Interaction of methotrexate metabolites with beef liver dihydrofolate reductase-I. Binary complex study

Area of Science:

• Biochemistry
• Enzymology
• Pharmacology

Background:

• Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism.
• Methotrexate (MTX) is a widely used antifolate drug that inhibits DHFR.
• Understanding the binding interactions of MTX and its metabolites is crucial for optimizing cancer therapy.

Purpose of the Study:

• To elucidate the differential inhibition of DHFR by MTX, 7-hydroxymethotrexate (7OH-MTX), and MTX polyglutamate derivatives (MTX-G1, MTX-G2).
• To characterize the binding parameters and thermodynamic properties of these interactions with beef liver DHFR.

Main Methods:

• Direct determination of binding parameters using fluorescence titrations to calculate association constants.
• Microcalorimetric measurements to assess thermodynamic parameters of enzyme-inhibitor interactions.

Main Results:

• MTX and its polyglutamate derivatives exhibit nearly identical binding parameters to DHFR.
• 7OH-MTX demonstrates distinct binding characteristics compared to MTX.
• Electrostatic forces play a less dominant role in 7OH-MTX binding.
• Complex formation with 7OH-MTX induces a greater conformational change in the enzyme.

Conclusions:

• The polyglutamation of MTX does not significantly alter its binding affinity or interaction thermodynamics with DHFR.
• 7OH-MTX exhibits altered binding kinetics and thermodynamics, suggesting a different inhibitory mechanism.
• These findings highlight the importance of metabolite structure in DHFR inhibition and drug efficacy.