Mitotic inhibitor
A mitotic inhibitor, microtubule inhibitor, or tubulin inhibitor, is a drug that inhibits mitosis, or cell division, and is used in treating cancer, gout, and nail fungus. These drugs disrupt microtubules, which are structures that pull the chromosomes apart when a cell divides. Mitotic inhibitors are used in cancer treatment, because cancer cells are able to grow through continuous division that eventually spread through the body. Thus, cancer cells are more sensitive to inhibition of mitosis than normal cells. Mitotic inhibitors are also used in cytogenetics, where they stop cell division at a stage where chromosomes can be easily examined.
Mitotic inhibitors are derived from natural substances such as plant alkaloids, and prevent cells from undergoing mitosis by disrupting microtubule polymerization, thus preventing cancerous growth. Microtubules are long, ropelike proteins, long polymers made of smaller units of the protein tubulin, that extend through the cell and move cellular components around. Microtubules are created during normal cell functions by assembling tubulin components, and are disassembled when they are no longer needed.
One of the important functions of microtubules is to move and separate chromosomes and other components of the cell for cell division. Mitotic inhibitors interfere with the assembly and disassembly of tubulin into microtubule polymers. This interrupts cell division, usually during the mitosis phase of the cell cycle when two sets of fully formed chromosomes are supposed to separate into daughter cells. Tubulin binding molecules have generated significant interest after the introduction of the taxanes into clinical oncology and the general use of the vinca alkaloids.
Examples of mitotic inhibitors frequently used in the treatment of cancer include paclitaxel, docetaxel, vinblastine, vincristine, and vinorelbine.
Colchicine and griseofulvin are mitotic inhibitors used in the treatment of gout and nail fungus, respectively.
Microtubules
s are the key components of the cytoskeleton of eukaryotic cells and have an important role in various cellular functions such as intracellular migration and transport, cell shape maintenance, polarity, cell signaling and mitosis. They play a critical role in cell division by their involvement in the movement and attachment of chromosomes during various stages of mitosis. Therefore, microtubule dynamics are an important target for the developing anti-cancer drugs.Structure
Microtubules are composed of two globular protein subunits, α- and β-tubulin. These two subunits combine to form an α,β-heterodimer which then assembles in a filamentous tube-shaped structure. The tubulin hetero-dimers arrange themselves in a head to tail manner with the α-subunit of one dimer coming in contact with the β-subunit of the other. This arrangement results in the formation of long protein fibres called protofilaments.These protofilaments form the backbone of the hollow, cylindrical microtubule, which is about 25 nanometers in diameter and varies from 200 nanometers to 25 micrometers in length. About 12–13 protofilaments arrange themselves in parallel to form a C-shaped protein sheet, which then curls around to give a pipe-like structure called the microtubule. The head to tail arrangement of the hetero dimers gives polarity to the resulting microtubule, which has an α-subunit at one end and a β-subunit at the other end. The α-tubulin end has negative charges while the β-tubulin end has positive charges. The microtubule grows from discrete assembly sites in the cells called Microtubule organizing centers, which are networks of microtubule associated proteins.
Two molecules of energy rich guanosine triphosphate are also important components of the microtubule structure. One molecule of GTP is tightly bound to the α-tubulin and is non-exchangeable whereas the other GTP molecule is bound to β-tubulin and can be easily exchanged with guanosine diphosphate. The stability of the microtubule will depend on whether the β-end is occupied by GTP or GDP. A microtubule having a GTP molecule at the β-end will be stable and continue to grow whereas a microtubule having a GDP molecule at the β-end will be unstable and will depolymerise rapidly.
Microtubule dynamics
Microtubules are not static but they are highly dynamic polymers and exhibit two kinds of dynamic behaviors : 'dynamic instability' and 'treadmilling'. Dynamic instability is a process in which the microtubule ends switches between periods of growth and shortening. The two ends are not equal; the α-tubulin ringed end is less dynamic while the more dynamic β-tubulin ringed end grows and shortens more rapidly. Microtubules undergo long periods of slow lengthening, brief periods of rapid shortening and also pauses in which there is neither growth nor shortening.Dynamic instability is characterized by four variables: the rate of microtubule growth; the rate of shortening; frequency of transition from the growth or paused state to shortening and the frequency of transition from shortening to growth or pause.
