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Published on: December 14, 2016
Mycobacterium tuberculosis dihydrofolate reductase reveals two conformational states and a possible low affinity
Marcio Vinicius Bertacine Dias1, Petros Tyrakis2, Romenia Ramos Domingues3
1Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QA, UK; Laboratório Nacional de Biociências (LNBio), Centro de Nacional de Pesquisa em Energia e Materiais, Campinas, (CNPEM), São Paulo 13083-100, Brazil.
Abstract:
Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.
Insights
Researchers investigated Mycobacterium tuberculosis dihydrofolate reductase (MtDHFR) inhibition. Structural and calorimetric studies reveal low affinity for classical antifolates, guiding new antibiotic design for tuberculosis.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Tetrahydrofolate (THF) biosynthesis inhibition is crucial for treating cancer and infections.
- Dihydrofolate reductase (DHFR) is a key enzyme in THF synthesis, but no DHFR inhibitors are approved for tuberculosis treatment.
Purpose of the Study:
- To structurally and functionally analyze Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors.
- To understand the basis for weak inhibition of MtDHFR by antifolate drugs.
- To provide insights for designing novel antibiotics targeting MtDHFR.
Main Methods:
- X-ray crystallography to determine the structures of MtDHFR-inhibitor complexes.
- Site-directed mutagenesis (Y100F mutant) to probe enzyme-inhibitor interactions.
- Isothermal titration calorimetry (ITC) to measure binding affinities.
Main Results:
- Structural and calorimetric analyses revealed low binding affinity of classical antifolates (trimethoprim, pyrimethamine, cycloguanil) to MtDHFR.
- The Y100F mutation further elucidated the low affinity of MtDHFR for these inhibitors.
- Key interactions within the MtDHFR active site were defined through complex structures.
Conclusions:
- Classical DHFR inhibitors exhibit weak inhibition against Mycobacterium tuberculosis DHFR.
- Understanding these interactions is critical for developing effective anti-tuberculosis drugs.
- The study provides a structural basis for designing improved MtDHFR-targeted antibiotics.
