Microtubule regulation by isotype expression, post translational modification, and by small molecules.

为了更好地了解药物等小分子实际上是如何作用于微管和微管阵列的，我们继续在生化、生物物理和结构水平上研究微管蛋白与药物的相互作用。这些研究包括分析可能具有更有利的作用光谱的新化合物，以及更容易的化学。在2020年的报告中，我们报道了新药rigosertib，Rigosertib是一种在秋水仙素位置与微管蛋白结合的化合物。这个结合位点多年来一直在这个项目中有重要的存在。现在已知的与这个部位结合的化合物的数量相当多，许多已经被研究用于各种疾病条件。Rigosertib是一种在秋水仙素部位与微管蛋白结合的化合物。我们证明了rigosertib有希望作为儿童横纹肌肉瘤的治疗药物，这种疾病预后很差，目前的治疗包括使用长春花碱，这种药物在不同的结合部位与微管蛋白结合，可能会显示出严重的毒性作为副作用。我们发现，虽然rigosertib可以抑制致癌的RAS信号，但其细胞毒活性是由于抑制了微管功能，如导致横纹肌肉瘤细胞系有丝分裂停止，导致细胞死亡。 我们通过对鸡红细胞微管蛋白上秋水仙碱位点的研究，扩展了我们对秋水仙碱位点配体的研究。这种微管蛋白含有最分化的β-微管蛋白(βVI，基因TUBB1)，其表达仅限于血细胞。为了研究这种微管蛋白与秋水仙碱结合部位的亲和力，并将其与更广泛表达的微管蛋白(大鼠脑内微管蛋白--主要用作微管蛋白的模型)进行比较，我们定量地测量了53种不同配体、一些活性使用中的药物、一些临床上正在开发的药物以及一些已知与脑微管蛋白秋水仙碱位点结合的简单配体的结合情况。为此，我们开发并使用了一种新的基于荧光的秋水仙素配体竞争分析方法，该方法使用了一种名为MDL的参考化合物。结果表明，对于大多数配体，特别是那些明显基于秋水仙素结构的配体，与血液微管蛋白的亲和力低于与脑微管蛋白的亲和力。以苯并咪唑为基础的配体有一个明显的例外。这些配体中的许多多年来一直在兽医和人类医学中用作抗寄生虫药物，其中一些现在正被重新用于治疗人类癌症。我们发现，这些化合物中的一部分与血液微管蛋白的结合比与脑微管蛋白的结合要好得多。这一发现开启了将这些化合物应用于血细胞疾病治疗的可能性，其中可能包括血细胞癌。 微管蛋白包含秋水仙素以外的结合位点，这些结合位点是通过它们第一次发现的或聚合的配体而知道的。其中一个这样的位点结合了一组高度修饰的多肽，这些多肽大多来自海洋来源。这个结合位点与长春新碱药物的结合位点重叠，在临床上长期使用。我们已经发表了许多关于VincA位点和多肽位点配体的研究，包括海洋脱脂多肽Cryptophycin。这种非常有效的细胞毒性药物与微管蛋白结合，并在原本是直的聚合物中诱导弯曲。这导致了仅由8个微管蛋白二聚体组成的紧密弯曲的环聚合物的形成，正如我们在以前的出版物中所显示的那样。我们在这一时期报道的研究展示了通过低温电子显微镜图像和重建获得的这种环聚合物的结构。该环的模型达到了3.3的分辨率，使人们能够前所未有地深入了解这种强效药物的机制。这为将这种强效药物用作抗体-药物结合物用于可能的人类癌症治疗提供了洞察力和指导。 通常，微管和微管阵列的调节以及小分子对这些阵列的影响的研究依赖于显微镜工具，无论是对纯化蛋白质的体外分析，还是对细胞内微管阵列的分析，以及通过对MT阵列的影响而介导的小分子对细胞生物学的影响。出于这个原因，我们继续研究使用荧光方法和先进显微镜应用来实现这些目标的新方法。我们之前已经发表了一些方法，使用基于肌红蛋白-mCherry结构的遗传编码的、基于FRET的氧传感器来绘制细胞内的氧水平，并显示它们是如何受到不同的细胞外氧水平的影响，以及如何将其与通过双光子寿命成像(FLIM)显微镜测量细胞质的氧化还原水平相结合。我们还报道了对细胞氧化损伤敏感的探针的开发，这种探针通过与(主要)来自活性氧物种(ROS)的氧化应激产生的蛋白质羰基发生共价反应而产生荧光来对细胞的氧化损伤敏感。我们还展示了新的荧光羰基探针在检测活细胞和肾脏疾病中的氧化损伤方面的应用。在目前的报告中，我们用一种新的遗传编码的两用探头来扩展这些方法，该探头可以在受控的外部氧气水平下测量活细胞中的氧水平和一氧化氮水平。这扩展了我们的分析能力，包括暴露在各种应激源下的活细胞的氧化和亚硝化应激，如低外部氧气、暴露于各种药物或其他化学挑战。

In pursuit of better understanding of how small molecules such as drugs actually work on microtubules and microtubule arrays, we have continued to study tubulin-drug interactions at biochemical, biophysical, and structural levels. These studies include analyzing new compounds with possibly more favorable spectra of actions as well as more facile chemistry. In the report of 2020, we reported on the novel drug rigosertib, Rigosertib is a compound that binds to tubulin at the colchicine site. This binding site has had significant presence in this project for many years. The number of compounds now known to bind to this site is quite large, and many have been investigated for use in various diseases conditions. Rigosertib is a compound that binds to tubulin at the colchicine site. We demonstrated that rigosertib has promise as a therapeutic for pediatric rhabdomyosarcoma, a disease with poor prognosis whose current treatment includes use of vinca alkaloids, drugs that bind to tubulin at a different binding site, and which can show severe toxicity as a side-effect. We show that, while rigosertib can inhibit oncogenic RAS signaling, its cytotoxic activity is due to inhibition of microtubule function, such as causing mitotic arrest in rhabdomyosarcoma cell lines, leading to cell death. We expanded our study of colchicine site ligands with a study of the colchicine site on chicken erythrocyte tubulin. This tubulin contains the most divergent beta-tubulin ( beta VI, gene TUBB1), which is restricted in expression to blood cells. To study the colchicine site affinity of this tubulin and compare it to more widely expressed tubulins (rat brain tubulin - which is mostly used as model for tubulin), we quantitatively measured the binding of 53 different ligands, some drugs in active use, some in development for possible