Inhibitors of Cell Cycle Progression and cell growth and division

1. By – Shina Khan
MSc. Biotechnology

2. Itraconazole
Inhibits cell cycle at G1 phase; also SMO antagonist
Itraconazole inhibits the Hedgehog signaling pathway
thereby inducing autophagy-mediated apoptosis of colon
cancer cells.
Itraconazole is an antifungal drug of the triazole class, with a high
bioavailability, broad spectrum and few side effects. It is widely used
for the prevention and treatment of systemic fungal infections5,6.
Recent studies have shown that itraconazole can induce autophagy
thereby inhibiting glioblastoma growth via downregulation of steroid
carrier protein 2 expression and redistribution of intracellular
cholesterol.
Mechanism: Itraconazole acts by inhibiting the fungal cytochrome P-450 dependent enzyme
lanosterol 14-α-demethylase. When this enzyme is inhibited it blocks the conversion of
lanosterol to ergosterol, which disrupts fungal cell membrane synthesis. Itraconazole exhibits
fungistatic (slows the growth) activity against yeast-like fungi and fungicidal (kills the fungus)
activity against Aspergillus spp.
In adult tissue, abnormal activation of the Hedgehog signaling
pathway is associated with the development of several cancer types
including breast, gastric, pancreatic and ovarian cancers as well as
hepatocellular carcinoma15–18. Binding of Sonic Hedgehog (shh) to the
Hedgehog receptor protein patched homolog 1 (PTCH1) initiates
activation of the Hedgehog pathway via reduction of smoothened
(SMO) repression, which in turn leads to the zinc-finger transcription
factor Gli family enabled transcription of downstream target genes19. It

3. is known that GANT61, a small molecule inhibitor of Gli1 and Gli2,
induces autophagy of human hepatocellular carcinomas20. Another
Hedgehog pathway inhibitor vismodegib, which is an antagonist of
SMO, induces autophagy of chronic myeloid leukemia cells, thereby
promoting apoptosis and decreased drug resistance21. Studies have
shown that itraconazole is not only effective for the treatment of
fungal infections, but also can inhibit cancer cell growth by
inactivating the Hedgehog pathway22. In the present study, the role of
itraconazole on Hedgehog pathway related autophagy has been
evaluated.
Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role
remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits
superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured
CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent
CDK7 inhibitor. YKL-5- 124 causes arrest at the G1/S transition and inhibition of E2F-driven gene
expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124,
demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to
RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted
by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential
redundancies in CDK control of gene transcription. These findings highlight the importance of
CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of
CDK7 may be useful for treatment of cancers marked by E2F misregulation.
YKL-5-124 Covalently Targets CDK7 Our initial efforts to develop a selective CDK7 inhibitor using
THZ1 (Figure 1A) as a lead compound were not productive. We turned our attention to PF-3758309,
a PAK4 inhibitor (Murray et al., 2010), since molecules from the series of compounds that gave rise
to PF-3758309 are reported to have strong CDK7 inhibitory activity (Rudolph et al., 2015). We
envisioned that the hybridization of the covalent warhead from THZ1 and the pyrrolidinopyrazole
core from PF-3758309 could lead to more selective CDK7 inhibitors. The first compound in the new
series, YKL-1-116 (Figure 1A) (Kalan et al., 2017), showed good selectivity for CDK7 but only
moderate potency, and had minimal anti-proliferative effects on cancer cell lines (Kalan et al., 2017).
Further iterative optimization has focused on tuning the acidity of the aminopyrazole core, gaining
additional hydrophobic interactions with the side-chain residue, and optimizing the length and
trajectory of the covalent warhead that targets C312. This focused medicinal chemistry campaign
yielded YKL-5-124, a potent and selective covalent CDK7 inhibitor, and YKL-5-167, an inactive analog
lacking the acrylamide reactive center and therefore incapable of forming a covalent bond with
CDK7- C312 (Figure 1A). Biochemical evaluation of YKL-5-124 using a fixed time-point, in vitro kinase
assay indicated that YKL-5-124 inhibited CDK7/ Mat1/CycH with an IC50 of 9.7 nM, while the other
CDKs tested biochemically, CDK2 and CDK9, had IC50 values of 1,300 nM and 3,020 nM, respectively.
To more precisely assay the contribution of covalent bond formation to compound inhibitory
activity, we measured kinact/Ki using a kinetic assay that monitors the shift in the electrophoretic
YKL 5-124
Potent and selective CDK7 inhibitor; induces cell cycle arrest at the G1/S transition

