure, atherosclerosis, myocardial infarction, congestive heart failure, peptic ulcer, anemias, hepatic disorders, tuberculosis, urinary tract infections and sexually transmitted diseases. Wherever applicable the molecular basis should be discussed.
Fundamentals of general pharmacology: Dosage forms and routes of administration, mechanism of action, combined effect of drugs, factors modifying drug action, tolerance and dependence; Pharmacogenetics; Principles of Basic and Clinical pharmacokinetics, absorption, Distribution, Metabolism and Excretion of drugs, Adverse Drug Reactions; Bioassay of Drugs and Biological Standardization; Discovery and development of new drugs, Bioavailability and bioequivalence studies; Pharmacology of Peripheral Nervous System: Neurohumoral transmission (autonomic and somatic), Parasympathomimetics, Parasympatholytics, Sympathomimetics, Adrenergic receptor and neuron blocking agents, Ganglion stimulants and blocking agents, Neuromuscular blocking Agents, Local anesthetic Agents.
Pharmacology of Central Nervous System: Neurohumoral transmission in the C.N.S., General Anesthetics, Alcohols and disulfiram, Sedatives, Hypnotics, Anti-anxiety agents and Centrally acting muscle relaxants, Psychopharmacological agents (anti-psychotics), anti-maniacs and hallucinogens, Antidepressants, Anti-epileptics drugs, Anti-Parkinsonian drugs, Analgesics, Antipyretics, Narcotic analgesics and antagonists, C.N.S. stimulants, Drug Addiction and Drug Abuse.
Pharmacology of Cardiovascular System: Drugs used in the management of congestive cardiac failure, Antihypertensive drugs, Anti-anginal and Vasodilator drugs, including calcium channel blockers and beta adrenergic antagonists, Anti-arrhythmic drugs, Anti-hyperlipedemic drugs, Drugs used in the therapy of shock.
Drugs Acting on the Hemopoietic System: Hematinics, Anticoagulants, Vitamin K and hemostatic agents, Fibrinolytic and anti-platelet drugs, Blood and plasma volume expanders.
Drugs acting on urinary system: Fluid and electrolyte balance, Diuretics. Autacoids: Histamine, Antihistaminic drugs, 5-HT- its agonists and antagonists, Prostaglandins, thromboxanes and leukotrienes, Angiotensin, Bradykinin and Substance P and other vasoactive peptides, non-steroidal anti-inflammatory and anti-gout agents.
Drugs Acting on the Respiratory System: Anti-asthmatic drugs including bronchodilators, Anti-tussives and expectorants, Respiratory stimulants.
Drugs acting on the Gastrointestinal Tract: Antacids, Anti-secretory and Anti-ulcer drugs, Laxatives and anti-diarrhoeal drugs, Appetite Stimulants and Suppressants, Emetics and anti-emetics, Miscellaneous: Carminatives, demulcents, protectives, adsorbents, astringents, digestants, enzymes and mucolytics.
Pharmacology of Endocrine System: Hypothalamic and pituitary hormones, Thyroid hormones and anti thyroid drugs, parathormone, calcitonin and Vitamin D, Insulin, glucagons, incretins, oral hypoglycemic agents and insulin analogs, ACTH and corticosteroids, Androgens and anabolic steroids, Estrogens, progesterone and oral contraceptives,
Drugs acting on the uterus & Chemotherapy: General Principles of Chemotherapy, Bacterial resistance; Sulfonamides and cotrimoxazole, Antibiotics- Penicillins, Cephalosporins, Aminoglycosides, Chloramphenicol, Macrolides, Tetracyclines, Quinolones, fluoroquinolones and Miscellaneous antibiotics; Chemotherapy of tuberculosis, leprosy, fungal diseases, viral diseases, HIV and AIDS, urinary tract infections and sexually transmitted diseases, malaria, amoebiasis and other protozoal infections and Anthelmentics. Chemotherapy of malignancy and immunosuppressive agents. Principles of Toxicology: Definition of poison, general principles of treatment of poisoning with particular reference to barbiturates, opioids, organophosphorous and atropine poisoning, Heavy metals and heavy metal antagonists.
Basic Concepts of Pharmacotherapy: Clinical Pharmacokinetics and individualization of Drug therapy, Drug delivery systems and their Biopharmaceutic & Therapeutic considerations, Drugs used during infancy and in the elderly persons (Pediatrics & Geriatrics), Drugs used during pregnancy, Drug induced diseases, The basics of drug interactions, General principles of clinical toxicology, Common clinical laboratory tests and their interpretation; Important Disorders of Organs, Systems and their Management: Cardio-vascular disorders- Hypertension, Congestive heart failure, Angina, Acute myocardial infarction, Cardiac arrhythmias. CNS Disorders: Epilepsy, Parkinsonism, Schizophrenia, Depression Respiratory disease-Asthma. Gastrointestinal Disorders- Peptic ulcer, Ulcerative colitis, Hepatitis, Cirrhosis. Endocrine Disorders- Diabetes mellitus and Thyroid disorders. Infectious Diseases- Tuberculosis, Urinary tract infections, Enteric infections, Upper respiratory infections. Hematopoietic Disorders- Anemias, Joint and Connective tissue disorders- Rheumatic diseases, Gout and Hyperuricemia. Neoplastic Diseases- Acute Leukaemias, Hodgkin’s disease. Therapeutic Drug Monitoring, Concept of Essential Drugs and Rational Drug use.
Figure 1. Metabolism of fluorinated pyrimidines by salvage and other enzymes. dR-P is deoxyribose 5′-phosphate, dR-1-P deoxyribose-1-phosphate, and R-1-P ribose-1-phosphate. Enzymes are 1, thymidylate synthase; 2, dihydrofolate reductase; 3, serine transhydroxymethylase; 4, thymidine kinase; 5, thymidine phosphorylase; 6, uridine phosphorylase; 7, uridine kinase; 8, uridylate kinase; 9, nucleoside diphosphate kinase; 10, ribonucleoside diphosphate reductase; 11, RNA polymerase.