clinical use, and some simply ligands known to bind to the colchicine site of brain tubulin. For this purpose we developed and used a new fluorescence-based competition assay for colchicine ligands using a reference compound known as MDL. The results showed that for most of the ligands, especially those obviously based on the colchicine structure, the binding to blood tubulin showed lower affinity than to brain tubulin. A significant exception was found for the ligands based on benzimidazole. Many of these ligands have been in use for years as anti-parasiticals in veterinary and human medicine, and some are now being repurposed for use in treatment of human cancers. We found that a subset of these compounds bind much better to blood tubulin than to brain tubulin. This discovery opens the possibility of applying these compounds to treatment of blood cell diseases, including possibly blood cell cancers. Tubulin contains binding sites other than the colchicine site, and these are known by their first discovered or paradigmatic ligands. One such site binds a group of highly modified peptides, mostly derived from marine sources. This binding site overlaps with the site for binding of the Vinca drugs, long in clinical use. We have published many studies of the Vinca site and the peptide site ligands, including the marine depsipeptide cryptophycin. This extremely potent cytotoxic drugs binds to tubulin and induces curvature in the otherwise straight polymers. This results in formation of tightly curved ring polymers composed of only 8 tubulin dimers, as we showed in previous publications. The study we reported in this period presents the structure of this ring polymer obtained from cryoelectron microscope images and reconstruction. The models of the ring achieve 3.3 resolution, allowing unprecedented insight into the mechanism of this potent drug. This provides insight and guidance into adapting this potent drug for use as an antibody-drug-conjugate for possible treatment of human cancers. Often study of the regulation of microtubules and microtubule arrays and the effects of small molecules on those arrays depends on tools of microscopy, both for in vitro assays with purified proteins and especially for assays that address microtubule arrays inside cells, and the effects of small molecules on the biology of cells mediated by effects on the MT arrays. For this reason we have continued our work on new methods that use fluorescence methods and advanced microscope applications to pursue these aims. We have previously published methods to use genetically encoded, FRET-based oxygen sensors based on a myoglobin-mCherry construct to map the intracellular oxygen levels and show how they are affected by varying extracellular oxygen levels, and how this can be combined with measurement of the cytoplasmic redox level by 2-photon lifetime imaging (FLIM) microscopy. We have also reported development of probes which are sensitive to oxidative damage to cells via producing fluorescence upon reacting covalently with (mostly) protein carbonyls produced by oxidative stress from Reactive Oxygen Species, or ROS. We also showed the utility of the new fluorogenic carbonyl probe for detection of oxidative damage in living cells and in kidney disease. In the current report, we expand these approaches with a new genetically-coded dual-purpose probe that can measure oxygen level and nitric oxide levels in living cells under exposure of controlled external oxygen levels. This extends our analytical capability to include oxidative and nitrosative stress in living cells exposed to various stressors, such as low external oxygen, exposure to various drugs, or other chemical challenges.