MICA: Novel mode of RNA polymerase inhibition by a new natural rifamycin, which is active against rifampicin-resistant RNA polymerases and bacteria

MICA：新型天然利福霉素抑制 RNA 聚合酶的新模式，对利福平耐药的 RNA 聚合酶和细菌具有活性

• 批准号: MR/T000740/1
• 负责人: Nikolay Zenkin
• 金额: $ 78.19万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FT000740%2F1
• 关键词: MICA; Novel; mode; RNA; polymerase

Rifamycins are a family of mainly semisynthetic antibiotics (including rifampicin, RIF) that inhibit bacterial RNA polymerase (RNAP), the essential enzyme accomplishing the first step of gene expression - transcription. RIF is a key drug in the treatment of tuberculosis - the leading cause of death by infectious disease, with 10.4 million new cases and 1.7 million deaths annually. Development of RIF-resistance by the causative agent of tuberculosis bacterium Mycobacterium tuberculosis is the major problem in treatment of tuberculosis, with 600,000 new RIF-resistant TB cases reported each year. Furthermore, RIF-resistant M. tuberculosis can also develop further resistance to other first, second and third line antibiotics (multi-drug-resistance (MDR-Mtb) and extensive-drug-resistance (XDR-Mtb)), resulting in 240,000 additional deaths per year. Therefore, overcoming RIF-resistance would be an important step in treatment of drug-resistant tuberculosis. However the development of new rifamycins has stalled, due to both RIF-resistance problems and the fact that the areas of the molecule known to allow semi-synthetic modification, without loss of activity, have been limited to those originally discovered in the 1950s. Previous attempts to introduce modifications on other positions resulted in inactivation of the antibiotic. Recently, MICA co-applicants and the Industrial Partner Demuris Ltd. discovered a new natural rifamycin, referred to as B1, which carries unique substituents in previously underexplored regions of the molecule. Remarkably, B1 is up to three orders of magnitude more effective against RIF-resistant RNA polymerases, with the most clinically frequent mutations having no significant effect on its efficacy, and also showed antibiotic activity against RIF-resistant MDR-Mtb. Furthermore, unlike any other rifamycins, that sterically block propagation of the growing nascent RNA through RNAP, B1 strongly inhibits synthesis of the initial dinucleotide, indicating that it has a different mode of action to previous rifamycins. However the mechanisms of inhibition of RNAP and of overcoming RIF-resistance by B1 are not known. Finally we found that B1 is also tolerant to ADP-ribosylation, a modification of the rifamycins that inactivates them leading to RIF-resistance - a poorly understood resistance mechanism adopted by some Mycobacteria. It is, however, not clear if B1 cannot be ADP-ribosylated or if its binding to RNAP is not affected by the modification. Understanding the mode of action of RNAP inhibitors at the molecular level has proven to be a powerful research tool in understanding functions of RNAP itself. We therefore propose a fundamental study into understanding the mechanisms of B1 action, and to use B1 (and its derivatives) as molecular probes to shed new light on the early steps of the complex process of initiation of RNA synthesis, plasticity of RNAP active centre, and basic principles of RIF-resistance. Employing a combination of organic chemistry, molecular biology and crystallography, we will modify unique groups of B1 and test the resulting derivatives in a very wide range of in vitro transcription experiments. Derivatives will be crystallised with transcription promoter open complex and RNAP holoenzymes carrying the clinically most frequent RIF-resistant mutations (which has already been successful with B1). Furthermore we will analyse in vitro ADP-ribosylation and its consequences on transcription. This information will help the understanding of the basic processes at the very first steps of RNA synthesis and the plasticity of RNAP active centre, providing essential knowledge towards overcoming RIF-resistance in the future. These studies may identify new targets for antimicrobials, and will be also important for future development of B1 or its derivatives as antibiotics against IRF-resistant Mtb by the Industrial Partner.

