Functional analysis of expressed peptides that bind yeast STE proteins Giordano Caponigro *, Majid Abedi, Alexander Kamb 677 7th Ave., Salt Lake City, UT 84103, USA Received 24 October 2002; received in revised form 28 April 2003; accepted 8 May 2003 Abstract Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide  /protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes. # 2003 Elsevier B.V. All rights reserved. Keywords: Peptides; STE; Two-hybrid 1. Introduction The process of modern drug discovery begins with protein targets that are the basis for high- throughput screens and rational design of small molecule compounds. Genomics research has produced an abundance of candidate targets chosen typically for sequence and/or expression properties. Due to the efficiency of the genomics process in fueling hypotheses about candidate targets, a downstream bottleneck in target valida- tion has ensued. There is great demand for methods that can rapidly test hypotheses about gene function in general and target suitability in particular. Such methods most often involve reverse genetics; that is, the assignment of physio- logical roles to genes via genetic manipulations of the candidate sequences. A reverse genetic approach that uses peptide inhibitors to test the physiological functions of proteins in vivo, and thereby validate potential drug targets, is potentially very powerful. Several strategies have been employed to identify such inhibitors, including the yeast two-hybrid method * Corresponding author. Tel.:  /1-801-595-0607. E-mail address: gmcaponigro@yahoo.com (G. Caponigro). Journal of Biotechnology 103 (2003) 213  /225 www.elsevier.com/locate/jbiotec 0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1656(03)00127-5