The regulation of cell cycle involves growth regulatory signals and also signals from proteins that monitor the genetic integrity to ensure the absence of any genetic damage. Cyclins are known to be very important cell cycle regulation. They are a group of related proteins and are of four basic types which are found in humans as well as most other eukaryotes. Those types are: G1 cyclins, G1/S cyclins, S cyclins, and M cyclins. Each cyclin is associated with a particular phase or set of phases in the cell cycle. The levels of different types of cyclins vary considerably through the cell cycle. A typical cyclin associated with a particular phase is present at low levels through most of the cell cycle, but the levels strongly increase when they are needed. In order to drive the cell cycle, a cyclin might have to activate and/or inactivate many target proteins inside of the cell. Cyclins do so by partnering with a family of enzymes called the cyclin dependent kinases (CDKs). CDK alone is inactive, but the binding of a cyclin activates it, thereby making it a functional enzyme and allowing it to modify target proteins. (“Cell cycle regulators”, n.d.) Cancer is characterized by aberrant cell activity. Unregulated activation of CDKs frequently found in case of human cancers provided the rationale for designing synthetic inhibitors of CDKs as anticancer drugs. The vast majority of human neoplasia have aberrations in one or more of its cell cycle components due to overexpression of positive regulators of CDK function and/or decrease in the negative regulators of CDK function, which eventually results in hyperactivation of CDKs.
Approaches for CDK inhibition:
CDKs have a complex nature of regulation and because of this, pharmacological modulation of CDK function was thought to be unlikely. However, with the advances in research such as advent of newer biological probes and techniques, modulating CDK activity can be approached through multiple modes for therapeutic purposes. Chemical inhibitors of CDK: CDKs are little kinases that show eleven common sub domains which are shared by all protein kinases. The ATP-binding site, present in a deep cleft between the two lobes of the protein, contains catalytic residues which are conserved across eukaryotic protein kinases. Small molecules that specifically act with ATP-binding site of CDKs represent the foremost opportunity to allow pharmacological design. A group of compounds are characterized that can occupy the ATP-binding pocket of the enzyme and are competitive with the ATP. Chemical CDK inhibitors can be subdivided into the following eight families: purine derivatives (including 6-DMAP, olomucine, roscovitine and purvanolol), polysulphates (including suramin), butyrolactone I, flavopiridols, staurosporine and derivatives, toyocamycin, 9-hydroxyellypticine and paullones. It must be noted that new kinds of inhibitors are constantly being discovered. The antiproliferative effects on the growth of several human cell lines by these compounds has been well documented. The effects of these compounds in vivo paralleled the in vitro efficiency and were further confirmed with the use of naturals CKIs and the microinjection of inactivating antibodies. Flavopiridol, the first CDK modulator tested in clinical trials, demonstrated clinical features which included cell cycle block at G1 and G2 (consistent with its inhibition of CDK1 and CDK2), promotion of differentiation, induction of apoptosis, inhibition of angiogenic processes and modulation of transcriptional events. Besides the direct effects, flavopiridol can also depletes cyclin D1 and D3 by transcriptional repression. This may be a consequence of the direct inhibition of CDK9-cyclinT which is also known as P-TEF (positive transcription elongation factor). P-TEF is a required cellular cofactor for the human HIV trans activator Tat. Flavopiridol blocked Tat trans activation and blocked HIV-1 replication in vitro assays. This biochemical effect shows that the flavopiridol should be tested in HIV malignancies including HIV-lymphomas. Initial clinical trials with infusional flavopiridol demonstrated activity in some patients with a variety of tumor types, including renal, colon and prostate cancers and non-Hodgkin’s lymphoma. The second CDK modulator which was tested in clinical trials is the staurosporine derivative UCN-01. It also blocks cell cycle progression and promotes apoptosis. Moreover, it may also abrogate checkpoints induced by genotoxic stress due to inhibition of Chk-1 kinase. UCN-01 showed a long plasma half-life due to binding to the alpha-1-acid-glycroprotein. Clinical activity was detected against lung cancer, melanoma, and non-Hodgkin’s lymphoma.
