Light‐Wavelength‐Based Quantitative Control of Dihydrofolate Reductase Activity by Using a Photochromic Isostere of an Inhibitor

Abstract

Photopharmacology has attracted research attention as an ew tool for achievingo ptical control of biomolecules, following the methods of caged compoundsa nd optogenetics. We have developed an efficient photopharmacological inhibitor-azoMTX-for Escherichia coli dihydrofolate reductase (eDHFR) by replacing some atoms of the originall igand, methotrexate, to achievep hotoisomerization properties. This fine molecular design enabled quick structural conversion between the active "bent" Z isomer of azoMTX and the inactive" extended" E isomer,a nd this property afforded quantitative control over the enzyme activity,d epending on the wavelength of irradiating light applied. Real-time photoreversible control over enzyme activity was also achieved.

Quantitative Imaging of Labile Zn2+ in the Golgi Apparatus Using a Localizable Small-Molecule Fluorescent Probe

“…Fractions containing the His-tagged HaloTag were collected and repeatedly ultra-filtered for buffer exchange to 50 mM Tris-HCl (pH 8.0) containing 4% glycerol. Cleavage of the His-tag sequence in the obtained protein and subsequent HisTrap HP column purification were conducted according to the previously described method using the His-tagged TEV protease (Mashita et al, 2019). Briefly, the His-tagged HaloTag (0.90 mM) was incubated with His-tagged TEV protease (9.0 mM) at 4 C for 25 h. The digestion solution was passed through a HisTrap HP column to remove any protein/peptide containing the His-tag.…”

Hypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors

“… 40 Moreover, modulating DHFR activity with light has seen promising results in previous studies. 8 , 20 , 21 …”

“…A related principle has been successfully applied to the azologization of methotrexate both in bacterial 20 and human DHFR. 21 …”

Comparative EPR Study on the Scavenging Effect of Methotrexate with the Isomers of Its Photoswitchable Derivative

“…PHX is a photoswitchable azobenzene analogue of MTX that has been synthesized and described by C. Matera et al [ 6 ] and by Mashita et al [ 12 ]. PHX contains a diazene stereogenic unit and its pharmacological activity is higher in its cis state than in the more thermodynamically stable trans state.…”