Development and validation of new reagents and assays to exploit the final steps of peptidoglycan construction

每年有数百万人死于细菌感染，数千万人遭受这些感染的后果。抗生素青霉素的发现曾经为治疗这些感染打开了大门。它通过阻止细菌在细胞壁中制造聚合物来将它们结合在一起。这种聚合物称为肽聚糖（PG），由糖和氨基酸（肽）链的联锁网络组成。专门的蛋白质（称为青霉素结合蛋白或PBPs，存在于所有细菌中）具有将糖骨架和肽缝合在一起的能力。由PBPs形成的肽交联是青霉素抑制的目标，青霉素阻止细胞壁的形成并杀死细菌。青霉素一直是一种很好的抗生素，尤其是因为它同时针对细菌内的多个PBPs。不幸的是，许多细菌不再被青霉素和其他攻击肽聚糖生产的抗生素杀死。细菌通过改变目标PBPs并产生降解抗生素的酶来逃避这些抗生素的作用。我们需要反击，探索PBP的新抑制剂的策略被广泛认为是一个重要的经过充分验证的选择。实现这一目标的进展一直受到阻碍，因为我们无法常规合成制造这种聚合物的关键化学成分。我们现在可以在沃里克这样做，并有一个特殊的记录，提供试剂研究肽聚糖生物合成的学术界在世界各地。在研究了这些试剂的前体如何由PG途径中的酶产生之后，我们打算利用我们发现的机会开发具有定制组分的全新试剂。这对学术界和工业界来说都是令人兴奋的，因为我们将能够为特定功能生产量身定制的中间体。例如，我们可以包括放射性糖或氨基酸，荧光标记，或以改变其聚合能力的方式修饰糖或氨基酸。这些试剂将使我们和更广泛的社区能够探索关于这些靶点以及细菌如何生长和控制肽聚糖生产的根本性重要未解问题。我们将能够使用这些试剂来开发雄心勃勃的新检测方法，不仅可以检测这些靶标的活性，还可以探索将这些检测方法转化为工业形式，用于寻找全新的抑制剂，克服目前对青霉素和相关抗生素的耐药性问题。为了实现这一目标，我们将利用我们在过去十年中与酶学家，化学家，工程师，数学家和物理学家的资助所积累的学术专业知识，并将其用于与工业界建立新的更紧密的伙伴关系。这种伙伴关系将为我们提供开放获取，以更广泛地开发工作，增加我们可以生产的试剂平台，将我们的能力扩展到新的检测方法中，以研究肽聚糖构建的复杂，困难的最后阶段。所有这些都将有助于从根本上获得新的生物学见解。它还将为进一步开发这些试剂和测定方法以供行业使用提供机会。要做到这一点，我们将不得不改进现有的方法，以扩大生产规模，并开发强大的工业质量检测。我们的合作伙伴关系由沃里克大学的科学家和技术支持组成，他们具有互补的技能和专业知识来完成这些任务，沿着阿斯利康，他们致力于支持开放获取这种新的基础技术，并帮助开发高通量筛选的新方法。这预示着一个学术界和工业界可以密切合作寻找新抗生素的时代。

Millions of people die each year from bacterial infections and tens of millions suffer from the consequences of these infections. The discovery of the antibiotic penicillin once opened the door to treat these infections. It did this by stopping bacteria from making the polymer in the cell wall that holds them together. This polymer, called peptidoglycan (PG), is made up of an interlocking network of sugars and strings of amino acids (peptides). Specialised proteins (called penicillin-binding-proteins or PBPs, which are present in all bacteria) either have the ability to stitch together the sugar backbone and peptides. The construction of peptide cross-links by PBPs is famously the target inhibited by penicillin which stops cell wall construction and kills the bacterium. Penicillin has been an excellent antibiotic, not least because it targets multiple PBPs simultaneously within a bacterium. Unfortunately, many bacteria are no longer killed by penicillin and other antibiotics that attack the production of peptidoglycan. Bacteria have changed by evading the action of these antibiotics by modifying the target PBPs and producing enzymes that degrade the antibiotic. We need to fight back and the strategy of exploring PBPs for new inhibitors is widely recognised as an important well validated option. Progress in achieving this has been hampered by our inability to routinely synthesise the key chemical components that make this polymer. We can now do this at Warwick, and have an exceptional track record of providing reagents to study peptidoglycan biosynthesis to academia worldwide. Having studied how the precursors of these reagents are produced by enzymes in the PG pathway, we intend to exploit the opportunities we have discovered to develop completely new reagents with bespoke components. This is exciting for both the academic and industrial communities as we will become able to produce tailor-made intermediates for specific functions. For example, we can include radioactive sugars or amino acids, fluorescent labels, or modifying sugars or amino acids in ways which alter their ability to polymerise. These reagents will enable us, and the wider community, to explore fundamentally important unanswered questions about these targets andhow bacteria grow and control the production of peptidoglycan. We will be in a position to use these reagents to develop ambitious new assays, not only to characterise the activities of these targets, but also to explore the translation of these assays into formats for industry to use them in the search for completely new classes of inhibitors, overcoming current problems of resistance to penicillin and related antibiotics. To achieve this we will use our academic expertise gathered over the past decade of funding with enzymologists, chemists, engineers, mathematicians and physicists, and use this in a new closer partnership with industry. This partnership will provide open access for us to develop the work more widely, to increase the platform of reagents we can produce, extend our capability into new assays to study the complex, difficult, final stages of peptidoglycan construction. All of this will work towards fundamentally new biological insights. It will also underpin opportunities to further develop these reagents and assays for use by industry. To do this we will have to refine current methods to scale up production and develop robust industry quality assays. Our partnership consists of scientists and technical support at Warwick University with complementary skills and specialist knowledge to acomplish these tasks, along with Astra Zeneca, who are committed to supporting open access to this new underpinning technology and helping to develop novel approaches to high throughput screens. This heralds an era where academics and industry can work closely together in the search for new antibiotics.