Screening of DHFR-binding drugs by MALDI-TOFMS

Abstract

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.

Novel Antifolates as Produgs for the Treatment of Melanoma

“…Since these first reports describing the inhibition of DHFR by tea polyphenols, several studies by us and other laboratories have reported that EGCG inhibits DHFR from a variety of biological sources (Navarro-Martínez et al, 2005;NavarroPerán et al, 2007;Hannewald et al, 2008;Kao et al, 2008;Spina et al, 2008;Sánchez-del-Campo et al, 2010a). Recently, a screening of DHFR-binding drugs by MALDI-TOFMS demonstrated that EGCG is an active inhibitor of DHFR and has a relative affinity between that of pyrimethamine and MTX (Hannewald et al, 2008). Fig.…”

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“…Pyrimethamine is a 248 Da lipophilic drug used to treat malaria for over 50 years and continues in this role today in 2021. It inhibits human dihydrofolate reductase (DHFR) [ 71 , 72 ]. Pyrimethamine’s Ki = 38 nM at DHFR is comparable to that of methotrexate, Ki = 2.3 nM, folinic acid Ki = 320 nM, and folic acid Ki = 830 nM [ 71 , 72 ].…”

Diagnosis of Biological Activities by Mass Spectrometry

“…The complex protein-ligand is analyzed by Mass Spectrometry and the direct detection of the ligand reflects its potential inhibition activity against the target. Screening tests have already been developed for the ligands of tubulin [2,3], dihydroxyfolate reductase (DHFR) [4] and recently for reversible and irreversible ligands of CDC25 phosphatases (isoforms A et C) [5]. This approach demonstrates its effectiveness with Madagascar periwinkle for tubulin, with Colchicum and green tea for DHFR and some promising plants are currently evaluated on CDC25s.…”