利福霉素是一类半合成抗生素（包括利福平、RIF），主要抑制细菌RNA聚合酶（RNAP）， RNAP是完成基因表达的第一步——转录的必需酶。RIF是治疗结核病的关键药物，结核病是传染病造成死亡的主要原因，每年有1 040万新病例和170万人死亡。结核分枝杆菌致病菌对rif产生耐药性是结核病治疗中的主要问题，每年有60万新的rif耐药结核病病例报告。此外，耐rif结核分枝杆菌还可能对其他一线、二线和三线抗生素（耐多药和广泛耐药）产生进一步耐药性，每年造成24万人额外死亡。因此，克服rif耐药性将是治疗耐药结核病的重要一步。然而，新利福霉素的开发已经停滞不前，这既是由于rif耐药性问题，也是由于分子中已知允许半合成修饰而不丧失活性的区域仅限于20世纪50年代最初发现的那些区域。以前在其他位置引入修饰的尝试导致抗生素失活。最近，MICA的共同申请者和工业合作伙伴Demuris有限公司发现了一种新的天然利福霉素，称为B1，它在分子中以前未被开发的区域携带独特的取代基。值得注意的是，B1对rif耐药RNA聚合酶的有效性提高了3个数量级，临床上最常见的突变对其疗效没有显著影响，并且对rif耐药MDR-Mtb也显示出抗生素活性。此外，与其他利福霉素通过RNAP立体阻断生长的新生RNA的传播不同，B1强烈抑制初始二核苷酸的合成，表明它与以前的利福霉素具有不同的作用模式。然而，B1抑制RNAP和克服rif耐药的机制尚不清楚。最后，我们发现B1对adp核糖基化也有耐受性，这是利福霉素的一种修饰，使它们失活，导致rif耐药，这是一些分枝杆菌采用的一种尚不清楚的耐药机制。然而，目前尚不清楚B1是否不能被adp核糖化，或者它与RNAP的结合是否不受修饰的影响。在分子水平上了解RNAP抑制剂的作用模式已被证明是了解RNAP本身功能的有力研究工具。因此，我们建议对B1的作用机制进行基础研究，并利用B1（及其衍生物）作为分子探针来揭示RNA合成起始复杂过程的早期步骤、RNAP活性中心的可塑性以及rif抗性的基本原理。结合有机化学、分子生物学和晶体学，我们将对B1的独特基团进行修饰，并在非常广泛的体外转录实验中测试所得到的衍生物。衍生物将与转录启动子开放复合体和RNAP全酶结晶，这些全酶携带临床上最常见的rif抗性突变（这已经在B1上取得了成功）。此外，我们将分析体外adp核糖基化及其对转录的影响。这些信息将有助于了解RNA合成的基本过程和RNAP活性中心的可塑性，为今后克服rif抗性提供必要的知识。这些研究可能确定抗菌剂的新靶点，并且对工业合作伙伴未来开发B1或其衍生物作为抗耐红外辐射结核分枝杆菌的抗生素也很重要。

项目成果

Transcription and chromatin-based surveillance mechanism controls suppression of cryptic antisense transcription.

• DOI: 10.1016/j.celrep.2021.109671
• 发表时间: 2021-09-07
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Heo DH;Kuś K;Grzechnik P;Tan-Wong SM;Birot A;Kecman T;Nielsen S;Zenkin N;Vasiljeva L
• 通讯作者: Vasiljeva L

Inhibition of RNA Polymerase by Rifampicin and Rifamycin-Like Molecules.

• DOI: 10.1128/ecosalplus.esp-0017-2019
• 发表时间: 2020-04-01
• 期刊: EcoSal Plus
• 作者: Mosaei, Hamed;Zenkin, Nikolay
• 通讯作者: Zenkin, Nikolay

Kanglemycin A Can Overcome Rifamycin Resistance Caused by ADP-Ribosylation by Arr Protein.

• DOI: 10.1128/aac.00864-21
• 发表时间: 2021-11-17
• 期刊: Antimicrobial agents and chemotherapy
• 影响因子: 4.9
• 作者: Harbottle J;Mosaei H;Allenby N;Zenkin N
• 通讯作者: Zenkin N

Structural Diversity of Perylenequinones Is Driven by Their Redox Behavior.

• DOI: 10.1021/acs.joc.1c02639
• 发表时间: 2022-03-04
• 期刊: The Journal of organic chemistry
• 作者: Al Subeh ZY;Waldbusser AL;Raja HA;Pearce CJ;Ho KL;Hall MJ;Probert MR;Oberlies NH;Hematian S
• 通讯作者: Hematian S

Ribosome reactivates transcription by physically pushing RNA polymerase out of transcription arrest.

• DOI: 10.1073/pnas.1919985117
• 发表时间: 2020-04-14
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Stevenson-Jones, Flint;Woodgate, Jason;Zenkin, Nikolay
• 通讯作者: Zenkin, Nikolay