BIOLOGICAL CHARACTERIZATION OF NEUROKININ ANTAGONISTS DISCOVERED THROUGH SCREENING OF A COMBINATORIAL LIBRARY

Recent advances in combinatorial chemistry have resulted in the rapid screening of libraries against biological targets, Another advance in biological screening is the ability to design and utilize novel, autom ated, nonradioactive assays for targets of pharmaceutical interest, Us ing encoding technology and europium time-resolved fluorescence, we ha ve designed primary receptor binding assays to define active compounds against the neurokinin-1 and neurokinin-2 receptor subtypes, In addit ion, a secondary, cell-based, functional assay measuring intracellular calcium flux with calcium sensitive fluorophores was used to determin e receptor agonist or antagonist activities, We adapted a cuvette base d assay to a CCD camera and digital imaging system that allowed us to demonstrate functional receptor antagonist activity in all 96 wells of a microtiter plate simultaneously, The screening of a 20,000 member l ibrary using europium labeled neurokinin ligands resulted in the ident ification of 43 active compounds for neurokinin-l and 27 for neurokini n-2, Through medicinal chemistry and structure-activity relationships, a compound was synthesized with balanced dual antagonist activity at both neurokinin receptors with greater than 100-fold less activity aga inst the neurokinin-3 receptor subtype, The structure-activity relatio nships generated from this initial library can now be used to design a new focused library to improve on neurokinin-1/neurokinin-2 receptor potency and selectivity.