4. mobility of a CDK7 peptide substrate following incubation with YKL-5-124 (Blackwell et al., 2009; Tan
et al., 2017). YKL-5-124 and THZ1 displayed similar ki values (1.9 nM and 2.1 nM, respectively)
showing that they achieved nearly equivalent inhibition of CDK7 (Figures 1B, S1A, and S1B).
However, YKL-5-124 exhibited a faster kinact of 103 ms1 nM1 as compared with kinact of 9 ms1 nM1
for of THZ1, demonstrating that YKL-5-124 covalently modifies CDK7 approximately 11-fold faster
than THZ1 (Figures 1B, S1A, and S1B).
10058-F4
Arrests cell cycle at G0/G1
A small-molecule c-Myc inhibitor, 10058-F4, induces cell-cycle arrest, apoptosis, and
myeloid differentiation of human acute myeloid leukemia. Exp Hematol.
The protooncogene c-Myc plays an important role in the control of cell proliferation,
apoptosis, and differentiation, and its aberrant expression is frequently seen in multiple
human cancers, including acute myeloid leukemia (AML). As c-Myc heterodimerizes with Max
to transactivate downstream target genes in leukemogenesis. Inhibition of the c-Myc/Max
heterodimerization by the recently identified small-molecule compound, 10058-F4, might be
a novel antileukemic strategy.
10058-F4, a c-Myc inhibitor, markedly increases valproic acid-induced cell death in Jurkat
and CCRF-CEM T-lymphoblastic leukemia cells
ABT 263
Induces G1/G0 phase arrest; Bcl-2 family inhibitor
ABT-263, a new BH3 mimetic, is a potent Bcl-2 family inhibitor that antagonizes Bcl-2
family members (Bcl-2, Bcl-xL and Bcl-w) [9]. It was found safe and effective against
some leukemia, lymphoma, small cell lung cancer, and other malignancies
ABT-263 induces G1/G0-phase arrest, apoptosis and
autophagy in human esophageal cancer cells in vitro
ABT-263 (5–20 μmol/L) dose-dependently induced G1/G0-phase arrest
in the 3 cancer cell lines and induced apoptosis evidenced by
increased the Annexin V-positive cell population and elevated levels of
cleaved caspase 3, cleaved caspase 9 and PARP. We further
demonstrated that ABT-263 treatment markedly increased the
expression of p21Waf1/Cip1 and decreased the expression of cyclin D1 and

5. phospho-Rb (retinoblastoma tumor suppressor protein) (Ser780)
proteins that contributed to the G1/G0-phase arrest. Knockdown of
p21Waf1/Cip1 attenuated ABT-263-induced G1/G0-phase arrest. Moreover,
ABT-263 treatment enhanced pro-survival autophagy, shown as the
increased LC3-II levels and decreased p62 levels, which counteracted
its anticancer activity. Our results suggest that ABT-263 exerts
cytostatic and cytotoxic effects on human esophageal cancer cells in
vitro and enhances pro-survival autophagy, which counteracts its
anticancer activity.
Artesunate
Arrests cell cycle in G2/M; antimalarial
Artesunate caused excessive mitochondrial ROS to induce cell senescence and inhibit
cell proliferation. (A) Artesunate arrested cell cycle at G0/G1 phase in SW480 and
HCT116.
Although artesunate has been reported to be a promising candidate
for colorectal cancer (CRC) treatment, the underlying mechanisms and
molecular targets of artesunate are yet to be explored. Here, we report
that artesunate acts as a senescence and autophagy inducer to exert
its inhibitory effect on CRC in a reactive oxygen species (ROS)-
dependent manner. In SW480 and HCT116 cells, artesunate treatment
led to mitochondrial dysfunction, drastically promoted mitochondrial
ROS generation, and consequently inhibited cell proliferation by
causing cell cycle arrest at G0/G1 phase as well as subsequent p16-
and p21-mediated cell senescence. Senescent cells underwent
endoplasmic reticulum stress (ERS), and the unfolded protein
response (UPR) was activated via IRE1α signaling, with upregulated
BIP, IRE1α, phosphorylated IRE1α (p-IRE1α), CHOP, and DR5. Further
experiments revealed that autophagy was induced by artesunate
treatment due to oxidative stress and ER stress. In contrast, N-
Acetylcysteine (NAC, an ROS scavenger) and 3-Methyladenine (3-MA,
an autophagy inhibitor) restored cell viability and attenuated
autophagy in artesunate-treated cells. Furthermore, cellular free
Ca2+ levels were increased and could be repressed by NAC, 3-MA, and
GSK2350168 (an IRE1α inhibitor). In vivo, artesunate administration
reduced the growth of CT26 cell-derived tumors in BALB/c mice. Ki67
and cyclin D1 expression was downregulated in tumor tissue, while
p16, p21, p-IRE1α, and LC3B expression was upregulated. Taken

6. together, artesunate induces senescence and autophagy to inhibit cell
proliferation in colorectal cancer by promoting excessive ROS
generation.
artesunate (ART) inhibited the growth of MCF-7 and MDA-MB-231 breast cancer cells. ART
arrested the cell cycle in the G2/M phase, which was accompanied by an upregulation of
p21. ART upregulated the expression of Beclin1, an initiator of autophagy (type II
programmed cell death). In addition, ART stimulated the aggregation of LC3, which is
considered to be a marker of autophagosome formation. We further verified the
transformation of LC3 from type I into type II. 3-MA, a classical autophagy inhibitor,
attenuated ART-induced autophagosome formation, cell growth repression, G2/M arrest,
and p21 upregulation. Autophagy induction and p21 upregulation were also repressed by
knockdown of Beclin1. Furthermore, ART sensitized breast cancer cells to the
chemotherapeutic agent epirubicin through an autophagy-dependent cascade. Our study
showed that ART induced autophagy in breast cancer cells and indicated that the anticancer
effects of ART were exerted through an autophagy pathway. Moreover, ART sensitized breast
cancer cells to epirubicin chemotherapy