Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

Abstract

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation ofO-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

“…In order to further refine the structure-activity relationships (SAR) around this class of derivatives, we herein report the design and synthesis of a series of cyclopropane-1,2-dicarboxylic acids variously functionalized. Although the binding properties of the previously reported analogs [24][25][26] could not be improved, the set of compounds herein presented provides interesting insight into the mode of binding of small molecules to OASS enzymes. In particular, saturation transfer difference NMR (STD-NMR) was used to further characterize the molecule/enzyme interactions for both OASS-A and B. Interestingly, most of the compounds induce a several fold increase in fluorescence emission of the pyridoxal 5 0 -phosphate (PLP) coenzyme upon binding to either OASS-A or OASS-B, likely due to changes in the polarity of the active-site microenvironment.…”

“…The binding affinity of ligands to OASS was determined by monitoring the increase in fluorescence emission of the PLP coenzyme following excitation at 412 nm 24,32 . Emission spectra were recorded as a function of ligand concentration in a solution containing 0.5-1.0 mM OASS, 100 mM Hepes buffer, pH 7.0, at 20 C. Fluorescence measurements were carried out using a FluoroMax-3 fluorometer (HORIBA), equipped with a thermostatted cell holder, and spectra were corrected for blank contribution.…”

“…Over the years, along with other groups 20,21 , we have investigated structural and functional properties of OASS isozymes with the aim of further characterizing the biological features of these proteins and to explore the possibility of their inhibition by small molecules. The rational design of the first inhibitors was based on the structure of SAT, that physiologically inhibits OASS activity upon formation of the cysteine synthase bioenzyme complex 22,23 ; in particular, it was considered that the carboxylic moiety of Ile267 of SAT is essential for the interaction between the two proteins [22][23][24] . Therefore, combining a structure-based with a ligand-based drug design approach, we planned and synthesized a series of cyclopropanecarboxylic acid derivatives that bind to both OASS isoforms at nanomolar concentrations [25][26][27] .…”

Open challenges in structure-based virtual screening: Receptor modeling, target flexibility consideration and active site water molecules description

“…FLAP (Fingerprint or Ligands and Proteins), based on the GRID Molecular Interaction Fields [112,272,273], allows the user to perform docking simulations automatically calculating the probability of crystallographic or predicted water molecules to be retained upon ligand binding to the protein target . The possibility of including or removing waters during docking was also implemented in DOCK 3.5.54 [260] and in GOLD [241], where a 2 kJ/mol penalty is associated to each water displaced from the binding site.…”

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein

“…Because of the central role played by cysteine in bacterial physiology, it has been proposed that enzymes involved in its biosynthesis [40, 41], and OASS isozymes in particular [42, 43], could be exploited as targets for the development of new antibiotics. However, high levels of intracellular cysteine are potentially toxic to the cell.…”