Targeting protein-protein interactions through directed evolution of lanthipeptid

描述（由申请人提供）：据估计，人类基因组中10%至30%的基因可能是治疗疾病的靶标(1)。然而，其他估计表明，只有不到10%的人类蛋白质组可以被传统的小分子药物药物治疗，而这两种情况的交集只剩下2%到5%的人类蛋白质既涉及疾病又可药物治疗(2)。这些因素突出了对新型药物分子的需求，这些药物分子能够扩大治疗当前难治性疾病的可药物靶点的范围。一类通常被认为难以干扰的靶标是蛋白质-蛋白质相互作用(3)。直观地说，多肽是破坏特定蛋白质-蛋白质相互作用的理想分子。事实上，筛选肽库的高通量方法，如噬菌体、核糖体和酵母展示，已经成功地在体外鉴定出可以破坏蛋白质-蛋白质相互作用的肽（4-6）。然而，作为药物分子，多肽有一些缺点，包括易被蛋白质水解和构象柔韧性(7)。尽管如此,

DESCRIPTION (provided by applicant): It has been estimated that between 10 and 30 percent of genes in the human genome may be targets for the treatment of disease (1). However, other estimates suggest that less than 10 percent of the human proteome is druggable by conventional small molecule drugs and that the intersection of these two conditions leaves as little as 2 to 5 percent of human proteins both involved in disease and druggable (2). These factors highlight the need for new classes of drug molecules capable of expanding the scope of druggable targets for the treatment of currently intractable diseases. One class of targets that is generally considered challenging to perturb is protein- protein interactions (3). Intuitively, peptides would be ideal molecules for disrupting specific protein- protein interactions. Indeed, high throughput methods of screening peptide libraries, such as phage, ribosome, and yeast display, have been successful at identifying peptides that can disrupt protein- protein interactions in vitro (4-6). However, as drug molecules peptides have drawbacks including susceptibility to proteolysis and conformational flexibility (7). Nevertheless, nature has evolved mechanisms for the production of biologically active small molecules based on peptides. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of natural products that are currently being investigated for the treatment of conditions ranging from bacterial and parasite infections to cancer (8). The most studied class of RiPPs is the lanthipeptides, which contain lanthionine or methyllanthionine thioether crosslinks (9). These crosslinks can provide resistance to proteolytic cleavage and confer conformational stability on lanthipeptides (10-13). One route for the installation of these crosslinks involves a bifunctional enzyme generically named LanM, which can dehydrate serine or threonine residues and catalyze the Michael-type addition of a cysteine residue onto these dehydrated residues to produce the thioether crosslink (9). Studies have identified a particular LanM enzyme in the planktonic marine cynaobacterium Prochlorococcus MIT9313, called ProcM, which is capable of processing 29 endogenous sequence-diverse lanthipeptide precursors (14) as well as a lanthipeptide precursor from a different genera of bacteria (15), suggesting that it is quite tolerant with respect to its substrate. As lanthipeptides are genetically encoded, they lend themselves to the facile synthesis of large libraries through combinatorial DNA library synthesis. This proposal will focus on the development of a yeast display system for lanthipeptides, the design of peptide libraries that are capable of being cyclized by ProcM, and the evolution of lanthipeptides towards the disruption of specific protein-protein interactions that have been implicated in human disease using fluorescent-activated cell sorting (FACS).