Time-resolved Studies of Endothelial Nitric Oxide Synthase Catalytic Mechanism Using Photoactive NADPH Analogues – NOSTIME

We will use a photoactive molecule with the ability to trigger the catalysis by ultrafast electrons injection to the protein. The targeted protein is the endothelial nitric oxide synthase (eNOS), a key enzyme in the cardiovascular system generating NO, a potent vasodilator. This protein is active as a homodimer, each monomer being composed by a catalytic heme domain linked by calmodulin binding domain to a reductase domain with binding site for NADPH and flavins. The structure of the hemodomain of this enzyme is known but structure of its reductase domain initiating the catalysis is lacking. We propose to examine the fundamental mechanism and dynamics of eNOS catalysis at the molecular level using novel photoactive NADPH analogues, called nanotriggers (NTs). Specifically, we propose that:
(1) the binding of NT that targets the NADPH site of selected flavoproteins (e.g., eNOS) will stabilize them in a closed conformation, thus facilitating crystallization. The structure of the enzyme/NT complex will capture the conformation of the enzyme during the hydride transfer reaction from NADPH to FAD and possibly the electron transfer from FAD to FMN;
(2) the crystal structure of the protein-ligand complex will allow elucidation of time-resolved mechanistic structure/function relationships at the molecular level by time-resolved absorption solution studies and at the atomic level by X-ray crystallography. Laser excitation of the NADPH analogues bound to the protein induces electron injection that triggers enzymatic catalysis in a synchronized manner;
(3) the crystal structures will also enable designing isoform-specific protein recognition of the NADPH analogues by grafting specific moiety defined by computational methods.
(4) the intrinsic imaging properties of our NADPH derivatives will allow monitoring in real time eNOS trafficking in living cells by two photon excitation. Spectroscopic and modeling studies also fall within the scope of this call.

A new series of photoactivable NADPH mimics bearing one or two O-carboxylmethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (F) are dependent on the solvent polarity, with a red-shift in a more polar environment (?max,em = 460-467 nm, F>0.53 in DMSO, and ?max,em = 475-491 nm, F<0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3’ position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.
We have also shown that several NTs are able to induce NOS-dependent membrane blebbing/cell death through electron transfer to NOS. To improve the specificity for the endothelial eNOS, we have designed a hook targeting variable residues of its NADPH site, yielding the NT3a,b derivatives. Based on modeling suggesting that the rigidity of the triazole moiety likely distorts the planarity of the chromophore, an additional CH2 was added to the linker between the chromophore and the nucleotide moiety to reduce the strain, giving rise to the NT3c derivatives. We have synthesized 11 novel nanotriggers NT3a-c. These compounds showed similar photophysical properties as the NT2a-c, with improved stability, however without improved affinity towards eNOS and nNOS.

Modeling will be undertaken to define the precise structure of NT3,4 so as to improve their specificity towards eNOS based on crystal structure. Synthesis of more soluble phosphate derivatives is ongoing. With NTs bearing improved chemical, photophysical properties and enzyme affinity, cellular imaging of eNOS, crystal structure determination of NT-protein (NOS and POR constructs) complexes by conventional macromolecular crystallography, mechanistic insights of eNOS catalytic intermediates in solution as well as crystal structure determination of NT-protein complexes by time-resolved crystallography will be realized.

1. Convergent synthesis and properties of novel photoactive NADPH mimics targeting nitric oxide synthases, N.-H. Nguyen, N. Bogliotti, R. Chennoufi, E. Henry, P. Tauc, E. Salas, L. J. Roman, A. Slama-Schwok, E. Deprez, J. Xie, Org. Biomol. Chem. 2016, 14, 9519-9532.

The aim of this proposal is to develop new photoactive tools able to isolate and trigger a specific catalytic event upon irradiation with a laser pulse. The laser pulse occurs at zero time, allowing for synchronization of initiation of catalysis, which can be monitored in a time-resolved manner. To date, caged compounds in which the active moiety is released from the caging group following laser excitation were primarily used to trigger reactions. The subsequent diffusion of the active moiety to the protein may be too slow and unsuited to time-resolved studies of proteins with fast turnover. This proposal represents a new approach to synchronize an ensemble of enzymes in solution, using a probe directly bound to the protein with the ability to trigger catalysis by ultrafast electrons injection to the protein.
Overview: This project focuses on the use of novel photoactivable NADPH analogues, called nanotriggers (NTs), targeted to the NADPH site of nitric oxide synthase (NOS) isoforms and cytochrome P450 reductase (POR), to stabilize the enzymes in a closed conformation, facilitating crystallization. This project will: (1) determine the X-ray structure of various NT-protein complexes; (2) elucidate time-resolved structure/function studies of the first catalytic steps by kinetics studies in solution and by time-resolved X-ray crystallography; (3) design by in silico simulations and synthesis of novel isoform-specific eNOS activators; and (4) monitor eNOS trafficking in cells by biphotonic excitation, due to the intrinsic imaging properties and specificity of the NT probes.
Broader Impacts: The photoactivation process of NT in which light absorption modulates the redox potential of the activated molecule (i.e., NT bound to NOS) and mediates electron flow is inspired from nature (i.e., photosynthesis), allowing the opening of new perspectives, especially in the field of artificial photosynthesis, a possible source of clean energy. In addition, the ability to trigger the activity of specific enzymes, particularly activation of eNOS, attenuated activity of which is a hallmark of endothelial dysfunction, open new therapeutic avenues for treatment of human diseases such as hypertension, diabetes, and degenerative neurological processes.
Value of International Collaboration: The strength of this proposal lies in the novel collaborative multidisciplinary skills gathered in this consortium that combines all the expertise required for its realization through the complementarity of the French partners: J. Xie, chemical synthesis, A. Slama-Schwok, drug design and biophysics, E. Deprez, imaging and time-resolved fluorescence, and the American partners: L. Roman and J.J. Kim, biochemistry and structure of NOS and P450, respectively. Drs. Roman and Kim are long-time collaborators in their studies of NOS, and their respective expertise in NOS enzymology, protein chemistry, and molecular biology and X-ray crystallography approaches together with the French groups, chemical, synthetic, spectroscopic and computational expertise will yield extremely high synergy.