Learning Objectives: Nucleic Acid therapeutics Reading - Blackburn

Learning Objectives:

Nucleic Acid therapeuticsReading - Blackburn

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 biosynthesisSome vaccines for viruses (Hep B) but doesn’t help those already infected so need treatmentsAnother problem results from latency of viruses

Most common viral diseases influenza virusrhinovirus (cold) change rapidly so vaccine

doesn’t work well

enterovirus

Nucleotides & Nucleic AcidsBiosynthesis and Degradation

Biosynthesis:De novo pathways - uses metabolic precursors (AAs, ribose 5-phosphate, CO2, NH3)Salvage pathways - recycle free bases and nucleosides released from nucleic acid breakdown

Nucleotides & Nucleic AcidsBiosynthesis:De novo pathways

Nucleotides & Nucleic Acids

Biosynthesis:De novo pathways

Nucleic Acid therapeuticsAnti-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 DNASuicide inhibitor blocks abstraction of H from C5 in enzyme-cofactor-substrate complex of thymidylate synthetase

cofactor

Used to treat common solid tumorsToxic, suppresses immune system


MethotrexatePrevents 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


Methotrexate and 5-fU


5-fU


CyclophosphamideAlkylating agent (lots of other chemo drugs too)Thought to crosslink DNA and interfere with replicationNeeds enzymatic oxidation (in liver) to form active speciesSelective for tumor cells which may be due to more efficient transport of active metabolite or lack of enzymatic degradation in tumor cellsToxicity, patient prone to viral & bacterial infections

Alkylating agent

Nucleic Acid therapeuticsAnti-viral chemotherapy

Every virus is different (membrane tags, viral replication cycle) No agent for ALL viruses like with bacterial antibioticsDrug discovery takes long time (exception: AZT)Very few antiviral drugs

lots for herpesvirusall but 3 are nucleosides/analogs


AcyclovirActivated in the body, used to treat herpesvirusHerpesviruses 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


AcyclovirNo metabolism of acyclovir in normal cellsHowever 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-deoxyacyclovirProdrug that gets converted into acyclovirHas greater solubility and so get higher levels in plasma


5’-substituted pyrimidine 2’-deoxynucleosides5’-iodo-deoxyuridine was first anti-viral nucleoside drug to be marketedMode of action still not completely know but it is a substrate for cell & viral TKMay 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


RibavirinUsed in aerosol form against RSV only even though it is active against 85% of all viruses studied5’-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 pol3. Competitive inhibitor of viral-

specific mRNA capping enzyme, so no viral protein syn.


Phosphonoformic / phosphonoacetic acidAntiherpes drug, selective inhibitor of viral DNA polAnalog of pyrophosphateNo activation needed so have to rely on different affinities of viral & cellular DNA pols


Retroviral inhibitors HIV - lentivirus, viral Reverse transcriptase Target viral RT and replicationChain 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


Retroviral inhibitors Structure of RT solvedactive form is a heterodimer having 1 pol and 1 RNase H active site


Retroviral inhibitors Specific inhibitors of RT found

nevirapine - binds near RT pol active siteCombo therapy used


Drug designtarget adhesion of virus to host celltarget virus uptaketarget uncoating of nucleic acidtarget release of viriontarget protease dimerization site

HIV - use of protease enzyme/dimerization inhibitors

Influenza virus - use of amantadine (ion channel inhibitor)

Learning Objectives: Nucleic Acid therapeutics Reading - Blackburn

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