Molecular basis for diaryldiamine selectivity and competition with tRNA in a type 2 methionyl-tRNA synthetase from a Gram-negative bacterium.

Gram-negative bacteria are responsible for a variety of human, animal, and plant diseases. The spread of multidrug-resistant Gram-negative bacteria poses a challenge to disease control and highlights the need for novel antimicrobials. Owing to their critical role in protein synthesis, aminoacyl-tRNA synthetases, including the methionyl-tRNA synthetases MetRS1 and MetRS2, are attractive drug targets. MetRS1 has long been exploited as a drug target in Gram-positive bacteria and protozoan parasites. However, MetRS1 inhibitors have limited action upon Gram-negative pathogens or on Gram-positive bacteria that produce MetRS2 enzymes. The underlying mechanism by which MetRS2 enzymes are insensitive to MetRS1 inhibitors is presently unknown. Herein, we report the first structures of MetRS2 from a multidrug-resistant Gram-negative bacterium in its ligand-free state and bound to its substrate or MetRS1 inhibitors. The structures reveal the binding mode of two diaryldiamine MetRS1 inhibitors that occupy the amino acid-binding site and a surrounding auxiliary pocket implicated in tRNA acceptor arm binding. The structural features associated with amino acid polymorphisms found in the methionine and auxiliary pockets reveal the molecular basis for diaryldiamine binding and selectivity between MetRS1 and MetRS2 enzymes. Moreover, we show that mutations in key polymorphic residues in the methionine and auxiliary pockets not only altered inhibitor binding affinity but also significantly reduced enzyme function. Our findings thus reinforce the tRNA acceptor arm binding site as a druggable pocket in class I aminoacyl-tRNA synthetases and provide a structural basis for optimization of MetRS2 inhibitors for the development of new antimicrobials against Gram-negative pathogens.

Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites.

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites.

Phenotypical Screening of an MMV Open Box Library and Identification of Compounds with Antiviral Activity against St. Louis Encephalitis Virus.

St. Louis encephalitis virus (SLEV) is a neglected mosquito-borne Flavivirus that may cause severe neurological disease in humans and other animals. There are no specific treatments against SLEV infection or disease approved for human use, and drug repurposing may represent an opportunity to accelerate the development of treatments against SLEV. Here we present a scalable, medium-throughput phenotypic cell culture-based screening assay on Vero CCL81 cells to identify bioactive compounds that could be repurposed against SLEV infection. We screened eighty compounds from the Medicines for Malaria Venture (MMV) COVID Box library to identify nine (11%) compounds that protected cell cultures from SLEV-induced cytopathic effects, with low- to mid-micromolar potencies. We validated six hit compounds using viral plaque-forming assays to find that the compounds ABT-239, Amiodarone, Fluphenazine, Posaconazole, Triparanol, and Vidofludimus presented varied levels of antiviral activity and selectivity depending on the mammalian cell type used for testing. Importantly, we identified and validated the antiviral activity of the anti-flavivirus nucleoside analog 7DMA against SLEV. Triparanol and Fluphenazine reduced infectious viral loads in both Vero CCL81 and HBEC-5i cell cultures and, similar to the other validated compounds, are likely to exert antiviral activity through a molecular target in the host.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Discovery of Potent N-Ethylurea Pyrazole Derivatives as Dual Inhibitors of Trypanosoma brucei and Trypanosoma cruzi.

Trypanosoma brucei (T. brucei) and Trypanosoma cruzi (T. cruzi) are causative agents of parasitic diseases known as human African trypanosomiasis and Chagas disease, respectively. Together, these diseases affect 68 million people around the world. Current treatments are unsatisfactory, frequently associated with intolerable side-effects, and generally inadequate in treating all stages of disease. In this paper, we report the discovery of N-ethylurea pyrazoles that potently and selectively inhibit the viability of T. brucei and T. cruzi. Sharp and logical SAR led to the identification of 54 as the best compound, with an in vitro IC50 of 9 nM and 16 nM against T. b. brucei and T. cruzi, respectively. Compound 54 demonstrates favorable physicochemical properties and was efficacious in a murine model of Chagas disease, leading to undetectable parasitemia within 6 days when CYP metabolism was inhibited.

Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism.

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg2+ and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K+ ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K+ coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D 15N-1H HSQC experiments revealed that the binding of K+ perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K+. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K+ concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K+ inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K+ and highlights the role of ADK in connecting depletion of intracellular K+ to the regulation of purine metabolism.

Mutations in the tetramer interface of human glucose-6-phosphate dehydrogenase reveals kinetic differences between oligomeric states.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phoshate to 6-phospho-gluconolactone with the concomitant reduction of NADP+ to NADPH. In solution, the recombinant human G6PDH is known to be active as dimers and tetramers. To distinguish between the kinetic properties of dimers and tetramers of the G6PDH is not trivial. Steady-state kinetic experiments are often performed at low enzyme concentrations, which favor the dimeric state. The present work describes two novel human G6PDH mutants, one that creates four disulfide bonds among apposing dimers, resulting in a 'cross-linked' tetramer, and another that prevents the dimer to dimer association. The functional and structural characterizations of such mutants indicate the tetramer as the most active form of human G6PDH.