A photosensitizing fusion protein with targeting capabilities

Abstract The photodynamic treatment for antimicrobial applications or anticancer therapy relies on reactive oxygen species generated by photosensitizing molecules after absorption of visible or near-infrared light. If the photosensitizing molecule is in close vicinity of the microorganism or the malignant cell, a photocytotoxic action is exerted. Therefore, the effectiveness of photosensitizing compounds strongly depends on their capability to target microbial or cancer-specific proteins. In this study, we report on the preparation and preliminary characterization of human recombinant myoglobin fused to the vasoactive intestinal peptide to target vasoactive intestinal peptide receptor (VPAC) receptors. Fe-protoporphyrin IX was replaced by the photosensitizing compound Zn-protoporphyrin IX. Taking advantage of the fluorescence emission by Zn-protoporphyrin IX, we show that the construct can bind prostate cancer cells where the VPAC receptors are expressed

The Energy Landscape of Human Serine Racemase

Human serine racemase is a pyridoxal 5′-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available. The comparison of structures of serine racemase co-crystallized with or without ligands indicates the presence of at least one open and one closed conformation, suggesting that conformational flexibility plays a relevant role in enzyme regulation. ATP, Mg2+, Ca2+, anions, NADH and protein interactors, as well as the post-translational modifications nitrosylation and phosphorylation, finely tune the racemase and dehydratase activities and their relative reaction rates. Further information on serine racemase structure and dynamics resulted from the search for inhibitors with potential therapeutic applications. The cumulative knowledge on human serine racemase allowed obtaining insights into its conformational landscape and into the mechanisms of cross-talk between the effector binding sites and the active site

Human serine racemase is allosterically modulated by NADH and reduced nicotinamide derivatives

Serine racemase catalyzes both the synthesis and the degradation of d-serine, an obligatory co-agonist of the glutamatergic NMDA receptors. It is allosterically controlled by adenosine triphosphate (ATP), which increases its activity around 7-fold through a co-operative binding mechanism. Serine racemase has been proposed as a drug target for the treatment of several neuropathologies but, so far, the search has been directed only toward the active site, with the identification of a few, low-affinity inhibitors. Following the recent observation that nicotinamide adenine dinucleotide (reduced form) (NADH) inhibits serine racemase, here we show that the inhibition is partial, with an IC50 of 246 ± 63 μM, several-fold higher than NADH intracellular concentrations. At saturating concentrations of NADH, ATP binds with a 2-fold lower affinity and without co-operativity, suggesting ligand competition. NADH also reduces the weak activity of human serine racemase in the absence of ATP, indicating an additional ATP-independent inhibition mechanism. By dissecting the NADH molecule, we discovered that the inhibitory determinant is the N-substituted 1,4-dihydronicotinamide ring. Particularly, the NADH precursor 1,4-dihydronicotinamide mononucleotide exhibited a partial mixed-type inhibition, with a KI of 18 ± 7 μM. Docking simulations suggested that all 1,4-dihydronicotinamide derivatives bind at the interdimeric interface, with the ring positioned in an unoccupied site next to the ATP-binding site. This newly recognized allosteric site might be exploited for the design of high-affinity serine racemase effectors to finely modulate d-serine homeostasis

Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenases

Compounds based on the 3-Br-isoxazoline scaffold fully inhibit glyceraldehyde 3-phosphate dehydrogenase from Plasmodium falciparum by selectively alkylating all four catalytic cysteines of the tetramer. Here, we show that, under the same experimental conditions that led to a fast and complete inhibition of the protozoan enzyme, the human ortholog was only 25% inhibited, with the alkylation of a single catalytic cysteine within the tetramer. The partial alkylation seems to produce a slow conformational rearrangement that severely limits the accessibility of the remaining active sites to bulky 3-Br-isoxazoline derivatives, but not to the substrate or smaller alkylating agents

Magnesium and calcium ions differentially affect human serine racemase activity and modulate its quaternary equilibrium toward a tetrameric form

Serine racemase is the pyridoxal 5′-phosphate dependent enzyme that catalyzes both production and catabolism of D-serine, a co-agonist of the NMDA glutamate receptors. Mg2 +, or, alternatively, Ca2 +, activate human serine racemase by binding both at a specific site and – as ATP-metal complexes – at a distinct ATP binding site. We show that Mg2 + and Ca2 + bind at the metal binding site with a 4.5-fold difference in affinity, producing a similar thermal stabilization and partially shifting the dimer-tetramer equilibrium in favour of the latter. The ATP-Ca2 + complex produces a 2-fold lower maximal activation in comparison to the ATP-Mg2 + complex and exhibits a 3-fold higher EC50. The co-presence of ATP and metals further stabilizes the tetramer. In consideration of the cellular concentrations of Mg2 + and Ca2 +, even taking into account the fluctuations of the latter, these results point to Mg2 + as the sole physiologically relevant ligand both at the metal binding site and at the ATP binding site. The stabilization of the tetramer by both metals and ATP-metal complexes suggests a quaternary activation mechanism mediated by 5′-phosphonucleotides similar to that observed in the distantly related prokaryotic threonine deaminases. This allosteric mechanism has never been observed before in mammalian fold type II pyridoxal 5′-phosphate dependent enzymes

Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors