1. 近年来,随着抗体、核酸、工程蛋白等新型药物形式的蓬勃发展,制药研究的"不可成药"边界正在被不断推进,许多此前被认为难以成药的靶点正成为新的研究热点。在后Ro5时代,迫切需要系统梳理这一领域的研究现状和突破进展。
2. 大环肽 (macrocyclic peptides) 介于小分子药物与大型蛋白质类药物之间,在针对多种具有挑战性的靶点(如蛋白质-蛋白质相互作用 (PPIs)、内在无序蛋白 (IDPs) 等)时展现出独特优势。随着NELS等技术的快速发展,利用大环肽针对不可成药靶点的研究取得了一系列突破,亟需对相关进展进行系统总结,为后续研究提供参考。

1. 噬菌体展示 (phage display)、mRNA展示等早期NELS技术的建立和应用。作为最早建立的NELS平台,噬菌体展示和mRNA展示在20世纪90年代就已广泛应用于环肽类药物的筛选,并催生了首批针对难成药靶点的环肽类先导化合物的发现。
2. 遗传密码扩展技术的发展。进入21世纪,遗传密码扩展技术的快速发展(尤其是氨酰-tRNA合成酶 (aaRS)/tRNA 等系统的建立)极大拓展了NELS平台中可引入的非天然氨基酸种类,使得环肽库的结构多样性得到显著提升。
3. 多种新型环化/构象稳定策略的涌现。随着环肽药物研发的深入,越来越多新颖的环化和构象稳定策略被开发出来,如硫醚 (thioether) 交联、二硫键 (disulfide bridge)、点击化学 (click chemistry)、关环复分解(RCM) 等,极大丰富了大环肽类先导化合物的结构类型。
4. 针对典型不可成药靶点的突破性进展。2010年以来,利用NELS平台针对小GTP酶 (small GTPases)、PPIs、IDPs 等多个典型不可成药靶点取得了一系列关键突破,如靶向KRas、Myc等"最难啃的骨头"的环肽抑制剂相继被发现,充分彰显了该策略在难成药靶点领域的应用前景。
5. 组合化学等新型NELS技术的兴起。近年来,DNA编码库等组合化学衍生的NELS技术快速兴起,通过将NELS与片段合成等经典药物发现策略相结合,进一步拓展了针对不可成药靶点环肽类先导物的发现途径。

1. NELS平台的建立和技术拓展

A huge variety of npAAs can be introduced into the RaPID system, which is discussed further in section 2.4.

2. 环肽分子设计策略的不断丰富

2.1 环化策略

A plethora of staples have been reported, and a recent review by Hu and co-workers covers the topic in great depth. Generally, the four most common stapling strategies are lactamization, ring-closing metathesis (RCM), click-chemistry, and thioether formation.

图3

2.2 支架(scaffold)稳定策略

Clearly the most popular and successful stapling strategies recently have been all-hydrocarbon-stapled peptides (Figure 4A) and hydrogen bond surrogate (HBS) based stapled peptides, both prepared through RCM, pioneered by the Verdine lab, which have yielded an impressive array of potent PPI inhibitors.

图4. 代表性支架(scaffold)稳定环肽构象策略

2.3 非天然氨基酸引入

Of particular interest are npAAs that can strongly influence the secondary structures of cyclic peptides. For instance, cyclic β-amino acids are promising building blocks for peptide-based therapeutics due to their ability to induce strong helix/turn structures, enhance structural rigidity, and increase proteolytic resistance.

图6. 代表性非天然氨基酸引入策略

3. 针对代表性"不可成药"靶点取得突破

3.1 针对KRas等"不可成药"蛋白的抑制剂发现

A GTPase switch, KRas exists primarily in a GTP-bound "on" state and a GDP-bound "off" state. The G12D mutation results in a loss of GTPase activity, effectively locking KRas in its "on" state and leading to aberrant activity such as promoting tumor growth. With this in mind, the screening strategy focused on identifying a binder selective for the GTP-bound ("on") state over the GDP-bound ("off") state.

图8. 针对KRas的RaPID筛选策略

3.2 靶向蛋白-蛋白相互作用(PPIs)

The proposed model of "hot spots" stipulates that, while the absence of a conventional binding pocket precludes standard druggability, compounds can be designed that target multiple hot-spots through binding fragments connected to some sort of scaffold (Figure 9A).

图9. 针对PPIs的环肽抑制剂设计策略

3.3 调控内在无序蛋白(IDPs)

There are two prevalent strategies to target IDPs: natural binder-mimicry/hot-spot targeting and allosteric site hunting.

The Ivarsson lab has leveraged a modified NELS technique termed proteomic-peptide phage display (ProP-PD). They recognized that many IDPs act via short linear motifs (SLiMs) and that there is a severe dearth of information on the sequence identity, distribution, and interactivity of these motifs, leading to a significant gap in the mapping of the human interactome.

核心观点小结

1. 细胞通透性改善策略的进一步突破。这是当前环肽类药物面临的最主要瓶颈。作者认为,随着支架肽、环肽嫁接等新型分子设计策略的不断涌现,环肽的细胞通透性有望在不久的将来取得实质性改善。
2. 基于结构的环肽药物分子设计将成为可能。作者预测,随着环肽-靶点复合物结构信息的日益丰富,理性设计有望在环肽先导化合物优化中发挥更大作用,加速环肽类药物的优化和发展进程。

We foresee that beyond the continuous development of new design scaffolds for macrocyclic peptides and technical improvements for NELS methods, the emergence of design guidelines will enhance the optimization of peptide hits to drug candidates.

3. 联用策略将成为环肽开发不可成药靶点药物的重要途径。作者提到,将环肽与其他药物发现策略(如PROTAC、抗体偶联等)联用,有望在解决环肽类药物的局限性(如通透性差、体内半衰期短等)方面取得新的突破。
4. 环肽嫁接将是重要发展方向。将NELS获得的高亲和力环肽嫁接到特定蛋白质支架上,不仅可显著提升其构象稳定性和代谢稳定性,更有望通过融合功能基团(如细胞穿透肽)等方式赋予环肽新的药物属性。作者认为这一策略在针对胞内难成药靶点方面前景广阔。

Grafting of a CPP motif on exposed loops of engineered proteins has been reported, which could be combined with other grafting techniques to deliver grafted peptide binders.

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