Hijacking prenyl and geranyl transferases - A route to carry out click modifications and to enhance cellular permeability of peptides

生物学研究的最新进展使人们能够更好地了解许多疾病的病因，并确定了新的治疗靶点。理想的药物应该与特定的细胞靶点结合，而对其他靶点没有亲和力。最近确定的药物发现的挑战性靶点之一是蛋白质-蛋白质相互作用，其已被证明涉及许多难以治疗的疾病，例如免疫疾病和癌症。这些相互作用沿着大蛋白质的延伸表面发生，因此对于小分子药物是非常具有挑战性的。生物药物例如抗体是大分子并且可以破坏蛋白质-蛋白质相互作用，但是不能口服施用并且非常昂贵。大环肽是一类新兴的候选药物，具有破坏蛋白质-蛋白质相互作用的能力，例如免疫抑制剂，使移植手术成为可能的环孢菌素。它们体积更小，比生物制剂便宜得多。与它们的线性“非环状”对应物相比，它们对酶更稳定，并且是半刚性的，以更好地与它们的靶标配合，就像钥匙适合锁一样。阻碍许多这些化合物的开发的限制是它们穿过细胞膜和到达细胞内靶点的能力低。几种修饰的环肽通常存在于药用天然产物中。这些化合物是通过自然选择进化而来的，自然选择可能是由它们对特定分子靶标的药理学效力以及它们通过穿过一个或多个生物膜到达这些靶标的能力驱动的。在自然界中，几种修饰被引入到环肽中以增强膜渗透性。这些修饰旨在通过用疏水性侧链屏蔽来减少分子的亲水性（极性）表面，因此化合物可以容易地扩散通过疏水性（主要是脂质）细胞膜。理想情况下，这些修饰应应用于特定位点，以避免水溶解度大幅降低或分子三维形状变化，从而降低其与靶点结合的能力。最近的研究揭示了大量这些修饰的环肽是如何在其宿主体内产生的。在这个项目中，我将确定和招募新的修饰生物合成酶，将增加疏水性化学基团，如异戊二烯基和香叶基在环肽的特定位点。使用化学方法进行这些修饰非常具有挑战性，不环保，并且在大多数情况下需要完全重新合成，这是耗时的。我将确定这些酶的结构和生化特征，以确定其活性和特异性的关键残基。我将利用这些见解来设计和产生具有不同残基特异性和引入其他化学基团能力的酶变体。我将使用化学合成和工程酶来生成定制的生物活性环肽的修饰衍生物，并测试不同修饰对膜渗透性和作为目标亲和力基础的分子的三维形状的影响。这些数据将有助于生成一个计算模型来预测生物活性环肽的膜渗透性，这将是非常宝贵的肽开发成药物。

Recent advances in biological research have allowed a better understanding of the causation of many diseases and identified new targets for therapy. An ideal drug should bind to a specific cellular target and have no affinity to others. One of the recently identified challenging targets for drug discovery is the protein-protein interactions that have been proved to be involved in many difficult-to-treat diseases e.g. immune disorders and cancer. These interactions are taking place along the extended surface of large proteins and thus are very challenging for small molecule drugs. Biological drugs e.g. antibodies are large molecules and can disrupt protein-protein interactions but cannot be administered orally and are very expensive. Macrocyclic peptides are an emerging class of drug candidates that have the ability to disrupt protein-protein interactions e.g. the immune-suppressant, cyclosporin that made transplant surgery possible. They are smaller in size and are very much cheaper than biologics. In contrast to their linear "non-cyclic" counterparts, they are more stable against enzymes and are semi-rigid to fit better with their targets much like a key fits into a lock. A limitation that hampers the development of many of these compounds is their low ability to cross cellular membranes and to reach intracellular targets. Several modified cyclic peptides are commonly found in medicinal natural products. These compounds were evolved via natural selection which is presumably driven by their pharmacological potency against specific molecular targets as well as their ability to reach these targets that is, by crossing one or more biological membranes. In nature, several modifications are introduced to cyclic peptides to enhance membrane permeability. These modifications aim to reduce the hydrophilic (polar) surface of the molecule by shielding with hydrophobic side chains thus the compound can easily diffuse through the hydrophobic (mainly lipid) cellular membranes. Ideally these modifications should be applied to specific sites to avoid a large reduction in water solubility or the change of three dimensional shape of the molecule with subsequent decrease in its ability to bind to its target. Recent research revealed how a large group of these modified cyclic peptides is made inside their hosts. In this project, I will identify and recruit new modifying biosynthetic enzymes that will add hydrophobic chemical groups such as prenyl and geranyl groups at specific sites in cyclic peptides. Making these modifications using chemical methods is very challenging, not eco-friendly and in most cases entails total re-synthesis which is time consuming. I will determine the structure and biochemical features of these enzymes to identify the key residues that underlie their activity and specificity. I will use these insights to engineer and generate enzyme variants with different residue specificity and ability to introduce other chemical groups. I will use chemical synthesis and the engineered enzymes to generate modified derivatives of bespoke bioactive cyclic peptides and test the effect of different modifications on membrane permeability and the three dimensional shape of the molecule that underlies target affinity. These data will help to generate a computational model to predict membrane permeability of bioactive cyclic peptides that will be invaluable for development of peptides into drugs.