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 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.