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

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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.