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

通过同种型表达、翻译后修饰和小分子进行微管调节。

• 批准号: 10920197
• 负责人: Dan L Sackett
• 金额: $ 60.44万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10920197
• 关键词: Adopted; Affinity; Anti-Inflammatory Agents; Antiparasitic Agents; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Blood; Blood Cells; Bone Marrow; Brain; Cells; Cellular Morphology; Cellular biology; Chemistry; Chickens; Cilia; Clinical; Clinical Medicine; Code; Colchicine; Collaborations; Cryoelectron Microscopy; Curiosities; Cytoplasm; Cytoskeleton; Development; Disease; Drug Interactions; Drug Targeting; Drug resistance; Drug usage; Elements; Erythrocytes; Familial hypophosphatemic bone disease; Fanconi Syndrome; Fluorescence; Genes; Genetic Predisposition to Disease; Genome; Growth; Guanosine Triphosphate Phosphohydrolases; HIV; Hematopoietic Neoplasms; Human; Hyperopia; Kidney; Knowledge; Learning; Ligands; Macrolides; Malignant Neoplasms; Measures; Mediating; Medicine; Microtubule Stabilization; Microtubules; Mitosis; Mitotic spindle; Modeling; Molecular Conformation; Movement; Mutation; National Human Genome Research Institute; Natural Products; Organism; Paclitaxel; Parasites; Parents; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Polymers; Post-Translational Protein Processing; Property; Protein Subunits; Proteins; Rattus; Recycling; Regulation; Reporting; Research; Retina; Role; Site; Solid Neoplasm; Structure; Surface; Tail; Testing; Therapeutic; Time; Tissues; Tubular formation; Tubulin; Work; alpha Tubulin; benzimidazole; beta Tubulin; blood treatment; cancer cell; chemotherapy; combinatorial; copolymer; dimer; disease-causing mutation; hearing impairment; improved; insight; katanin; marine natural product; novel therapeutics; physical property; preclinical development; programs; protein aminoacid sequence; residence; resistance mechanism; rev Protein; small molecule; stoichiometry; trafficking; tumor

In pursuit of better understanding of how tubulin, MT, and MT arrays function in the biology of the cell and organism, and how small molecules such as drugs actually work on microtubules and microtubule arrays, we have continued to study MT, tubulin, and 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 2021, we reported on 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 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. This past year, we examined the possibility of using what we learned in the 2021 study of blood cell tubulin to take colchicine site drugs used in anti-parasite therapy and repurpose them for use in new aspects of clinical medicine, especially as cancr chemotherapy. Compounds with reduced affinity for blood cell tubulin (TUBB1) can and have been used to treat solid tumors, while sparing blood cells and bone marrow. On the other hand, compounds with increased affinity for blood cell tubulin might be useful in treatment of blood cancers, while targeting the activity so as to spare non-blood tissues. This re-purposing of known compounds is an exciting development that is finding relevance with many small compounds. Tubulin contains binding sites other than the colchicine site, and can destabilize MT, like colchicine-site ligands, or stabilize MT, like the widely employed cancer chemotherapeutic Taxol. A number of compounds, most natural products, have been discovered that improve on various aspects of Taxol therapy. A relatively new compound is Zampanolide, a marine natural product, which has similar potency to Taxol, but binds covalently to tubulin, can evade muti-drug resistance mechanisms, and possibly extend the residence time of the drug in a target tissue. The compound is currently in pre-clinical development. Zampanolide is a macrolide, and we were curious to test if the activity required this closed ring structure. We demonstrated that a linear molecule could be constructed that would adopt the appropriate conformation in solution to yield similar binding affinity to the parent compound. We have also examined the role of the outer surfact of the MT on interaction with other proteins. The CTT extend from the surface of the MT and these are recognized by the MT-breaking protein katanin. We found that the sequence differences between the CTT of different tubulin isotypes regulate the affinity of katanin for the tubulin tails, and therefore would alter the activity of katanin on breaking and recycling MT composed of differing CTTs, and therefore different CTT sequences. We also examined the MT- depolymerizing mechanism of the HIV protein Rev. We demonstrated this activity a number of years ago, but gained new insight into how Rev disassembles MT in a study using Cryo-EM. This allowed us to describe the basis for the ring-polymers of tubulin and Rev that form when the two proteins meet. Induction of this curvature in the MT wall causes peeling our of the component protofilaments. This will arrest MT growth and cause disassemble of MT and formation of ring co-polymers at sufficient stoichiometry of the two proteins. The function of MT and MT arrays is sensitive to small changes in tubulin sequence since it can alter the folding of tubulin subunits and interfere with assembly of MT and development of MT arrays. In collaboration with the Undiagnosed Diseases Program, NHGRI, we reported a patient who had a mutation in a particular beta-tubulin isotype, TUBB4B, which is a critical component of cilia. The patient presented a number of phenotypes, including hearing loss and hyperopia without retinal abnormalities, as well as two phenotypes of unknown genetic etiology, i.e., renal tubular Fanconi Syndrome (FS) and hypophosphatemic rickets (HR). We determined a potential structural explanation for the effect of the mutation on MT function, due to interference with tubulin GTPase activity.

