Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic ‘warhead’ to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.

共价结合大分子靶标的分子已作为基于活性的探针和不可逆结合药物得到广泛应用。然而，形成共价键所需的亲电子试剂的一般反应性使得选择性的控制变得困难。目前还没有快速、公正的筛选方法来从高度多样化的候选分子库中识别新类别的共价抑制剂。在这里，我们描述了一种噬菌体展示方法，可直接筛选通过共价键形成与蛋白质靶标结合的配体。这种方法利用反应性接头在噬菌体表面形成环肽，同时引入亲电“弹头”与靶标上的亲核试剂发生共价反应。使用这种方法，我们鉴定了能够不可逆地抑制半胱氨酸蛋白酶和丝氨酸水解酶的环肽，其具有纳摩尔效力和卓越的特异性。这种方法应该能够实现快速、公正的筛选，以确定针对不同分子靶点的新型高选择性共价抑制剂。