1. Introduction
Small molecules that regulate enzyme activity play an important role in many biological functions, and are crucial for drug discovery [1,2]. Screening libraries of small molecules, peptides, and nucleic acids has been widely used to discover ligands that bind to proteins and modulate their functions [3,4]. Peptides represent a promising class of potential enzyme modulators [5] due to their large chemical diversity [6] and the existence of well-established approaches for library synthesis [7]. Peptides and their derivatives are found to inhibit many important enzymes [8], such as dehydrogenases [9], protein kinases [10], and proteases [11]. Cell-permeable peptides are becoming more and more useful in blocking cellular signaling pathways [12,13]. Over the past few decades, peptide microarrays have been developed for the high-throughput screening of a library of peptides that can bind to biological targets and alter their functions [7]. These arrays can be made by printing pre-synthesized peptides [14], SPOT synthesis (spotting and synthesis) [15], and light-directed, spatially-addressable synthesis [16]. Hydrogel-coated peptide microarrays were also reported as a means of screening for enzyme activity that is modulated by specific protein–peptide interactions, and has made it possible to perform activity assays using high-density microarrays [17].
Here, we reported on a selection of cooperative peptide pairs for inhibiting enzymes by screening a library of peptides specifically for binding to the inhibited enzyme complex on a microarray. Using this approach, we selected new peptides that enhanced the inhibition of the target enzyme by using them together with a primary inhibitory peptide. Without the primary peptide, these new peptides showed little inhibition impact on the enzyme activity. We also demonstrated that some negatively charged peptides could recover the activity of inhibited peptide/enzyme complex.