Screening of DHFR-binding drugs by MALDI-TOFMS.
The class of antimetabolite chemotherapeutical agents has been used to treat cancers in humans for almost 50 years and gives significant results by binding dihydrofolate reductase (DHFR), a key enzyme in DNA synthesis. Therefore, finding new active compounds inhibiting DNA synthesis through their binding to DHFR is of prime interest. The aim of this work is to describe a protocol designed to study the binding of compounds to DHFR. This screening protocol involves matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) detection of target-bound compounds. Firstly, a screening protocol is developed and proves to be a simple, fast, and specific method to characterize the binding capability of a compound. Secondly, the possibility of determining the relative affinities of DHFR-binding compounds by comparing MALDI-TOFMS data is discussed. A ratio is calculated for a compound X such as R(X) = A.I.(denaturation)(X)/A.I.(direct)(X) (where AI(direct) and AI(denaturation) are the average absolute intensities of a binding compound X before and after denaturation of DHFR). It is shown that by using this protocol, one can characterize the strength of the binding of different compounds. These two strategies are then applied to screen green tea (Camellia sinensis) extracts for DHFR-binding compounds, and epigallocatechin gallate is shown to be an active compound with a relative affinity between those of pyrimethamine and methotrexate.
Laboratoire de Spectrométrie de Masse et de Chimie Laser, 1, Bd Arago, 57078, Metz Technopôle 2000 Cedex 03, France.
Paul
P
Hannewald
Benoît
B
Maunit
Jean-François
JF
Muller
D000970:Antineoplastic Agents / Q000737:chemistry; D002392:Catechin / Q000031:analogs & derivatives; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005493:Folic Acid Antagonists / Q000737:chemistry*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013662:Tea; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D013662:Tea; D002392:Catechin; C045651:epigallocatechin gallate; D013762:Tetrahydrofolate Dehydrogenase