Drugs that affect PURINE nucleotide synthesis:

· Name the three targets.

·         Glutamine phosphoribisyl amidotransferase

·         GAR and ALCAR transformylase

· IMP dehydrogenase

Drugs that affect PURINE nucleotide synthesis:

·         Glutamine phosphoribisyl amidotransferase

o   Formation of ______ to ______.

o   Is it committed or not?

o   What happens when we block this enzyme?

oHow can we get around this blockade?

·         PRPP to PRA

·         Yes, it is the first committed step.

·         Will not make IMP, GMP, or AMP; everything down stream is affected.

· Salvage pathway

Drugs that affect PURINE nucleotide synthesis:

·         GAR and ALCAR transformylase

·         Use ____________ ________- that donate a carbon as make a base without these derivatives can’t continue reaction.

· How many steps does it take to get from PRA to IMP?

·         Tetrohydrofolate

·9

Drugs that affect PURINE nucleotide synthesis:

·         IMP dehydrogenase (IMPDH)

o   This enzyme converts ____ to _____.

oIf block this enzyme what happens?

·         IMP to GMP

· It impacts the guanosine side.  Eventually want have GTP so adenosine side will shut off too.

Drugs that affect PURINE nucleotide synthesis:

·         IMP dehydrogenase (IMPDH)

o   _______ helps the adenosine side proceed.

o   If block this enzyme, _____ will build up.

o What happens when this builds up?

·         GTP

·         IMP

·When IMP builds up it acts like AMP and GMP.  AMP and GMP regulate first enzyme.  So when AMP and GMP build up they inhibit this enzyme.

Mercaptopurine:

·         Looks like ____________ base.

·What is the only difference?

·         Hypoxanthine

·6-Mercaptopurine contains a sulfur and Hypoxanthine contains an oxygen.

Mercaptopurine

·         Which of the three bases does it look like?

·  6- Mercaptopurine is a ____________ analog.

·         Hypoxanthine, guanine, adenine.

· Hypoxanthine

Mercaptopurine

·         To be active, must be in _________ form?

·         How can we convert it to this form?

·  HINT: Hypoxanthine-guanine _______ and __________.

·         Nucleotide

·         Take the base and make monophosphate done by HGPRT with PRPP.

· PRT (HGPRT) and PRPP

Mercaptopurine

·         Normally this enzyme, HGPRT converts hypoxanthine to ______.

·  6- Mercaptopurine is converted by HGPRT to 6-thio-inosine monophosphate which can also be further converted to _____________.

·         IMP

· 6-Thio GMP

·         These 6- Mercaptopurine monophosphates mimic which two natural purine monophosphates?

o   6-Thio-IMP?

o 6-Thio-GMP?

·         IMP

· GMP

·         What enzymes in purine de novo synthesis do you think 6- Mercaptopurine targets?

·         What effect would this have on cancer cell proliferation?

·   Do you expect the effect to be limited to cancer cells?

·         Glutamine phosphoribisyl amidotransferase, PRPP sythetase and IMP DH (inhibit the natural enzymes that the monophophates inhibit because will imitate IMP and GMP)

·         Reduce/block it

·  No

Additional effects associated with 6-mercaptopurine

·         6-thio-GMP can be further metabolized to _________ and get incorporated into _______ and affect protein synthesis.

·  Can also be converted to 6-thio-deoxyGTP and get incorporated into _____.

·         6-thio-GTP, RNA

·         DNA

·         With 6-thio-dGTP have _____ present instead of _______.

·  What does this do?

·         Sulfur instead of oxygen

·  The sulfur is more reactive, have no enzymatic methylation of sulfur that causes base pair mismatching and triggers repair mechanisms.  Happens in cancer cells and normal proliferation cells.

6-mercaptopurine degradation and elimination:

·         6-thio-IMP and 6-thio-GMP are degraded to the respective nucleoside by _________ __________.

·         6-thio-inosine and 6-thio-guanosine are converted to the respective bases by ________________________.

· 6-mercaptopurine (Hypoxanthine) and 6-thio-guanine are converted to 6-thio-_____________ by xanthine oxidase.

·         5’-nucleotidase

·         Purine nucleoside phosphorylase (PNP)

·  Uric acid

·         6-mercaptopurine (Hypoxanthine) and 6-thio-guanine are converted to 6-thio-uric acid by xanthine oxidase.

· What is the problem with this?

·         THF derivatives form _________.

·         N¹⁰ – formyl THF it is a ________ derivative and _____ donor.

· Name an antifolate.

