Learning Objectives: Nucleic Acid therapeutics ReadingChapter 4, p from Blackburn & Gait Know the general mechanisms of the anti-cancer drugs that are nucleoside analogs 5-fluoroU, methotrexate, cyclophosphamide Know the general mechanisms of the anti-viral drugs that are nucleoside analogs acyclovir, 5’-substituted pyrimidines, ribavirin, phosphonoformic acid, phosphonoacetic acid, AZT, ddI, ddC, nevirapine Structure of HIV RT and how drugs inhibit it

Nucleic Acid therapeutics Review nucleic acid biosynthesis - de novo and salvage pathways Cancers and viruses treated by targeting NA biosynthesis Some vaccines for viruses (Hep B) but doesn’t help those already infected so need treatments Another problem results from latency of viruses Most common viral diseases influenza virus rhinovirus (cold) change rapidly so vaccine doesn’t work well enterovirus

Nucleotides & Nucleic Acids Biosynthesis and Degradation Biosynthesis: De novo pathways - uses metabolic precursors (AAs, ribose 5- phosphate, CO 2, NH 3 ) Salvage pathways - recycle free bases and nucleosides released from nucleic acid breakdown

Nucleotides & Nucleic Acids Biosynthesis: De novo pathways

Nucleotides & Nucleic Acids Biosynthesis: De novo pathways

Nucleic Acid therapeutics Anti-cancer chemotherapy Rate of replication in cancer cells is high compared to normal cells so many chemo drugs target DNA replication 5-Fluorouracil 5-fU is converted to a nucleotide in the body Potent inhibitor of thymidylate synthetase and so it prevents de novo synthesis of dTMP, dTTP and DNA Suicide inhibitor blocks abstraction of H from C5 in enzyme-cofactor- substrate complex of thymidylate synthetase cofactor Used to treat common solid tumors Toxic, suppresses immune system

Nucleic Acid therapeutics Anti-cancer chemotherapy Methotrexate Prevents de novo synthesis of dTTP by inhibiting biosynthesis of tetrahydrofolate (required as cofactor and donor of -CH3) Selectivity of methotrexate for cancer cells due to its preferential uptake of the drug

Nucleic Acid therapeutics Anti-cancer chemotherapy Methotrexate and 5-fU

Nucleic Acid therapeutics Anti-cancer chemotherapy 5-fU

Nucleic Acid therapeutics Anti-cancer chemotherapy Cyclophosphamide Alkylating agent (lots of other chemo drugs too) Thought to crosslink DNA and interfere with replication Needs enzymatic oxidation (in liver) to form active species Selective for tumor cells which may be due to more efficient transport of active metabolite or lack of enzymatic degradation in tumor cells Toxicity, patient prone to viral & bacterial infections Alkylating agent

Nucleic Acid therapeutics Anti-viral chemotherapy Every virus is different (membrane tags, viral replication cycle) No agent for ALL viruses like with bacterial antibiotics Drug discovery takes long time (exception: AZT) Very few antiviral drugs lots for herpesvirus all but 3 are nucleosides/analogs

Nucleic Acid therapeutics Anti-viral chemotherapy Acyclovir Activated in the body, used to treat herpesvirus Herpesviruses 1. dsDNA viruses that cause cold sores, eye infections, genital sores, chicken pox, shingles, mononucleosis 2. Latent virus (after infection, virus goes into latent state in nerve endings from where it can be reactivated by stress, UV other viruses 3. Virus codes for many enzymes involved in its own repl (DNA pol, TK) 4. Virus vulnerable b/c properties of virally encoded enzymes are slightly different than corresponding host cell enzymes 5. Virus relies on salvage pathways for production on dTTP for DNA syn.- virus encodes its own thymidine kinase (TK) 6. Viral TK not so specific so it phosphorylates many analogs which once activated can inhibit viral replication Acyclovir - purine analog (missing 2’ and 3’-OH) phosphorylated by HV TK at 5’-OH

Nucleic Acid therapeutics Anti-viral chemotherapy Acyclovir No metabolism of acyclovir in normal cells However in virally infected cell, phosphorylated acyclovir is recognized by guanylate kinase and it gets to the triphosphate stage - now it’s a substrate for HV DNA pol and it is incorporated into viral DNA, then it acts as a chain terminator, inhibiting DNA synthesis 6-deoxyacyclovir Prodrug that gets converted into acyclovir Has greater solubility and so get higher levels in plasma

Nucleic Acid therapeutics Anti-viral chemotherapy 5’-substituted pyrimidine 2’-deoxynucleosides 5’-iodo-deoxyuridine was first anti-viral nucleoside drug to be marketed Mode of action still not completely know but it is a substrate for cell & viral TK May cause an effect b/c it is incorporated into DNA or b/c it may inhibit viral DNA pol 5’-vinyl-deoxyU (4.21b) more potent than 5’-iodo in cells but in animals no effect (E)-5-(2-bromovinyl)-2’-deoxyU (BVDU) (4.21c) effective against HSV-1 b/c substrate for viral TK; triphosphate of BVDU is substrate for & inhibitor of viral DNA pol

Nucleic Acid therapeutics Anti-viral chemotherapy Ribavirin Used in aerosol form against RSV only even though it is active against 85% of all viruses studied 5’-triphosphate form needed in cells 1. thought to inhibit inosine monophosphate dehydrogenase, which causes depletion of cell’s GTP pools 2. Competitive inhibitor of viral RNA pol 3. Competitive inhibitor of viral-specific mRNA capping enzyme, so no viral protein syn.

Nucleic Acid therapeutics Anti-viral chemotherapy Phosphonoformic / phosphonoacetic acid Antiherpes drug, selective inhibitor of viral DNA pol Analog of pyrophosphate No activation needed so have to rely on different affinities of viral & cellular DNA pols

Nucleic Acid therapeutics Anti-viral chemotherapy Retroviral inhibitors HIV - lentivirus, viral Reverse transcriptase Target viral RT and replication Chain terminator drugs 3’-azido-2’,3’-dideoxyT (AZT) - found to be least toxic 5’-phos needed for active AZT ddI, ddC - toxicity and need to be phosphorylated

Nucleic Acid therapeutics Anti-viral chemotherapy Retroviral inhibitors Structure of RT solved active form is a heterodimer having 1 pol and 1 RNase H active site

Nucleic Acid therapeutics Anti-viral chemotherapy Retroviral inhibitors Specific inhibitors of RT found nevirapine - binds near RT pol active site Combo therapy used

Nucleic Acid therapeutics Anti-viral chemotherapy Drug design target adhesion of virus to host cell target virus uptake target uncoating of nucleic acid target release of virion HIV - use of protease inhibitors Influenza virus - use of amantadine (ion channel inhibitor)