Photosubstitutionally active ruthenium complexes show high potential as prodrugs for the photoactivated chemotherapy (PACT) treatment of tumors. One of the problems in PACT is that the localization... Show morePhotosubstitutionally active ruthenium complexes show high potential as prodrugs for the photoactivated chemotherapy (PACT) treatment of tumors. One of the problems in PACT is that the localization of the ruthenium compound is hard to trace. Here, a ruthenium PACT prodrug, [Ru(3)(biq)(STF-31)](PF6 )2 (where 3 = 3-(([2,2':6',2″-ter- pyridin]-4'-yloxy)propyl-4-(pyren-1-yl)butanoate) and biq = 2,2'-biquinoline), has been prepared, in which a pyrene tracker is attached via an ester bond. The proximity between the fluorophore and the ruthenium center leads to fluorescence quenching. Upon intracellular hydrolysis of the ester linkage, however, the fluorescence of the pyrene moiety is recovered, thus demonstrating prodrug cellular uptake. Further light irradiation of this molecule liberates by photosubstitution STF-31, a known cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, as well as singlet oxygen via excitation of the free pyrene chromophore. The dark and light cytotoxicity of the prodrug, embedded in liposomes, as well as the appearance of blue emission upon uptake, were evaluated in A375 human skin melanoma cells. The cytotoxicity of the liposome-embedded prodrug was indeed increased by light irradiation. This work realizes an in vitro proof-of-concept of the lock-and-kill principle, which may ultimately be used to design strategies aimed at knowing where and when light irradiation should be realized in vivo. Show less
Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better... Show moreOpsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize QuasAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light. Longer red-light exposure resulted in the formation of a long-lived photoproduct similar to pink membrane, previously found to be a photoproduct of the BR O intermediate with a 9-cis retinylidene chromophore configuration. However, unlike QuasAr2 whose O-like state is stable in the dark, NovArch exhibits an O-like state which slowly partially decays in the dark to a stable M-like form with a deprotonated Schiff base and a 13-cis,15-anti retinylidene chromophore configuration. These results reveal a previously unknown complexity in the photochemistry of OTVSs including the ability to optically switch between different long-lived states. The possible molecular basis of these newly discovered properties along with potential optogenetic and biotechnological applications are discussed. Show less
Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals.... Show moreOpsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT-Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all-trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single-/double-bond system similar to the O-intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13-cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR-OTVSs, especially for applications such as deep brain imaging. Show less
Proteorhodopsin is a light‐driven proton pumping membrane protein related to bacteriorhodopsin. It contains an all‐trans retinal A1 chromophore covalently bound to a lysine residue via a protonated... Show moreProteorhodopsin is a light‐driven proton pumping membrane protein related to bacteriorhodopsin. It contains an all‐trans retinal A1 chromophore covalently bound to a lysine residue via a protonated Schiff base. In this study, we exploited density functional theory (DFT) calculations to investigate the retinal binding pocket in the dark state and after mimicking photoisomerization. The model of the binding pocket is constructed incrementally by adding the residues near the retinal that are necessary to ensure a stable protonated Schiff base. The presence of a few water molecules near the Schiff base turns out to be an essential feature of the model. The absorption properties are then studied using time‐dependent DFT (TDDFT) and compared to experimental data to further validate the structural model and to assess the accuracy of the computational setting. It is shown that TDDFT is able to reproduce the main absorption peak accurately and to quantitatively determine the spectral shift induced by substituting the native all‐trans retinal A1 chromophore with different retinal analogues. Moreover, ab initio molecular dynamics simulations are performed to investigate the vibrational spectra of our models before and after isomerization. Specific differences in the vibrational spectra are identified that provide further insight into experimental FTIR difference spectra. Show less
Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has... Show moreCalcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450 kJ m(-2) of UVA-1 (340-400 nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, gamma-interferon, IL-4 and IL-10 that are controlled by the Ca(2+)-Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors. Show less
Smit, N.; Musson, R.; Romijn, F.; Rossum, H. van; Pelt, J. van 2010
Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has... Show moreCalcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450kJm−2 of UVA-1 (340–400nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, -interferon, IL-4 and IL-10 that are controlled by the Ca2+–Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors. Show less
Smith, S.O.; Groot, H.J.M. de; Gebhard, R.; Lugtenburg, J. 1992
