Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.

蛋白质-蛋白质相互作用（PPI）控制着细胞内生命，而鉴定PPI抑制剂则具有挑战性。天然PPI的弱结合亲和力导致检测开发的障碍阻碍了该领域的发展。我们推测通过蛋白质工程增强天然PPI的结合亲和力将有助于测定方法的开发和命中发现。这项原理研究针对线性泛素链和NEMO UBAN域之间的PPI，后者激活NF-κB信号传导。使用噬菌体展示，我们生成了泛素变体，它们以高亲和力与功能性UBAN表位结合，可作为竞争性抑制剂，并在结构上维持现有的PPI界面。当用于分析开发时，变体可以生成用于化学筛选的可靠的基于细胞的分析。使用这种方法鉴定出的主要化合物可直接与UBAN结合并抑制NF-κB信号传导。这项研究说明了在命中鉴定中整合蛋白质工程和化学筛选的优势，我们预计这一发展将在药物发现中得到广泛应用。