What do you guys think of this? Trying to figure out how this enzyme fits into possible life extension. This enzyme seems to be resveratrol main target. Even tho SIRT1 is a target of resveratrol you are actually targeting this enzyme much more. It is interesting to note that "Melatonin" has shown to increase the life span of mice.
may increase longevity; it has been shown to increase the average life span of mice by 20% in some studies.[35][36][37]
NRH:quinone reductase 2: An enzyme of surprises and mysteries
Fanny Vellaa, Gilles Ferrya, Philippe Delagrangeb and Jean A. Boutina,
, 
aPharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125, Chemin de Ronde 78290 Croissy-sur-Seine, France
<a name="aff2">bDépartement des Sciences Expérimentales, Institut de Recherches Servier, 11, rue des Moulineaux 92150 Suresnes, France
Received 6 July 2005; accepted 8 September 2005. Available online 25 October 2005.
Quinone reductase 2 has been discovered in 1961 and rediscovered in 1997. Because of its sequence homology with quinone reductase 1, it has been suspected to detoxify quinones. Ten years later, evidences begin to point to a versatile role of this enzyme. Indeed, QR2 is strongly suspected to be the molecular target of anti-malarian drugs such as chloroquin or paraquine, and of red wine-derived resveratrol that might be responsible for the so-called French paradox. It also is identical to the melatonin binding site MT3, and might therefore be a rationale explanation for the antioxidant role of melatonin. Finally QR2 might be implicated in the toxicity, in vivo, of quinones such as menadione. The present commentary attempts to summarize this information and discusses a series of hypotheses.
Keywords: Quinone reductase 2; Quinone reductase 1; Melatonin; Resveratrol; Chloroquine; Molecular pharmacology
4. A role for QR2 QR1 has a clear role in detoxification. For QR2, though, a similar role would be difficult to established on the basis of the currently available experiments.
4.1. Human genetic studiesTwo studies [60] and [61] reported an association between polymorphisms of QR2 and Parkinson's disease and schizophrenia. The polymorphism was in the form of an insertion/deletion of 29 nucleotide base pairs (bp) in the promoter region of QR2. According to the sequence involved, the authors speculated that this polymorphism would result in decreased production of the enzyme and may lead to an excess of the cathecholamine-derived o-quinones in the brain. Nevertheless, another group recently considerably modulated these conclusions [62]. It has been also reported that the promoter containing the 29-bp insertion polymorphism presented lower QR2 expression than that with the deletion [63]. Indeed, the insertion introduced a binding site to the transcription factor Sp3, which in this case acts as a repressor. Consequently, the deletion associated with either Parkinson's disease or schizophrenia leads to an excess in the production of QR2. This hypothesis has been confirmed in human fibroblasts from individuals with or without the deletion. The QR2 activity was statistically increased in the fibroblasts with the deletion. The authors concluded that higher QR2 activity might make individuals more susceptible to Parkinson's disease.
Similarly, Strassburg et al. [22] reported that in hepatocellular and biliary tissues, QR2 was down regulated, while QR1 was up-regulated, suggesting not only a differential regulation, but also, potentially a different role in carcinogenesis. QR2 might generate an harmful signal in cells, rendering its activity an activation/toxifying process. The impact of this activation process might become key on some disease developments. The availability of cellular models in which QR2 can be modulated will decipher the nature of the role of this enzyme, in particular in menadione-induced toxicity [57].
4.2. Mice deleted for QR2 Our initial KO mouse strain is currently transferred onto stable genetic backgrounds C57Bl/6 and also C3H/He, according to the fact that C57Bl/6 synthesize very low levels of melatonin. Meanwhile, we confirmed on the mixed genetic background (129Sv/C57BL/6) that organs from these QR2−/− mice were deprived of the melatonin binding site MT3 [59]. Long et al. [24] reported a major experiment for the field on their own KO mouse strain (on an unspecified genetic background). These mice, once challenged by toxic doses of menadione, survived, while treated under the same conditions, their wild-type littermates died. The difference was even more significant when the mice were co-treated with the co-substrate of QR2, NRH. The same type of challenge had been previously done by the same group on their own QR1−/− mice, and they reported the expected and opposed result, that menadione was far less toxic to wild-type mice than to QR1 KO ones [64]. These data clearly indicated that the earlier hypothesis according to which QR2 – by analogy to QR1 – was a detoxification enzyme can be challenged. A discrepancy remains in the literature, though. It is the remarkable gain of sensitivity to benzo(a)pyrene-induced skin carcinogenesis of QR2−/− mice [65]. These KO mice seem to respond with an increased development of skin tumors when benzo(a)pyrene or benz(a)anthracene were topically applied to their skin, as compared with the wild-type animals. Hence, the QR2 deletion conferred a greater susceptibility to carcinogenesis induced by these compounds. In wild-type mice, it is difficult to understand how to link the QR2 activity with the carcinogenesis of the compound, since benzo(a)pyrene inhibits the enzyme [10] and [13]. In QR2−/− knock-out mice, the apparent enhanced activity of the carcinogen cannot be linked to the enzyme, since it is not present. Nevertheless, it has been hypothesized that the deletion of the enzyme in mice would induce other antioxidant mechanisms of defence [65].
