Influence of inhibitors on plant dry mass production

and 46) showed increased dry mass production; among those were 6 phthalazinone derivatives and 4 quinazolinone derivatives.

There was no general dose dependency detectable, besides for 64, which showed decreased dry masses for higher concentrations. This effect could not be generalized, e.g. the correlation coefficients between 1 and 10 µM, between 10 and 25 µM and between 25 and 50 µM were 0.11, 0.43, and 0.30, respectively. That means that one cannot derive the dry mass production from one concentration to the next higher concentration. There was no correlation between the IC50 values of the inhibitors and the shoot dry mass growths found (with R²=0.04, 0.08, 0.18 and 0.17 for the four concentrations of 1, 10, 25 and 50 µM, data not shown). This indicates that PARP is not the only player in the network of drought stress response.

Currently there is only one whole-plant assay published that measures effects of compounds on drought stress and could be used for high or medium throughput screening. This Lemna minor plants assay was developed by our group in which which the growth rate of a treated plant is compared to the untreated plant’s growth rate upon drought stress application via PEG.²⁰⁷ All 52 compounds for which IC50 values for AtPARP1 inhibition have been determined were subjected to this assay and the observed plant growth of those compounds was compared to their IC50 for AtPARP1. Using a simple linear regression model, there was no correlation found between Lemna minor growth and the IC50 values (R²<0.001, data not shown). This result could be explained by the assumption that AtPARP1 inhibitors are no inhibitors of Lemna minor PARP (which would be equivalent to the term Lemna minor decoys) or these compounds inhibit AtPARP1 and Lemna minor PARP through different modes of action. It might also be that PARP is not a relevant arget, at all or that secondary effects as compound metabolism interfere with effects that have been observed in AtPARP in vitro / in planta studies. If one assumes that those compounds inhibit both enzymes through the same mode of action, there is no evidence that all compounds reach their target at the concentration at with they were applied to the medium in the Lemna minor assay. Lemna growth might also very likely be influenced by acting of AtPARP1 inhibitors on other targets, especially on those with similar active sites to PARP or on those that use NAD⁺ as substrate or co-substrate.

4 Summary and outlook

In this work, an in silico characterisation of the Arabidopsis thaliana Poly-(ADP-ribose)-Polymerase (AtPARP1) and the first virtual screening study for a plant PARP enzyme was conducted, which resulted in the identification of 52 AtPARP1 inhibitors.

Using a broad range of molecular modelling tools, the catalytic domain of AtPARP1 was characterised in silico. This characterisation encompassed the investigation of protein stability from which it was concluded that the three-dimensional shape of this conserved PARP domain is of high similarity to the HsPARP catalytic domain. Furthermore, there is overwhelming evidence provided by MD simulation of the AtPARP1 catalytic domain in complex with the natural substrates (or substrate analogues) NAD⁺ and CNA (an ADP-ribose analogue) that AtPARP1 binds its substrates in an analogous manner as it is described for HsPARP and as it is observed for ADPR-transferases like Diphtheria Toxin. Based on the results of that work, the role of a conserved glutamate essential for the catalytic reaction in PARP is the same in AtPARP1 as was shown for other ADPRT.

As for the in silico characterisation of the catalytic domain of AtPARP1, the virtual screening for AtPARP1 inhibitors involved the use of statistical tools like receiver operator characteristics (ROC) curves and power analysis to guide the VS process and improve its quality.

Based on the identification of AtPARP1 inhibitors but also from proposed compounds which proved not to be AtPARP1-active, general characteristics (descriptors) of the structures were used to derive binary quantitative structure-activity relationship (QSAR) that could help to understand the structural requirements that are responsible for AtPARP1 inhibition.

This work contributes to an understanding of the role of AtPARP1. Since PARP are implicated as a first responder to drought stress (by depleting the NAD⁺ pools of the plant that leads to disturbances in energy homeostasis upon drought stress), inhibitors of these enzymes might increase the drought stress tolerance of plants. To further test this hypothesis, application of identified inhibitors on crop plants like Zea mais would be desirable. During this study, some of the inhibitors showed increased dry mass production in Lolium perenne giving first hints that these inhibitors do increase the drought stress tolerance in plants;

although in this study the effects could not solely be related to PARP enzymes. Further investigations also could involve the ability to selectively inhibit plant PARP enzymes. In

Arabidopsis, it is still not completely investigated if selective PARP inhibitors increase the drought stress tolerance more than unselective ones.

In the same context the selectivity of plant PARP inhibitors with respect to human PARP is also of importance because human PARP inhibitors are promising compounds to treat severe conditions like breast and ovarian cancer or ischemia-reperfusion injury. Since a lot of molecular modelling tools have already been used to study human PARP, computer-aided drug design in the context of the development of selective plant PARP inhibitors would be a useful tool to elucidate the role of PARP and drought stress. Furthermore, from the identified inhibitors, lead compounds could be derived with higher potency or ADME(T) characteristics, using molecular modelling tools in close collaboration with medicinal chemistry.

5 Appendix