ABSTRACT
The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1. Of the 48 ligands identified, 24 exhibit a decided preference to bind hTR-P2b RNA rather than A-site rRNA and 10 do not bind A-site rRNA at all. Binding affinity was measured by monitoring RNA imino proton resonances for some of the compounds that showed hTR binding preference.
Subject(s)
Computer Simulation , Databases, Factual , Drug Discovery/methods , RNA/chemistry , RNA/metabolism , Telomerase/chemistry , Telomerase/metabolism , Aminoquinolines/chemistry , Aminoquinolines/pharmacology , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Ligands , Magnetic Resonance Spectroscopy , Molecular Structure , Molecular Weight , Phenothiazines/chemistry , Phenothiazines/pharmacology , Polycyclic Aromatic Hydrocarbons/chemistry , Polycyclic Aromatic Hydrocarbons/pharmacology , Reference Standards , Structure-Activity RelationshipABSTRACT
We have synthesized a series of phenothiazine derivatives, which were used to test the structure-activity relationship of binding to HIV-1 TAR RNA. Variations from our initial compound, 2-acetylphenothiazine, focused on two moieties: ring substitutions and n-alkyl substitutions. Binding characteristics were ascertained via NMR, principally by saturation transfer difference spectra of the ligand and imino proton resonance shifts of the RNA. Both ring and alkyl substitutions manifested NMR changes upon binding. In general, the active site, while somewhat flexible, has regions that can be capitalized for increased binding through van der Waals interactions and others that can be optimized for solubility in subsequent stages of development. However, binding can be nontrivially enhanced several-fold through optimization of van der Waals and hydrophilic sites of the scaffold.
