Investigations into aryl nitriles for protein modification via an untapped mode of reactivity

肽和蛋白质的受控化学修饰是化学生物学和生物医学科学中许多快速发展领域的关键基础技术。应用包括：抗体-药物偶联物（ADC）和相关靶向治疗剂;用于成像的放射性标记蛋白质;药物发现中的共价抑制剂;作为稳定治疗剂的钉合肽;以及许多纳米颗粒偶联物/其他纳米技术。化学反应的发现、开发或再利用，以新的方式构建这种缀合物，可能是该领域创新的关键驱动力。最近授予2022年诺贝尔化学奖，以表彰“点击化学”的发展，这有力地说明了这一点，该化学在实现多样化应用方面发挥了关键作用。在这个项目中，我们假设一个特定类别的试剂，含有腈官能团，可以通过其反应性的未开发模式，即它们与两个硫醇连续反应，使新的蛋白质修饰模式。一旦我们证明了这些反应的可行性，效率和范围，我们将探索新的机会。一个关键的焦点应用将是能够构建ADC，证明这些腈提供了一种独特的动态机制途径，以获得高度同质的位点选择性抗体缀合物。ADC代表了最令人兴奋的新型靶向抗癌疗法之一，目前已有13种ADC获得临床批准。它们旨在通过将细胞毒性药物特异性地递送到癌细胞来克服现有化疗药物的局限性，从而减少与损害健康组织相关的副作用。为了最大限度地提高ADC实现其治疗潜力的机会，必须改进其设计和化学结构。因此，本项目中开发的方法旨在提供一种非常方便和有效的方法，以高度受控的方式进行抗体的化学附着。此外，我们正在开发的试剂类别将是第一种能够简单地通过调整分子设计来控制抗体表面上两种不同氨基酸的附着的试剂;通过称为“二硫键钉合”和“半胱氨酸至赖氨酸转移”的策略。这也将使我们能够进行迭代的控制化学修饰，访问多功能的共轭物，代表了一个诱人的前景为下一代的抗体为基础的therapeutics.In第二个关键演示的应用程序所启用的化学方法在这个项目中开创的，我们的目标是显示腈试剂也可以提供一个独特的能力，选择性地抑制特定类别的酶。这种共价抑制剂是药物发现的一个增长领域，因为它们提供了延长治疗效果的前景。然而，在这些战略中，选择性至关重要。我们的目的是表明，试剂，形成稳定的加合物，只有当它们与两个近端的半胱氨酸氨基酸反应，形成一个新的机械类别的酶的选择性抑制剂。我们在这个项目中的总体方法是探索和调整一个未开发的，迷人的化学反应，并证明它在蛋白质修饰中提供了重要的新机会。通过这样做，该项目将提供从基础化学到治疗开发的见解。

The controlled chemical modification of peptides and proteins is a crucial underpinning technology for a number of rapidly growing areas in chemical biology and the biomedical sciences. Applications include: antibody-drug conjugates (ADCs) and related targeted therapeutics; radiolabelled proteins for imaging; covalent inhibitors in drug discovery; stapled peptides as stabilised therapeutics; and numerous nanoparticle conjugates/other nanotechnologies. The discovery, development or repurposing of chemical reactions to enable the construction of such conjugates in new ways can be a key driving force behind innovations in the area. This is powerfully illustrated by the recent award of the 2022 Nobel Prize in Chemistry for the development of 'Click Chemistry' which has played a key role in dramatically enabling diverse applications. In this project we are hypothesising that a particular class of reagents, containing a nitrile functional group, could enable new modes of protein modification via an untapped mode of their reactivity, i.e. their reaction with two thiols consecutively. Once we have demonstrated the viability, efficiency and scope of these reactions, we will explore the new opportunities enabled. A key focus application will be to enable the construction of ADCs, demonstrating that these nitriles offer a unique dynamic mechanistic pathway to highly homogenous, site-selective antibody conjugates. ADCs represent one of the most exciting classes of new targeted anti-cancer therapeutics, with 13 ADCs now having achieved clinical approval. They aim to overcome the limitations of existing chemotherapeutics by delivering the cytotoxic drug specifically to the cancer cells, and thus reducing the side-effects associated with damaging healthy tissue. To maximise the chances for ADCs achieving their therapeutic potential, their design and chemical construction must be improved. As such, the methods developed in this project aim to provide an extremely convenient and efficient approach to carry out the chemical attachment to antibodies in a highly controlled manner. Furthermore, the reagent class we are developing would be the first of a kind in enabling controlled attachment to two different amino-acids on the surface of antibodies simply by tuning the molecular design; by strategies known as 'disulfide stapling' and 'cysteine-to-lysine transfer'. This will also enable us to carry out iterations of controlled chemical modification, accessing multifunctional conjugates which represent an enticing prospect for the next generation of antibody-based therapeutics.In a second key demonstration of applications enabled by the chemical methods pioneered in this project, we aim to show that nitrile reagents could also offer a unique ability to selectively inhibit specific classes of enzymes. Such covalent inhibitors are a growth area in drug discovery, as they offer the prospect of prolonged therapeutic effects. However, selectivity is vital in such strategies. We aim to show that reagents which form stabilised adducts only when they react with two proximal cysteine amino-acids, form a new mechanistic class of selective inhibitors for enzymes. Our overall approach within this project is to explore and tune an untapped, fascinating chemical reaction, and to demonstrate that it offers significant new opportunities in protein modification. In so doing, this project will offer insights stretching from fundamental chemistry to therapeutic development.