Combination of Pharmacophore Matching, 2D Similarity Search, and In Vitro Biological Assays in the Selection of Potential 5‐HT₆ Antagonists from Large Commercial Repositories

Abstract

Rapid in silico selection of target-focused libraries from commercial repositories is an attractive and cost-effective approach. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compound databases, but the generated library requires further focusing. We report here a combination of the 2D approach with pharmacophore matching which was used for selecting 5-HT6 antagonists. In the first screening round, 12 compounds showed >85% antagonist efficacy of the 91 screened. For the second-round (hit validation) screening phase, pharmacophore models were built, applied, and compared with the routine 2D similarity search. Three pharmacophore models were created based on the structure of the reference compounds and the first-round hit compounds. The pharmacophore search resulted in a high hit rate (40%) and led to novel chemotypes, while 2D similarity search had slightly better hit rate (51%), but lacking the novelty. To demonstrate the power of the virtual screening cascade, ligand efficiency indices were also calculated and their steady improvement was confirmed.

Structure-Based Consensus Scoring Scheme for Selecting Class A Aminergic GPCR Fragments

“…We selected fragments at sum-vote cutoff ≥ 6. The 36 virtual screening hits were subjected to antagonist activity measurements in a cell-based assay against 5-HT 6 receptor. , We have identified eight in vitro hits with remarkable inhibitory levels (biological activities are summarized in Table ), two of them appeared to be PAINS frequent hitters (phenolic Mannich-bases). The remaining six fragment hits, corresponding to a hit rate of 16.7% possess pharmacophore features of typical aminergic compounds.…”

“… a 5-HT 6 serotonin receptor inhibition assay, antagonist efficacy at 50 μM final concentration 100%; same inhibition with the control antagonist, 0%; no inhibition (agonist only); negative control 1% DMSO; positive control 10 μM SB271046. …”

“…Characteristic interactions are Table 5. Antagonist Efficacy of FrACS Hits against 5-HT 6 Receptor a a 5-HT 6 serotonin receptor inhibition assay, 42 The benzene ring of the indoline scaffold occupies the same hydrophobic pocket, like the benzene-sulfonyl part of the reference ligand. The fluorophenyl part of compound 4 is oriented toward the same hydrophobic pocket (Phe284 6.51 , Phe285 6.52 , and Trp281 6.48 ) occupied by the quinoline-ring of SBP-742457.…”

“…CHO-K1 cells expressing stably the mitochondrially targeted aequorin (luminescent indicator) and Gα16 were used for the assay. 42 The principle of the assay is that aequorin (derived from Aequorea victoria) complex emits blue light while binding Ca 2+ ions. The aequorin and Gα16 expressing CHO-K1 cells were transiently transfected with plasmid harboring the gene expressing human 5-HT 6 or CB 1 respectively using Roche X-treme GENE HP DNA Transfection Reagent.…”

Rapid Identification of Potential Drug Candidates from Multi-Million Compounds’ Repositories. Combination of 2D Similarity Search with 3D Ligand/Structure Based Methods and In Vitro Screening

“…First, the reference space was defined by collecting 49 known 5-HT 6 antagonists (‘seeds’) representing various chemotypes (cca. 25) from available literature [ 71 ].…”