4.3. QR2 = MT3? While attempting to characterize the third melatonin binding site, MT3, Nosjean et al. purified QR2 by affinity chromatography and characterized some of its functions [58] and [66]. Because of the plasticity of phase 2 enzymes, particularly UGTs [67], we wondered if the apparent affinity of QR2 for melatonin was the reflect of an actual role of melatonin in the QR2 function. Using a series of different approaches, including the development of inhibitors [43] and [56] and QR2−/− mice [59], we attempted to understand this relationship. We demonstrated that the binding of melatonin in QR2 was possible at two distinct sites of the enzyme, including the binding site. As stated above (Section 4.2), all tissues from QR2−/− mice are depleted of melatonin MT3 binding sites [59]. Altogether, these data demonstrate that QR2 is indeed MT3. More questions, though, remain to be answered, among which the key one: is the specific interaction of melatonin with QR2 could explain the antioxidant properties of this hormone?
4.4. QR2 and malaria In two outstanding publications from the same group, Kwiek et al. [23] and Graves et al. [42] reported on the search of the molecular target(s) of the anti-malarian quinolines, chloroquine and quinacrine. Two enzymes were identified by both proteomic studies, and by affinity purification onto a quinoline-immobilized column. In a manner similar to resveratrol, recently (see below), or to melatonin, some time ago (see above), these authors identified QR2 as the major (and in some case unique) target of these compounds. Furthermore, the authors tested in several different ways the capacity of these compounds to inhibit the catalytic activity of QR2. In particular, using the naturally occurring co-substrate, N-methyldihydronicotinamide, they found chloroquine to have a Ki in the micromolar range, and quinacrine, in the 0.5 micromolar range, either with a recombinant or a blood cell-purified QR2. Furthermore, using an assay based on the quenching of the natural FAD-absorbance at 450 nm, they also reported these compounds as being very potent inhibitors. These authors suggested that inhibiting QR2 activity may affect the redox status of red cells. By enhancing the production of reactive oxygen species derived from endogenous quinones – normally detoxified by QR2 – the inhibitor would create an inhospitable environment for the parasite, as it is sensitive to reactive oxygen species [68]. This particular hypothesis was proposed in the context of QR2 being a detoxifying enzyme. This might be a major breakthrough in the search of anti-malarian medicines.
4.5. QR2 = resveratrol main target? A recent publication showed that using resveratrol [70] as bait in an affinity chromatography step, it was QR2 that was fished out from biological sources. As for quinolines or for melatonin, whether these observations are specific or linked to the function(s) of QR2 remains to be clarified. These reports [57] and [70] on resveratrol, were extremely convincing that this compound, believed to be at least in part responsible of the so-called 'French paradox' [71] might, by its inhibition of QR2, protect mammals from over-exposure to activated reactive oxygen species. These reports were based on a series of observations, particularly the co-crystallisation of QR2 with resveratrol as well as the construction of a K562 cell line stably expressing a specific RNAi against QR2. These cells were resistant to menadione stress, as were the wild-type cells when treated by resveratrol while the naïve cells were killed by this menadione treatment. All this information convincingly pointed out to a central role of QR2 in the action of resveratrol. Even more recent data from the same group [72] showed that resveratrol might be an inducer of QR2, a feature that would complicate further the picture, since the same compound would be an inducer (enhancing the expression of the protein) and an inhibitor (impairing the enzyme catalytic activity).
4.6. Is QR2 a nitroreductase? Amongst the surprising compounds specifically substrates of QR2, is the cytotoxic pro-drug, CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. This compound seemed to be specifically activated by QR2 into a potent cytotoxic compound [45] and [73] R.J. Knox, P.J. Burke, S. Chen and D.J. Kerr, CB 1954: from the Walker tumor to NQO2 and VDEPT, Curr Pharm Des 9 (2003), pp. 2091–2104. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (16)[73]. The level of specificity is so high, that only added co-substrates of QR2 rendered CB1954 cytotoxic. This compound entered phase 1 study for cancer therapy [74]. In addition, several lines of evidence suggest a strong relationship between menadione, ubiquinone and nitroreductase, as can be extrapolated from Grimaldi et al. [75].