为了更好地了解微管蛋白、微管蛋白和微管阵列在细胞和生物体生物学中的功能，以及药物等小分子实际上如何作用于微管和微管阵列，我们继续在生物化学、生物物理学和结构水平上研究微管蛋白和微管蛋白-药物相互作用。这些研究包括分析可能具有更有利的作用谱和更容易的化学性质的新化合物。在2021年的报告中，我们报告了我们对秋水仙碱位点配体的研究，并对鸡红细胞微管蛋白上的秋水仙碱位点进行了研究。这种微管蛋白含有最趋异的β-微管蛋白（β VI，基因TUBB 1），其表达仅限于血细胞。为了研究这种微管蛋白的秋水仙碱位点亲和力，并将其与更广泛表达的微管蛋白（大鼠脑微管蛋白-主要用作微管蛋白的模型）进行比较，我们定量测量了53种不同配体的结合，一些药物正在积极使用，一些正在开发用于可能的临床用途，以及一些已知与脑微管蛋白的秋水仙碱位点结合的简单配体。为此，我们开发并使用了一种新的基于荧光的秋水仙碱配体的竞争试验，使用称为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碱结构的配体，与血微管蛋白的结合亲和力低于与脑微管蛋白的结合亲和力。一个显着的例外，发现基于苯并咪唑的配体。这些配体中的许多已经在兽医和人类医学中作为抗寄生虫药使用多年，并且一些现在被重新用于治疗人类癌症。我们发现，这些化合物的子集结合更好地血液微管蛋白比脑微管蛋白。 这一发现开启了将这些化合物应用于治疗血细胞疾病（可能包括血细胞癌症）的可能性。在过去的一年里，我们研究了使用我们在2021年血细胞微管蛋白研究中学到的东西来服用抗寄生虫治疗中使用的秋水仙碱位点药物的可能性，并将其重新用于临床医学的新方面，特别是作为抗寄生虫化疗。对血细胞微管蛋白（TUBB 1）具有降低亲和力的化合物可以并且已经用于治疗实体瘤，同时保留血细胞和骨髓。另一方面，对血细胞微管蛋白具有增加的亲和力的化合物可用于治疗血癌，同时靶向活性以节省非血液组织。这种对已知化合物的重新利用是一个令人兴奋的发展，它与许多小化合物有关。 微管蛋白含有秋水仙碱位点以外的结合位点，并且可以使MT不稳定，如秋水仙碱位点配体，或稳定MT，如广泛使用的癌症化疗剂紫杉醇。许多化合物，大多数天然产物，已被发现，改善紫杉醇治疗的各个方面。一种相对较新的化合物是Zampanaltine，一种海洋天然产物，其具有与紫杉醇相似的效力，但与微管蛋白共价结合，可以逃避多药耐药机制，并可能延长药物在靶组织中的停留时间。该化合物目前处于临床前开发阶段。Zampanaltine是一种大环内酯，我们很想测试这种活性是否需要这种闭环结构。我们证明了可以构建线性分子，其在溶液中采用适当的构象以产生与母体化合物相似的结合亲和力。 我们还研究了MT外表面与其他蛋白质相互作用的作用。CTT从MT的表面延伸，并且这些被MT断裂蛋白Katanin识别。我们发现，不同微管蛋白同种型的CTT之间的序列差异调节katanin对微管蛋白尾部的亲和力，因此将改变katanin对由不同CTT组成的MT的破坏和再循环的活性，因此改变不同CTT序列。我们还研究了HIV蛋白Rev的MT解聚机制。我们在几年前证明了这种活性，但在使用Cryo-EM的研究中获得了对Rev如何分解MT的新见解。这使我们能够描述当两种蛋白质相遇时形成的微管蛋白和Rev的环状聚合物的基础。MT壁中这种弯曲的诱导会导致组成原丝的剥落。这将阻止MT生长，并导致MT分解和在两种蛋白质的足够化学计量下形成环状共聚物。 MT和MT阵列的功能对微管蛋白序列的微小变化敏感，因为它可以改变微管蛋白亚基的折叠并干扰MT的组装和MT阵列的形成。 在与未诊断疾病项目NHGRI合作中，我们报告了一名在特定β-微管蛋白同种型TUBB 4 B中发生突变的患者，TUBB 4 B是纤毛的关键成分。该患者表现出多种表型，包括听力损失和无视网膜异常的远视，以及两种未知遗传病因的表型，即，肾小管Fanconi综合征（FS）和低血磷性佝偻病（HR）。我们确定了一个潜在的结构解释的突变MT功能的影响，由于干扰微管蛋白GT3活性。

