NMR determination of Siderocalin ligand binding properties

The aim of this project is to determine, by nuclear magnetic resonance (NMR), the structural and dynamic features involved in ligand binding properties of siderocalins, a potential target for new anticancer agents. In contrast to existing methodology which concentrate on ground (major) state and employs conventional structural biology methods, the approach taken in the project will apply (and extend) different NMR-dispersion experiments to investigate the structural and dynamical features of low populated protein conformations (minor or excited states). It thus represents the first systematic approach to investigate the relevance of protein excited states for structure-based drug design programs. Siderocalins are small secreted eukaryotic proteins belonging to the lipocalin superfamily. Siderocalins are characterized by their ability to capture small iron binding molecules, called siderophores, with a very high affinity (<0.5 nM). The function(s) of siderocalins is still unclear but they seem to be involved in an iron delivery pathway particularly active during cells growth and differentiation. Growing evidences indicate a role of siderocalin in cell proliferation and cancer progression. Lipocalin Q83 is expressed in chicken embryos and in quail oncogenic fibroblasts, which suggest a role of Lipocalin Q83 in cell growth and/or differentiation. We have shown (Lise Meitner project M1049-B05) that Q83 binds a bacterial siderophore called enterobactin with a very high affinity (0.5 nM). Consequently, Q83 belongs to the siderocalin class. Moreover, we observed that Q83 binds unsaturated fatty acids, such as arachidonic acid (AA), with a low nanomolar affinity (<25 nM). More puzzling, we determined by NMR that enterobactin and AA are able to bind to Q83 simultaneously to form a ternary complex. These striking binding properties have never been observed so far. Considering that AA is the precursor of leukotrienes and prostaglandins, our astonishing results strengthen the potential role of lipocalin Q83 and siderocalins in cell growth/differentiation and cancer progression. Finally, our backbone dynamics data show that binding of enterobactin to Q83 increases the milli-to-microsecond dynamics at the AA binding site, potentially enhancing AA binding to Q83, similarly to an allosteric mechanism. Considering their involvement in cancer progression, siderocalins are targets of a choice for the conception of new potent anticancer drugs. In addition, the surprising ability of these proteins to bind two ligands simultaneously would allow the design of fused ligands as suggested by the fragment-based drug discovery (FBDD) approach. In that context, it is critical to fully understand the binding properties of siderocalins, which will be the main concern of the current proposal. To achieve this goal, we will determine, by NMR, the structural and dynamic features involved in lipocalin Q83 ligand binding properties. We will particularly focus on the sidechain dynamic changes induced by the binding of the different ligands. In addition, we will characterize the structure of the different excited states generated by ligand binding. We will also investigate the ligand specificity of lipocalin Q83, since siderocalins do not seem to be absolutely specific despite their high ligand affinity. Finally, we will develop new (and/or extend existing) NMR tools, to characterize protein structural dynamics involved in protein- ligand binding. All together, these data will address the potentiality of siderocalins as novel drug targets. First, the detailed investigation of the striking binding properties of Q83 is of a fundamental interest to understand the structural and dynamic features involved in general protein/ligand interactions. Moreover, by elucidating the ligand specificity of siderocalins, this project will contribute to clarify the biological function of siderocalins. Finally, when bringing together, our results will allow the design of fused ligands (in a FBDD approach) exhibiting high affinity and specificity with high potential as novel anticancer drugs.

Proteins are natures robots. These large molecules perform the tasks necessary to our cells maintenance and growth in normal healthy conditions but also in pathological ones. Therefore, they are the targets of many drugs used in therapeutic interventions. Until recent years, the common view about the way drugs interact with their protein targets was the lock and key model. In this model the protein is a rigid lock and the drug is a key that fits the protein lock and triggers a response (usually the inhibition of the proteins function). It has become increasingly evident that proteins are actually not rigid at all but are rather flexible and undergo a lot of movements. We call these shape fluctuations protein dynamics. It has also been increasingly evident that taking into account this protein dynamics during the conception and development of new drugs would improve the drugs efficiency.We use a particular protein, Siderocalin, which seems to be involved in cancer progression, as a model system to study the importance of protein dynamics for drug development strategies. Using powerful spectroscopic methods (Nuclear Magnetic Resonance), we were indeed able to show that siderocalins undergo large dynamics, and that this dynamics is essential for their abilities to interact with ligands (drug like molecules). This is an important finding that suggests that drug development strategies could be improved by taking protein dynamics into account.Additionally, drug design strategies usually need to know the 3D structure of the protein (the shape of the lock) in order to design efficient drugs (complementary keys). In the context of this project we also developed a new strategy, combining computer prediction algorithms and spectroscopic methods, to design drugs without the knowledge of the 3D structure of the protein. We applied these methods to Siderocalins and were able to design new ligands (drugs) for this protein. We of course anticipate widespread application of these new methods.