·         Folates

·         THF, Carbon

·  Methotrexate

·         The antifolates, such as methotrexate (MTX) have two effects:

1.      Interfere with the formation of ________-__________

2.  Mimic ________-_________ at the enzyme binding site, preventing the _________ transfer.

·         THF-cofacters

· THF-cofactors, carbon

·         If block DHFR (dihydrofolate reductase) what happens?

·         MTX inhibits the dihydrofolate reductase (DHFR) and blocks the formation of ________.

·  What is the difference in the structure of Folic acid and MTX?

·         THF

· Folic acid contains an H on N and MTX does not have an H present.

·         MTX inhibits the dihydrofolate reductase (DHFR) and blocks the formation of THF

o   This effects the formation of THF _________.

§  N⁵, N¹⁰ methylene THF- NO ______.

§  N¹⁰ formyl THF- NO _____.

§  N⁵ methyl THF

·         Derivatives

·         TMP

· IMP

·         Without THF derivatives, the formation of _________ is blocked.

·  Formation of what else is affected by inhibition of DHFR?

·         IMP

· TMP

 Mycophenolate mofetil: IMP dehydrohenase (IMPDH):

·         Why is this a better choice to use verse Azothioprin?

· Mycophenolate mofetil is an _____________ used for _____ ________.

·         Because Mycophenolate mofetil is more specific for lymphocyte IMPDH, so less off target hitting occurs (there are two forms of IMPDH, this drugs is specific for the ones associated with immune cells)

· Immunosuppressant, organ transplant

·         IMPDH

·         GMP;

·         NOTE: GDP à GTP

        GDPà dGDPà dGTP

        Without dGTP can’t make DNA

Drugs that affect Thymidine monophophates synthesis  (Pyrimidines)     

·         Name the two targets

·  Both of these enzymes are key for formation of __________?

1.      Dihydrofolate reductase (DHFR)

2.      Thymidylate Synthase

·  TMP

Drugs that affect Thymidine monophophates synthesis  (Pyrimidines)

·         If don’t have THF, can’t make derivative and therefore can’t convert _______ to _________.

· See Picture in notes

Antifolates: dihydrofolate reductase (DHFR):

·         Methotrexate and trimethoprim (component of bactrim) target ______________ blocking the formation of __________ and __________.

o   Net effect, conversion of ____ to _____ is blocked.

o  How does this effect DNA sythesis?

·         DHFR, THF, THF dervivatives

·         dUMP to TMP

·  No TMP, no TTP, no DNA (Need TMP to make TTP)

·         In addition to blocking DHFR, MTX can also bind directly to Thymidylate synthase, mimicking methylene THF.  This is related to the use of MTX in the treatment of?

5-Fluorouracil and Thymidylate synthase:

·         5-Fluorouracil (5-FU) is an analog of ________ and ________.

· 5-FU targets Thymidylate synthase…. What form do you think 5 FU needs to be in to accomplish this?

·         Thymine and Uracil

·  Has to look like dUMP (5-F-dUMP)

5-F-deoxyuridine monophosphate (5-F-dUMP):

·         Reminds you of __________.

·         So in the reaction catalyzed by thimidylate synthase, 5-F-dUMP mimics _______.

· Explain why 5-F-dUMP is a suicide inhibitor.

·         dUMP

·         dUMP

· There is a F present (instead of CH3) enzyme gets stuck and can’t displace the F to add CH3.  The complex can’t fall apart.

5FU and TMP formation:

·         _________ ________ is not able to methylate 5-F-dUMP

o   Sustained inhibition of the ________.

o   Decreased production of TMP, which means what with respect to TTP?

o And thus we stop _______ synthesis

·         Thymidylate Synthase

·         Enzyme

·         No TMP, no TTP, no DNA

·  DNA (will hear that cells experience a Thymidine less death)

5-FU activation pathway: uses pyrimidine salvage enzymes:

·         Realize that the salvage pathway is taking ______ to ________, this is the same for 5FU.

·         What is the active metabolite?

·         In the end everything leads to ____________

·         What does 5-F-dUtP have the potential to get incorporated into?

· What does 5-FUTP have the potential to get incorporated into?

·         Uracil to UMP

·         5-F-dUMP

·         5-F-dUMP

·         DNA

·         RNA

5-FU degradation and elimination: follows the same path as discussed with ________.

·         This is where ________ patients come into play?

·         Can cancer cells get around 5FU?

·  Patients without ______ will have increased 5FU.

·         Uracil

·         Polymorphic

·         Yes

·  DPD (dihydropyrimidine dehydrogenase)

5-FU degradation and elimination

·         Patients that lack or have limited DPD activity will not clear _____ effectively.  These people are at risk for significant and potentially life threatening effects.

·  What impact would high DPD activity have?

·         5FU

· Have increased metabolism so therapy is decreased.