Protein and peptide based inhibitors: CKIs combined with adenovirus vectors for vehicles for delivery and expression are a powerful approach to examine therapeutic applications of CDK inhibiton. Introduction of p16INK4a in tumor cells with functional pRb induces growth arrest of the cells at G1 phase. In a breast derived cell line, the chimera containing antennapedia peptide. and the carboxyl-terminal residue of pcip/wafl, had higher specificity for cdk4/cyclin D than for cdk2/cyclin E and arrested the cells in G1 phase. However, in vitro, the chimera containing amino terminal peptides of pcip/wafl, inhibited both CDK1 and CDK2, and the cells were arrested in all phases of the cell cycle. A 20 amino acid peptide, identified by use of a combinatorial library, specifically binds CDK2 and inhibits its activity at low nanomolar concentration in vitro. This peptide could act by blocking the interaction of the catalytic subunit with substrates or cyclin cofactors.
Altering regulatory pathways: The depletion of cyclin partner leads to CDK activity inhibition. Depletion of cyclin D1 from tumor cell lines with antisense fragments induced antiproliferative effects that were synergistic with other drugs. Inhibiton of either cyclin A or E synthesis or activity through microinjection of plasmids encoding antisense cyclin cDNA or affinity-purified anti-cyclin antibodies during G1 phase inhibited DNA sysnthesis, thereby providing a basis for the use of this trait as a therapeutic approach. Other than the use of antisense technologies to deplete the tumor cells from specific cyclins and/or CDKs, several compounds can inhibit tumor progression by modulating the levels of cell cycle proteins. In breast carcinoma cells, antiestrogens and retinoids inhinit the expression of cyclin D and other cell cycle realted proteins inhibiting CDK activity. In some systems rapamaycin treatment was associated with a decline in cyclin D1 and prevents IL-2-stimulayed degradation of p27kip1. Other molecules, such as lovastanin, block cells at G1 concomitant with p21cip1/waf1 and p27kip1 induction and CDK inactivation.
CDC25: It is a dual-specificity phosphate, which removes inhibitory phoshorylations on Thr14 and Tyr15 residues on the ATP anchor motif of CDKs, activating the kinase. CDC25, therefore, has been involved in cell transformation and tumorigenesis, checkpoint control and apoptosis. Hence, based on this information, the CDC25 family has been the subject of a screen for inhibitory compounds.
Targeting other cell cycle proteins: Proteasome inhibition: The ubiquitin-proteasome pathway is responsilbe for the degradation of eukaryotic cellular proteins. This ATP dependent process is crucial for normal cell cycling, fucntion, and survival, thereby making proteasome inhibition a novel therapeutic agent in cancer. Bortezomib was the first inhibitor of the ubiquitinin-proteasome pathway to enter clinical studies. Sequential turnover of certain cell cycle regulators, including cyclins and CKIs p21cip1/waf1 and p27kip1, are mediated by the 20S proteasome, which promotes proteolytic degradation through the ubiquitin/proteasome pathway. Increased turnover of these cyclins are associated with the loss of CDK activity. On the contrast, the inhibition of p21cip1/waf1 or p27kip1 specific degradation could induce CDK inhibition through accumulation of the CKI. DNA damage checkpoints and WEE1 inhibitors: Cell cycle check points are essential for halting the cell cycle progression in response to DNA damage, thereby allowing time for DNA repair. Inhibiton of CHK1 or WEE1 in cancer cells prevents cell cycle arrest during S or G2 phase and allows cell proliferation despite of presence of DNA damage. This can lead to cell death by a process sometimes referred to as mitotic catastrophe during mitosis. This strategy applies to cancer cells with compromised G1 checkpoint due to loss of p53 function, as these cells depend crucially on the G2 checkpoint, especially in the presence of DNA damage inducing drugs. B8776ecause of this, inactivation of p53 renders cancer cells sensitive to inhibition of CHK1 and WEE1, an example of so called synthetic lethality. MK-8776 exhibits high potency and selectivity for CHK1, and when cancer cells were treated with it, accumulation of DNA double-strands breaks was caused which lead to apoptotic cell death in vitro. Further, it also synergized with gemcitabine, hydroxyurea and cytabrine in causing apoptosis of AML and breast cancer cells in vitro, as well as with gemacitabine in ovarian and pancreatic cancer xenografts. On the basis of these studies, the first clinical phase 1 trials were initiated with MK-8776 in combination with gemcitabine for pateints with advanced solid tumors. The trial showed preliminary activity and little toxicity.
The success of future cell cycle regulatory protein targeted therapies will largely depend on indentification of specific vulnerablities of cancer cells. (Otto & Sicinski, 2017, p. 111) A combination of several protein inhibitors may provide a good response in targeting cancer cells, but they may not be tolerated by the pateints. Further advancements in research for cell cycle regulatory proteins and their inhibitors as well as inducers offer hope for developing a novel class of medicines that can particularly target aberrant proliferation.