Flavins photoactivation of prodrugs: let’s shine a light

Title:  Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

Authors: Silvia Alonso-de Castro, Aitziber L. Cortajarena, Fernando López-Gallego, Prof. Luca Salassa

Year: 2018

Journal: Angewandte Chemie international Edition

DOI: 10.1002/anie.201800288

In my last bite, we looked at the possible uses of nanographene as photodrug. Here we explore another photodrug produced within the cell via a “bioorthogonal” reaction.

Bioorthogonal chemistry is a new and exciting branch of chemistry,[1] studying selected chemical reactions happening within the cellular environment without any interferences from other biological components. In fact, when a drug is introduced into a cell, it is important that it doesn’t react with components that it is not supposed to. This could change its activity and give undesired results, as will soon be described for cisplatin.

In this paper, we are shown an example of these exciting reactions. The authors explore the selective activation of organometallic prodrug complexes — molecules that act as a drug once activated by flavoproteins like FAD (flavin adenine dinucleotide).

Flavins are ubiquitous molecules, usually associated (cofactors) with some proteins (flavoproteins), intervening in many enzymatic processes within the cells. The cofactors are small molecules that are necessary to the activity of an enzyme. The basic unit of flavins is called isoalloxazine with R=H: the derivatization of the R group gives origin to multiple species that are involved in redox reactions within the organism (Figure 1).

Figure 1: Equilibrium between the oxidized and reduced forms of flavins

An example is FAD that can exist in multiple forms, according to its oxidation. FAD can accept 1 or 2 electrons (and 1 or 2 protons) to give FADH and FADH2, respectively. It can, therefore, oxidize multiple species and it is exploited in a vast number of reactions like the electron transport, during cellular respiration.

In this study, flavins are used as catalysts to activate some metal complexes after being irradiated at 460 nm (blue light) with an LED. This starts a reaction that, with the help of an electrondonor, can liberate the drug molecule from its scaffold (Figure 2). MES (2-(N-morpholino)ethanesulfonic acid) buffer at pH 6 and NADH (nicotinamide adenine dinucleotide) in PBS buffer at pH 7 were explored as electrondonors (the suggested mechanism is shown in Figure 2).


Figure 2: suggested reaction mechanism of  drug activation

After a preliminary study on different types of prodrugs activated by FAD, 2 were chosen for further experiments with flavoproteins (Figure 3).

Figure 3: Prodrugs that have been explored in this study

The two molecules are the precursors of cisplatin and, after the irradiation of the flavin, cisplatin is released and can perform its activity (produced in Figure 2). Cisplatin is a chemotherapy medication and works by binding to the DNA of the target cell (cancerous cell, in principle) and blocking its replication. The controversial issue is that it is not cell-selective, also killing the natural cells and having numerous side effects. Selective irradiation of the cancer cells after injection of the prodrug could help overcome them.

Within the cells, flavins are bound to some proteins either covalently or with Van der walls bonds. These bonds and the specific protein environment can change the redox properties of the flavins and their activity. With this in mind, the authors explored four flavoproteins with different flavin-binding pockets and verified how this characteristic could influence the activation of 1 and 2.

The four proteins and their characteristics are listed below:

  1. miniSOG uses flavin mononucleotide (FMN) to photogenerate singlet O2. The pocket is near the protein surface and there is a positive charge surrounding the FMN.
  2. NOX oxidizes NADH while generating H2O2 from O2. The pocket is neutral and on the surface of the protein.
  3. GOX oxidizes glucose to H2O2 and has a buried, negatively charged pocket.
  4. GR reduces glutathione in presence of NADH. Its flavin-binding pocket is buried and neutral. Moreover, it does not generate any reactive oxygen species.

Probably due to the binding pocket being so buried in the proteins, GOX and GR show the lowest catalytic activity towards compounds 1 and 2.

MiniSOG, upon photoirradiation, converted 1 and 2 in their photoproducts in the presence of MES and NADH without any discrimination on the substrate.

NOX showed the most interesting properties: it exhibited activity towards both substrates in the presence of MES after irradiation, being 7 times more reactive towards 2. With NADH it was also active in the dark completely converting 1 in less than 7.5 minutes. This behavior is influenced by the concentration of NADH and to the presence of O2 -that is NOX natural substrate.

Being NADH naturally present within the cell, this results can suggest that some flavoproteins may be involved in the conversion of the prodrug into the active species.

Notably, NOX and miniSOG maintained similar reactivity towards 1 also when incubated in cell cultural medium, effectively acting in a bioorthogonal way.

Although not exactly elucidating the mechanism of the prodrug activation by flavoproteins, the authors of this paper showed how the protein scaffold can influence these reactions, and how cellular components can selectively be exploited for chemotherapy.


[1] Acc. Chem. Res.201144 (9), pp 666–676

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