Medicinal Chemistry of Methotrexate Flashcards
Describe the structure of folic acid (folate/vitamin B9).
- Pterin (bicyclic heteroaromatic ring; double ring)
- (para) p-aminobenzoic acid
- glutamate tail (x10 glutamic acid residues; amide linkages bonding monomers together)
Humans cannot biosynthesise Vitamin B9 (folic acid/folate); how is it obtained?
- Through our diet
- Vital as precursor for thymidylate (DNA synthesis), guanylate/adenylate (DNA + RNA synthesis) and methionine (AA; protein synthesis)
How is cellular uptake of folic acid (folate/vitamin B9) achieved, given its v. polar glutamate tail?
- Polar molecules not normally able to cross cell membranes (CO2- in folic acid)
- Reduced Folate Carrier (RFC) is able to actively transport folic acid across cell membranes into the cell
How is folic acid (vitamin B9/folate) retained within cells once imported by RFCs?
Via Folyl Polyglutamyl Synthase (FPGS; enzyme)
- Adds further glutamic acid monomers onto glutamate tail; extending polymeric tail
- With each additional glutamic acid unit added by FPGC, folic acid becomes more polar (one more CO2- with each monomer)
»> Thus less likely to efflux (RETAIN)
What is folic acid (vitamin B9) converted to once in the cell? By what? What is the structural difference?
- Folic acid (folate) is reduced to THF (tetrahydrofolate; has 4 hydrogenated Nitrogens)
- Via DHFR (dihydrofolate reductase)
- Catalyses reduction of pterin ring; nitrogens of ring on the right are reduced (+H), ring becomes saturated (from prior 2x N = C’s)
What is the most important structural feature of THF that allows it to carry out its role?
- Nitrogens bridging pterin ring and p-aminobenzoic acid act as ‘pincers’ and are able to grab hold of C atoms
- THF thus able to act as a carrier for 1-C units/atoms in the biosynthesis of many molecules
What is the role of THF? Where is the 1-C unit it can carry go to?
Biosynthesis of:
- Thymidylate (nucleotide; monomer for DNA synthesis); C for methyl group
- Guanylate/adenylate (nucleotides; monomers in DNA + RNA synthesis etc); C’s for purine rings
- Methionine (AA; protein synthesis); C for methyl group
Describe the Biosynthesis (cycle) of Thymidylate (conjugate base of thymidine).
dUMP + THF = dTMP + DHF
- dUMP (deoxyuridine monophosphate) is converted to dTMP (deoxythymidine monophosphate)
- Reductive methylation of dUMP catalysed by Thymidilate Synthase (TS) enzyme
- N5, N10-methylene THF (THF w/C pincered) provides the carbon (C) for methyl attachment to 6-membered ring
- DHF (dihydrofolate) produced as by-product after C atom donation
- DHF is recycled (is not a 1-C carrier; no other use)
- DHFR reduces DHF back to THF (recycled; the same DHFR that converts folic acid to THF; which is a reduction that normally yield 2x hydrogenations)
- THF can now pick up another C (carbon), forming N5, N10-methylene THF, which can now reduce dUMP again to give it a methyl (reductive methylation) to form dTMP
What is the name of the THF form that has a carbon ‘pincered’?
N5, N10-methylene THF
What is the role of DHFR? How does it bind folate (vitamin B9/folic acid)?
- Biosynthesis of and recycling of THF
- Folate is of an L-shape configuration when bound in DHFR at the junction of the pterin ring and p-aminobenzoic acid
What is MTX, methotrexate?
- Antimetabolite
- Potent competitive inhibitor of DHFR
»> Binds to DHFR 10^4 times more strongly than its substrate (folate/folic acid, DHF/dihydrofolate)
What is MTX potent-af?
- MTX has a pKa of 5.7; folate a pKa of 2.4
- Thanks largely to its amino group on pterin-like ring of MTX; where folate has a C=O (carbonyl) group
- Pterin-like ring much more BASIC as a result; hence pKa, thus significant protonation of MTX (nitrogens) occur at pH 5.7 (or lower); where in folate, pH would have to drop to 2.4 first
- Protonation results in hydrogen bond formation w/Glu residue of DHFR active site
- MTX is protonated in DHFR active site, folate isn’t (due to MTX’s increased basicity; protonated at higher pH; DHFR active site environment)
- pTerin-like ring of MTX adopts different conformation when bound to DHFR active site too
- Produced ‘charge-assisted’ H-bonding; not just ordinary ones (- charge on Glu of DHFR, + charge on MTX’s pterin-like ring)
»> Much stronger interaction as a result (much greater affinity)
What is the purpose of the pTerin ring normally in folate binding to DHFR?
- pTerin ring is anchored to the DHFR enzyme via-H bonding
- Stabilises folate in active site (H-bonding does) for main conversion of folate to THF (2x hydrogenations = TetraHydroFolate)
MTX is bants yh and binds to DHFR with 10^4 affinity and all; but how does it even get into cells, given its high polarity?
- MTX is v. polar (O- as well as CO2- in folate)
- BUT, MTX is also taken up by RFC (Reduced Folate Carrier); hijacking mechanism of folate
- Due to similarity in structure
How does MTX stay in the cell to do its 10^4 binding affinity to DHFR ting?
- Glutamate tail of MTX (like folic acids’) is ALSO substrate for FPGS (folyl polyglutamyl synthase)
- Thus adds further glutamic acid residues to the MTX chain, further increasing its polarity (and decreasing its likelihood of efflux; thus accumulation of MTX in-cell)
- Due to its structural similarity (as per RFC cellular uptake)