项目成果

Clinical, genetic, and structural characterization of a novel TUBB4B tubulinopathy.

新型TUBB4B肾小管病的临床，遗传和结构表征。

• DOI: 10.1016/j.ymgmr.2023.100990
• 发表时间: 2023-09
• 期刊: MOLECULAR GENETICS AND METABOLISM REPORTS
• 影响因子: 1.9
• 作者: McFadden, Jason R.;Tolete, Christina Deanne P.;Huang, Yan;Macnamara, Ellen;Sept, David;Nesterova, Galina;Gahl, William A.;Sackett, Dan L.;Malicdan, May Christine V.
• 通讯作者: Malicdan, May Christine V.

Using in vivo fluorescence lifetime imaging to detect HER2-positive tumors.

• DOI: 10.1186/s13550-018-0384-6
• 发表时间: 2018-04-04
• 期刊: EJNMMI research
• 影响因子: 3.2
• 作者: Ardeshirpour Y;Sackett DL;Knutson JR;Gandjbakhche AH
• 通讯作者: Gandjbakhche AH

Total synthesis and biological evaluation of tubulysin U, tubulysin V, and their analogues.

• DOI: 10.1021/jm8013579
• 发表时间: 2009-01-22
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Balasubramanian R;Raghavan B;Begaye A;Sackett DL;Fecik RA
• 通讯作者: Fecik RA

Taurine Is Covalently Incorporated into Alpha-Tubulin.

• DOI: 10.1021/acs.jproteome.0c00147
• 发表时间: 2020-08-07
• 期刊: Journal of proteome research
• 影响因子: 4.4
• 作者: Olson MT;Yergey AL;Mukherjee K;Pergande MR;Bane SL;Cologna SM;Sackett DL
• 通讯作者: Sackett DL

Targeting mitochondrial hexokinases increases efficacy of histone deacetylase inhibitors in solid tumor models.

• DOI: 10.1016/j.yexcr.2018.12.012
• 发表时间: 2019-02-15
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: McDonald AJ;Curt KM;Patel RP;Kozlowski H;Sackett DL;Robey RW;Gottesman MM;Bates SE
• 通讯作者: Bates SE