Drugs that mimic endogenous nucleotides (nucleotide analogs):

·         These agents interfere with endogenous nucleotide usage:

o   Can be incorporated into _____ as “false” nucleotides, causing DNA chain termination.

o   Can inhibit ______ _______ functions, including DNA ________.

o Can inhibit _______ _______.

·         DNA

·         DNA polymerase, repair

·         RR

· Or can have any of the three combinations

Some examples of drugs that mimic endogenous nucleotides:

·        _________ and ________; anti-virals

·        ________ and ________; anti-cancer

·        Based on structure, these agents are __________ analogs.

·         Zidovudine (HIV), Acyclovir (Herpes)

·         Gemcitabine and clofarabine

·         Nucleoside

· Nucleotide

·        Have to phosphorylate.  First phosphorylation step is rate-limiting by specific kinases.

·        MPà DPà TP

Zidovudine

·        Converted to Zidovudine-MP by _______ _______.

·        Sequential phosphorylation to the _______, the active form.

·        Thymidine Kinase

·        Triphosphate

Zidovudine

·        HIV reverse transvriptase (RNA directed DNA polymerase), uses Zidovudine-TP as if it were ________ and incorporates it into the growing viral DNA.

·        TTP

Acyclovir: antiviral: herpes virus:

·        Acyclovir is a ________ _________

·        What type of analog?

·        Converted to Acyclovir-MP by _________ _______ _________.

·        Sequential phosphorylation to the _______, the active form.

·        Chain Terminator

·        Guanisine or deoxyguanise, but acts as if it were deoxyguanise

·        Herpes Thymidine kinase

Acyclovir: antiviral: herpes virus:

·        Herpes viral DNA polymerase uses acyclovir-TP as if it were ________ and incorporates it into the growing viral DNA.

·        dGTP

Gemcitabine: Cancer

·        What type of analog?

·        Most like what base?

·        Converted to gemcitabine-MP by _________ _________.

·        Sequential phosphorylation to the _______, the active form.

·        Could be a ribo or deoxy analog, but acts most like deoxy.

·        Cytidine

·        Deoxycytidine Kinase (DCK)

Gemcitabine: Cancer

·        Inhibits DNA polymerase: gemcitabine-TP mimics ______________.

·        Additional effects:

o       Inhibits _______ _______

o       Inhibits _________ _________

o       Gemcitabine-DP inhibits _______ ______

o Will it stop gain elongation?

·        dCTT (deoxy Cytidine triphosphate)

·        dCMP deaminase (dCMPàdUMP), protects from elimation

·        CTP synthetase (UTPà CTP), protects from competetion

·        RR, makes difficult for cell to overcome drug.

Clofarabine: Cancer

·        Analog of ___________.

·        Converted to clofarabine-MP by _________ _______.

·        Sequential phosphorylation to the _______, the active form.

·        Will chain elongation still occur?

·        Deoxyadenosine

·        Deoxycytidine kinae (DCK)

·        Triphosphate

Clofarabine: Cancer

·        Inhibits DNA polymerase, clofarabine-TP mimics _______

·        Additional effects:

o       Clofarabint-TP inhibits _____________ ________

·        dATP (master switch for RR)

·        RR

[image]

The above structure is that of tenofovir, an antiviral used in the treatment of HIV infection.  Based on the structure, you would classify as?

Both gemcitabine and clofarabine inhibit ribonucleotide reductase yet gemcitabine only affects formation of

Clofarabine mimics dATP;

review regulation of ribonucleotide reductase to understand the relationship between dATP and formation of all deoxynucleotides 

The active metabolite of 5-FU is _____________.

The active metabolite of 5-FU is 5-F-deoxyuridine monophosphate.

This metabolite inhibits formation of thymidine monophosphate by mimicking deoxyuridine monophosphate 

A cancer patient being treated with mercaptopurine develops gout.  Why should allopurinol be used with

By blocking xanthine oxidase, allopurinol inhibits the elimination of mercaptopurine

thereby enhancing activation of mercaptopurine; puts the patient at risk to experience significant toxicity

6) Dihydropyrimidine dehydrogenase activity is involved in the degradation and elimination of the anti cancer

5-F-uracil 

False; Make sure you understand why this is false 

Purine nucleotides 

Guanosine and deoxyguanosine nucleotides;

synthesis of RNA and DNA will also be affected 

leucovorin is used to “rescue” patients from excessive toxicity associated with methotrexate.  Based on

Review the mechanism by with methotrexate (MTX) affects the synthesis of tetrahydrofolate (THF) and

consequently, the effect on IMP and TMP synthesis.  How would leucovorin restore IMP and TMP synthesis?