Optimized Translocator Protein Ligand for Optical Molecular Imaging and Screening

Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (<b>29</b>, <i>K</i>_d = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an <i>n</i>-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand–protein interference. Probe <b>29</b> was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe <b>29</b> quantified by fluorescence and ³H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand <b>29</b> for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties

Identification of Metabotropic Glutamate Receptor Subtype 5 Potentiators Using Virtual High-Throughput Screening

Selective potentiators of glutamate response at metabotropic glutamate receptor subtype 5 (mGluR5) have exciting potential for the development of novel treatment strategies for schizophrenia. A total of 1,382 compounds with positive allosteric modulation (PAM) of the mGluR5 glutamate response were identified through high-throughput screening (HTS) of a diverse library of 144,475 substances utilizing a functional assay measuring receptor-induced intracellular release of calcium. Primary hits were tested for concentration-dependent activity, and potency data (EC₅₀ values) were used for training artificial neural network (ANN) quantitative structure−activity relationship (QSAR) models that predict biological potency from the chemical structure. While all models were trained to predict EC₅₀, the quality of the models was assessed by using both continuous measures and binary classification. Numerical descriptors of chemical structure were used as input for the machine learning procedure and optimized in an iterative protocol. The ANN models achieved theoretical enrichment ratios of up to 38 for an independent data set not used in training the model. A database of ∼450,000 commercially available drug-like compounds was targeted in a virtual screen. A set of 824 compounds was obtained for testing based on the highest predicted potency values. Biological testing found 28.2% (232/824) of these compounds with various activities at mGluR5 including 177 pure potentiators and 55 partial agonists. These results represent an enrichment factor of 23 for pure potentiation of the mGluR5 glutamate response and 30 for overall mGluR5 modulation activity when compared with those of the original mGluR5 experimental screening data (0.94% hit rate). The active compounds identified contained 72% close derivatives of previously identified PAMs as well as 28% nontrivial derivatives of known active compounds

Discovery, Synthesis, And Structure-Based Optimization of a Series of <i>N</i>‑(<i>tert</i>-Butyl)-2‑(<i>N</i>‑arylamido)-2-(pyridin-3-yl) Acetamides (ML188) as Potent Noncovalent Small Molecule Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL Protease

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (<b>16-(<i>R</i>)</b>, (<i>R</i>)-<i>N</i>-(4-(<i>tert</i>-butyl)­phenyl)-<i>N</i>-(2-(<i>tert</i>-butylamino)-2-oxo-1-(pyridin-3-yl)­ethyl)­furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, <b>16-(<i>R</i>)</b> is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure–activity relationships within S_1′, S₁, and S₂ enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with <b>16-(<i>R</i>)</b> was instrumental in guiding subsequent rounds of chemistry optimization. <b>16-(<i>R</i>)</b> provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action