The other dynamic behavior called treadmilling is the net growth of the microtubule at one end and the net shortening at the other end. It involves the intrinsic flow of tubulin sub-units from the plus end to the minus end. Both the dynamic behaviors are important and a particular microtubule may exhibit primarily dynamic instability, treadmilling or a mixture of both.
Mechanism of action
Agents which act as inhibitors of tubulin also act as inhibitors of cell division.A microtubule exists in a continuous dynamic state of growing and shortening by reversible association and dissociation of α/β-tubulin heterodimers at both the ends. This dynamic behavior and resulting control over the length of the microtubule is vital to the proper functioning of the mitotic spindle in mitosis i.e., cell division.
Microtubules are involved in different stages of the cell cycle. During the first stage or prophase, the microtubules required for cell division begin to form and grow towards the newly formed chromosomes,forming a bundle of microtubules called the mitotic spindle. During prometaphase and metaphase this spindle attaches itself to the chromosomes at a particular point called the kinetochore and undergoes several growing and shortening periods in tune with the back and forth oscillations of the chromosomes. In anaphase also, the microtubules attached to the chromosomes maintain a carefully regulated shortening and lengthening process. Thus a drug which can suppress the microtubule dynamics can block the cell cycle and result in the death of the cells by apoptosis.
Tubulin inhibitors thus act by interfering with the dynamics of the microtubule, i.e., growing and shortening. One class of inhibitors operate by inhibiting polymerization of tubulin to form microtubules and are called polymerization inhibitors like the colchicine analogues and the vinca alkaloids. They decrease the microtubule polymer mass in the cells at high concentration and act as microtubule-destabilizing agents. The other class of inhibitors operate by inhibiting the depolymerization of polymerized tubulin and increases the microtubule polymer mass in the cells. They act as microtubule-stabilizing agents and are called depolymerization inhibitors like the paclitaxel analogues. These three classes of drugs seems to operate by slightly different mechanism.
Colchicine analogues blocks cell division by disrupting the microtubule. It has been reported that the β-subunit of tubulin is involved in colchicine binding. It binds to the soluble tubulin to form colchicine-tubulin complex. This complex along with the normal tubulins then undergoes polymerization to form the microtubule. However the presence of this T-C complex prevents further polymerization of the microtubule. This complex brings about a conformational change which blocks the tubulin dimers from further addition and thereby prevents the growth of the microtubule. As the T-C complex slows down the addition of new dimers, the microtubule disassembles due to structural imbalance or instability during the metaphase of mitosis.
The Vinca alkaloids bind to the β-subunit of tubulin dimers at a distinct region called the Vinca-binding domain. They bind to tubulin rapidly, and this binding is reversible and independent of temperature. In contrast to colchicine, vinca alkaloids bind to the microtubule directly. They do not first form a complex with the soluble tubulin nor do they copolymerize to form the microtubule, however they are capable of bringing about a conformational change in tubulin in connection with tubulin self-association. Vinca alkaloids bind to the tubulin with high affinity at the microtubule ends but with low affinity at the tubulin sites present along the sides of the microtubule cylinder. The binding of these drugs at the high affinity sites results in strong kinetic suppression of tubulin exchange even at low drug concentration while their binding to the low affinity sites in relatively high drug concentration depolymerizes microtubules.
In contrast to colchicine and vinca alkaloids, paclitaxel enhances microtubule polymerization promoting both the nucleation and elongation phases of the polymerization reaction, and it reduces the critical tubulin sub-unit concentration. Microtubules polymerized in presence of paclitaxel are extremely stable. The binding mechanism of the paclitaxel mimic that of the GTP nucleotide along with some important differences. GTP binds at one end of the tubulin dimer keeping contact with the next dimer along each of the protofilament while the paclitaxel binds to one side of β-tubulin keeping contact with the next protofilament. GTP binds to unassembled tubulin dimers whereas paclitaxel binding sites are located only in assembled tubulin. The hydrolysis of GTP permits the disassembly and the regulation of the microtubule system; however, the activation of tubulin by paclitaxel results in permanent stabilization of the microtubule. Thus the suppression of microtubule dynamics was described to be the main cause of the inhibition of cell division and of tumor cell death in paclitaxel treated cells.