antifolates

• highlights of the folic acid cycle

  • folic acids, (dihydrofolate (DHF), tetrahydrofolate (THF), 5,10-CH2-THF, along w/ a number of others) are essential nutrients for the one-carbon transfer reactions in the synthesis of methionine and certain DNA bases
  • depletion of the folic acids will inhibt the synthesis of protein and DNA, and consequently will either inhibit the cell growth or lead to cell death (in fast growing cells)
  • eukaryotic cells can uptake folic acids
  • most bacteria cells cannot uptake folic acids; they have to make them
• prototype antifolate sulfonamides

  • basic of selective toxicity of sulfonamides on bacteria:
  • sulfonamides inhibit 2 enzymes for the biosynthesis of DHF, the source for all the bacterial folic acids
  • most bacteria cells cannot uptake DHF from the environment (lack of folate transporters)
  • biosynthesis of DHF is vital to their survival
  • eukaryotic cells take DHF from the environment (though diet)
  • they do not synthesize DHF for their survival
• mechanism of sulfonamides: inhibition of the synthesis of HDF/THF

  • mech #1: sulfonamides mimic the structure of PABA
  • they bind to and inhibit dihydropteroate synthetase
  • mech #2: in some strains of bacteria, a sulfonamide can be processed by dihydropteroate synthetase into a false metabolite which binds to and inhibits down-stream folic acid synthesis enzymes such as DHF synthetase
  • sulfonamides inhibits the metabolism of cells and are thus called anti-metabolites
  • since they inhibit folic acid-related metabolism, they are also examples of anti-folates
• dihydrofolate reductase (DHFR)

  • required in both microorganisms and human for the synthesis of THF and eventually 5,10-CH2-THF, the essential folate for one-carbon transfer reactions
  • good news: there is significant topological difference btwn human DHFR and those from microorganisms
• trimethoprim and pyrimethamine

  • a structural analog of hydrofolate
  • a potential inhibitor of bacterial dihydrofolate reductase (Ki = 5 - 15nm) but a very weak inhibitor of human DHFR (Ki - 3 * 10^5 nm)
  • can exhibit synergistic effect and reduce the chance of drug resistance if used in combinaiton w/ sulfonamide drugs (eg, trimethoprim-sulfamethoxazole)
  • pyrimethamine is another DHFR inhibitor
  • it has similar structure to trimethoprim and the same MOA
  • active against DHFR parasite
• dapsone, a "sulfone drug" structurally similar to sulfonamides

  • sulfones were studied after the success of sulfonamides
  • MOA: same as that of sulfonamides
  • both possess approximately the same range of anti-bacterial activity and both are antagonized by PBA
  • sulfone drugs are most important agents to heart leprosy
• trimethoprim-sulfamethoxazole (co-trimoxazole, bactrim, septra)

  • this is a classical example of combination therapy
  • rationale: inhibition of two sequential steps in a vital biochemical pathway to give synergistic antibacterial effect and less chance of resistance
• resistance to sulfa drugs

  1. expression of a mutated dihydropteroate synthetase w/ lowered affinity to sulfa drugs

  • plasmids encoding such a mutated enzyme can be passed from one strain to another (major)

  1. increased production of the substrate (PABA) to antagonize the competitive inhibition

  • eg, some resistant staphylococci synthesize 70 fold as much PABA as a sensitive strain (major)

  1. decreased drug permeation or increased efflux of the drug
  2. finding alternative pathways to obtain the folic acids or their precursors (less common)
• resistance to trimethoprim

  • expression of a mutated DHFR w/ lowered affinity to trimethoprim
  • plasmids encoding such a mutated enzyme can be passed from one strain to another
• pKa values for selected sulfonamides

  • a stronger electron withdrawing group leads to a lower pKa value of the sulfonamide nitrogen

  
• pKa values for selected sulfonamides (cont.)

  • sulfonamide drugs are HA type weak acids
  • the pKa of a sulfonamide drug has profound impact on its potency, biodistribution and solubility

  
• the unionized form (0 charged) of sulfonamides permeates across the lipid bilayers

  • many drugs are weakly acidic or weakly basic compounds
  • only their uncharged form (less hydrophilic and more lipophilic) can permeate the lipid bilayers
  • eg, absorption through the gut; penetration into the cells
  • otherwise a transporter or channel protein is needed
  • a low pH and a high pKa of sulfonamides (HA type drug) favor their penetration across bacteria cell membranes
• possible adverse effects of sulfonamide drugs

  • hypersensitivity (~ 5% of pts)
  • kidney damage cause by crytsalluria (precipitation of drug in the kidney)
  • hematological adverse
  • GI discomfort and nausea
  • today, sulfonamide drugs are used less than they used to be due to their adverse effects and the occurrence of resistance
• pH in different parts of the body

  • stomach: ~ 2
  • small intestine lumen: ~ 6
  • big intestine lumen: ~8
  • blood circulation: 7.32 - 7.42
  • extracellular fluid: ~ 7.4
  • cytosol: ~ 7.2
  • urine: ~ 6 in normal conditions
• SAR of sulfonamide drugs

  • the free aniline (benzyl amine) group is required for the enzyme binding and inhibition
  • any modifications on this group abolishes the anti-bacterial activities
  • a low pKa favors the binding of the drug to dihydropteroate synthetase
  • a high pKa favors the permeation through GI and bacteria cell membranes
  • a low pKa decreases the risk of crystallura
  • balancing all the above factors, newer sulfonamides that are more commonly used in the clinic have pKa values in range of 5 - 7.4
• approaches to reduce the risk of sulfa drug crystal formation in kidney

  • greatly increase urine flow
  • Increase the pH of urine by taking sodium bicarbonate, etc.
  • developing sulfonamide drugs w/ lower pKa values and hence higher water solubility
  • mixing different sulfonamides to achieve an appropriate total dose