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对许多疾病的病因有了更好的了解，并确定了新的治疗目标。理想的药物应该与特定的细胞靶点结合，而对其他细胞没有亲和力。最近发现的具有挑战性的药物发现靶点之一是蛋白质-蛋白质相互作用，这种相互作用已被证明与许多难以治疗的疾病有关，例如免疫疾病和癌症。这些相互作用是沿着大蛋白质的延伸表面发生的，因此对小分子药物来说非常具有挑战性。生物药物，如抗体是大分子，可以破坏蛋白质之间的相互作用，但不能口服，而且非常昂贵。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，例如免疫抑制剂环孢素，使移植手术成为可能。它们体积更小，而且比生物制剂便宜得多。与它们的线性“非环”对应物相比，它们对酶更稳定，并且是半刚性的，可以更好地适应它们的目标，就像钥匙能锁住锁一样。阻碍这些化合物发展的一个限制是它们穿过细胞膜和到达细胞内目标的能力较低。几种修饰的环肽在药用天然产物中很常见。这些化合物是通过自然选择进化而来的，这可能是由它们对特定分子目标的药理学效力以及它们通过穿过一个或多个生物膜达到这些目标的能力所驱动的。在自然界中，一些修饰被引入到环肽中以增强膜的渗透性。这些修饰的目的是通过疏水侧链屏蔽分子的亲水性（极性）表面，从而使化合物可以容易地通过疏水（主要是脂质）细胞膜扩散。理想情况下，这些修饰应该应用于特定的位点，以避免水溶性的大幅降低或分子三维形状的改变，从而降低其与靶标的结合能力。最近的研究揭示了这些修饰过的环状肽是如何在宿主体内产生的。在这个项目中，我将识别和招募新的修饰生物合成酶，将在环肽的特定位点添加疏水化学基团，如戊烯基和香叶基。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生成具有不同残基特异性和引入其他化学基团的能力的酶变体。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通透性的影响以及目标亲和力基础分子的三维形状。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通透性，这将对肽开发成药物具有宝贵的价值。

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.