5. Hypotheses Historically, QR2 has been considered to be a detoxifying enzyme, by analogy with QR1, and some initial experimental data supported this fact.
<h4 class="h4">5.1. Pro (QR2 protects)</h4>Several experimental reports argued for a protective role for QR2:
By inhibiting QR2, it seems that anti-malarian compounds such as quinacrine favour the red blood cell oxidative stress leading to the death of the parasite, which is very sensitive to it.By deleting QR2 it seems that mice become increasingly susceptible to polycyclic aromatic hydrocarbon-induced skin carcinogenesis.
By inducing QR2, resveratrol seems to stabilize p53, which in turn would contribute to the diminution of growth in melanoma cells [72].
5.2. Con (QR2 activates) Recently, data obtained from the KO mice (QR2−/−) seemed to indicate that without QR2 – but in a very complex system (a whole animal) – menadione was not toxic anymore. This fact is stressed when a co-substrate of QR2 was co injected to these mice. These data seemed to argue against a detoxifying role of QR2.
These data were further backed up by data on resveratrol since this compound inhibits the enzyme, rendering treated cells as poorly sensitive to menadione as cells in which QR2 has been deleted by RNAi [57]. The decreased sensitivity to menadione of cells depleted of QR2 has been confirmed in our lab [69]. A parallel might be established with melatonin to some extent, since the inhibiting capacity of this neurohormone is about 10–100 times less potent than resveratrol and seems to be co-substrate dependent.
5.3. Coda: what does QR2? In view of these data, it is hard to reconcile this increase susceptibility to skin carcinogenesis with a decrease sensitivity to menadione toxicity in the same QR2-deleted mice. Several factors might interfere with interpretation of the hard data: for instance, the substrate actually recognized in some situation by the enzyme (when it is still there). Not only it might not be the same, but its nature is still unknown. Even more perturbing is the exact nature of the co-substrate. It might be that NRH is mainly produced during NADH breakdown, in which case, the oxidative stress might be responsible of the awake of QR2 activity, if it helps cleaving NADH into NRH, while in standard situations, no co-substrate is available for QR2 to function.
A provocative hypothesis might be, in the red cells exposed to parasite, that QR2 renders the oxygen species produced during an oxidative stress, more aggressive and lethal for the cells. By limiting this process, QR2 inhibitors would limit the toxicity of the oxidative stress induced by parasite or other events leading to oxidative stresses. Obviously, this hypothesis needs to be confirmed by further hard data coming from KO animals, potent, and selective QR2 inhibitors [57], etc. For instance, it would be interesting to compare the capacity of these various QR2 inhibitors (such as S 26237, chrysoeriol, resveratrol, melatonin, quercetin, and quinacrine and more are becoming available), to limit or decrease the menadione-dependent toxicity in a unique cellular system. However, we should keep in mind that other explanations of the anti-malarial properties of quinolines, unrelated to the QR2 enzyme, have also been proposed [76].
Finally, and maybe most importantly, one should solve the co-substrate question. We can further tentatively hypothesise that QR2 behaviour differs as a function of its available co-substrate(s). We could question whether these co-substrate are similarly available in various cell types or/and in various physio-pathological conditions – ageing, oxidative stress, etc. By being present in a given condition, a given co-substrate would favor the formation, by QR2, of variously active and/or toxic molecular species.
6. Conclusion It might be difficult to conclude in a few sentences the various points raised along the present commentary. The main question, to our view point, remains the nature of the activity QR2 actually catalyses. As long as this question will not be clearly answered, it will remain complicated to gather all the various observations inside a unique frame according to which the role of QR2 would be explained in such various physio-pathological situations, as in the melatoninergic system, the malaria aetiology and the quinone-induced toxicity. Furthermore, Knox et al. <a name="bbib43">[43] stated that QR2 could be considered as a human NRH-dependent nitroreductase. To our knowledge, nitroreduction is not a function catalysed by mammals. The confirmation of this fact would be a complete change in perspective on how to study this enzyme. Despite a rather limited number of PubMed® entries, QR2 (unfortunately a.k.a. NQO2), remains a source of many questions: is QR2 a detoxification enzyme? As such, is its inhibition by either anti-malarian drugs or by resveratrol, a key explanation in the benefit of these compounds? Is melatonin binding a 'side feature', linked to the rather large size of the binding site (the catalytic site), or is melatonin acting like those compounds that provide some antioxidant protection? Obviously, the discovery of new chemical entities and the construction of new molecular tools (RNAi and KO cells, KO animals, directed mutagenesis, etc.) will help to answer those questions and build a comprehensible frame.
Edited by Hedgehog, 28 April 2008 - 04:21 PM.
