The insufficient selectivity of drugs is a bane of present-day therapies. This problem is significant for antibacterial drugs, difficult for antivirals, and utterly unsolved for anti-cancer drugs, which remain ineffective against major cancers, and in addition cause severe side effects. T he problem may be solved if therapeutic agents could acquire a multi-target, combinatorial selectivity, killing (or otherwise modifying) a cell if, and only if, it contains a predetermined set of molecular targets and lacks another predetermined set of targets. Recently, a solution to the problem of drug selectivity was proposed that is applicable to small (less than or equal to 1 kD) compounds (Varshavsky, A. PNAS 95, 2094-2099 (1998)). Two ideas, interference/co-dominance (IC) and anti-effectors, should allow a therapeutic regimen to possess combinatorial selectivity, in which the number of positively and negatively sensed macromolecular targets can be two, three, or more. The in vivo effects of such drugs would be analogous to the outcomes of the Boolean operations """"""""and"""""""", """"""""or"""""""" """"""""not"""""""", and combinations thereof. The proposed studies, to be supported by the present R21 application, will verify the principles of multi-target designs using the yeast Saccharomyces cerevisiae as a model organism.
Specific Aims 1) The ubiquitin fusion technique will be used to express and analyze, in the yeast S. cerevisiae, a set of multi-moiety linear peptides that act as IC- type compounds in this in vivo setting. 2) Bipartite, IC-type compounds that comprise the anhydrotetracycline (at) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety linked to at will be synthesized, and thereafter characterized in vitro (mutual exclusivity of binding to the purified TetR or DHFR) and in vivo (differential toxicity to S. cerevisiae that either lack or express the at- binding E. coli TetR repressor). 3) Bipartite, IC-type compounds containing the novobiocin (nov) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential cytotoxicity with S. cerevisiae that either lack or express the nov-binding B-subunit of E. coli DNA gyrates (GyrB). 4) Tripartite, IC-type compounds containing the at moiety, the beta- ecdysone (ec) moiety, and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential toxicity with S. cerevisiae that lack or express the corresponding macromolecular targets and their pairwise combinations.
