Zebrafish studies identify serotonin receptors mediating antiepileptic activity in Dravet syndrome.

Dravet syndrome is a life-threatening early-onset epilepsy not well controlled by antiepileptic drugs. Drugs that modulate serotonin (5-HT) signalling, including clemizole, locaserin, trazodone and fenfluramine, have recently emerged as potential treatment options for Dravet syndrome. To investigate the serotonin receptors that could moderate this antiepileptic activity, we designed and synthesized 28 novel analogues of clemizole, obtained receptor binding affinity profiles, and performed in vivo screening in a scn1lab mutant zebrafish (Danio rerio) model which recapitulates critical clinical features of Dravet syndrome. We discovered three clemizole analogues with 5-HT receptor binding that exert powerful antiepileptic activity. Based on structure-activity relationships and medicinal chemistry-based analysis, we then screened an additional set of known 5-HT receptor specific drug candidates. Integrating our in vitro and in vivo data implicates 5-HT2B receptors as a critical mediator in the mechanism of seizure suppression observed in Dravet syndrome patients treated with 5-HT modulating drugs

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 μM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development. </p

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography

Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three sub-Angstrom resolution X-ray crystal structures of CTX-M \u3b2-lactamase, representing three stages of the enzymatic pathway, apo protein (0.79 \uc5), pre-covalent complex (0.89 \uc5), and acylation transition state analog (0.84 \uc5). The binding of a non-covalent ligand induces a proton transfer from the catalytic Ser70 to the general base Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73. QM/MM reaction path calculations determined the proton transfer barrier between Ser70 and Lys73 to be 1.53 kcal/mol, further confirming the presence of a LBHB. This LBHB is absent in the other two structures. Our data represents the first evidence of a direct and transient LBHB stabilizing a nucleophilic serine, as hypothesized by Cleland and Kreevoy. These results have important implications for the study of enzyme mechanisms as well as protein-inhibitor interactions

From Tethered to Freestanding Stabilizers of 14-3-3 Protein-Protein Interactions through Fragment Linking

Small-molecule stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. However, the systematic discovery of PPI stabilizers remains a largely unmet challenge. Herein we report a fragment-linking approach targeting the interface of 14-3-3 and a peptide derived from the estrogen receptor alpha (ERα) protein. Two classes of fragments—a covalent and a noncovalent fragment—were co-crystallized and subsequently linked, resulting in a noncovalent hybrid molecule in which the original fragment interactions were largely conserved. Supported by 20 crystal structures, this initial hybrid molecule was further optimized, resulting in selective, 25-fold stabilization of the 14-3-3/ERα interaction. The high-resolution structures of both the single fragments, their co-crystal structures and those of the linked fragments document a feasible strategy to develop orthosteric PPI stabilizers by linking to an initial tethered fragment.</p

Beta-lactamase inhibitors, formulations, and uses thereof

Provided herein are heterocyclic compounds and pharmaceutical formulations that can be used to treat bacterial infections. Also provided herein are methods of making and using the heterocyclic compounds and pharmaceutical formulations

Compositions, methods of use, and methods of treatment

Embodiments of the present disclosure, in one aspect, relate to a beta-lactamase inhibitor, pharmaceutical compositions including a beta-lactamase inhibitor, methods of treatment of a condition (e.g., infection) or disease, methods of treatment using compositions or pharmaceutical compositions, and the like

Compositions, methods of use, and methods of treatment

Embodiments of the present disclosure, in one aspect, relate to a beta-lactamase inhibitor, pharmaceutical compositions including a beta-lactamase inhibitor, methods of treatment of a condition (e.g., infection) or disease, methods of treatment using compositions or pharmaceutical compositions, and the like

Heteroaryl Phosphonates as Noncovalent Inhibitors of Both Serine- and Metallocarbapenemases

Gram-negative pathogens expressing serine β-lactamases (SBLs) and metallo-β-lactamases (MBLs), especially those with carbapenemase activity, threaten the clinical utility of almost all β-lactam antibiotics. Here we describe the discovery of a heteroaryl phosphonate scaffold that exhibits noncovalent cross-class inhibition of representative carbapenemases, specifically the SBL KPC-2 and the MBLs NDM-1 and VIM-2. The most potent lead, compound 16, exhibited low nM to low μM inhibition of KPC-2, NDM-1, and VIM-2. Compound 16 potentiated imipenem efficacy against resistant clinical and laboratory bacterial strains expressing carbapenemases while showing some cytotoxicity toward human HEK293T cells only at concentrations above 100 μg/mL. Complex structures with KPC-2, NDM-1, and VIM-2 demonstrate how these inhibitors achieve high binding affinity to both enzyme classes. These findings provide a structurally and mechanistically new scaffold for drug discovery targeting multidrug resistant Gram-negative pathogens and more generally highlight the active site features of carbapenemases that can be leveraged for lead discovery