Small molecule inhibitors of cyclin-dependent kinase 9 for cancer therapy

Abstract Cyclin-dependent kinase 9 (CDK9) plays a vital role in transcription through regulation of short-lived anti-apoptotic genes required for cancer cell survival. Therefore, targeting CDK9 with small molecule inhibitors has emerged as a potential cancer therapy. This article reviews the most recent CDK9 patent literature (2012–2020) related to small molecule inhibitors in cancer along with their selectivity profile and biological results in preclinical studies.


Introduction
Since the discovery of imatinib in 2001, the first kinase inhibitor to obtain Food and Drug Administration (FDA) approval for treatment of chronic myeloid leukaemia, there has been a great interest in kinases as therapeutic target in cancer, especially for those malignant conditions that currently have limited treatment options.
Cyclin-dependent kinases (CDKs) form heterodimers with a specific family of proteins called cyclins. These functional CDK-cyclin complexes regulate cell cycle progression and gene transcription 1 . CDK9 is a member of the CDK family; it dimerises with cyclin T to form the positive transcription elongation factor b (p-TEFb) complex 2,3 . This complex stimulates transcription elongation through phosphorylation of the C-terminus domain (CTD) subunit of RNA polymerase II at Ser2 4 . CDK9 plays a vital role in controlling the transcription of a number of genes, including Myc, a proto-oncogene that regulates processes required for cell growth and cell cycle progression, and Mcl-1, an anti-apoptotic member of the Bcl-2 family that enhances cell survival 5 . Therefore, CDK9 inhibition reduces messenger RNA (mRNA) transcription and prevents the expression of target genes (e.g. Myc and Mcl-1), which together regulate proliferation and cancer cells survival.
There is a sufficient evidence to support CDK9 as a valid therapeutic target in cancer through promotion of cell proliferation and regulation of anti-apoptotic proteins such as Mcl-1 and Myc that initiate cancer cell immortality. A dysregulated CDK9-related pathway has been established as a major component for initiation and/or progression of a number of malignancies, including lymphomas, prostate cancer, breast cancer and others [5][6][7][8][9][10][11] . Multiple studies have reported that dysregulated CDK9 signalling is associated with pathogenesis of a number of haematological malignancies 9,11,12 . Elevated Mcl-1 expression has been linked to the development of acute myeloid leukaemia in human cells 13 . High p-TEFb activity has been found in a number of pathological diseases, such as lymphomas and Hodgkin's disease 14 . CDK9 plays a vital role in prostate cancer. Initially, androgens stimulate growth and survival of prostate cells, and most castration-sensitive prostate cancer responds to androgen deprivation. However, 20% of prostate cancer patients develop castrate-resistant prostate cancer (CRPC), which is unresponsive to conventional therapy and associated with a poor prognosis. Recently, CDK9 has been identified as a key component in CRPC through modulating the activity of the androgen receptor 6,7,9,15 . Although there is a diverse genetic background in breast cancer, alteration in CDK9 expression is one molecular pathway in the development of the disease through its interaction with proto-oncogenes 16 . A study found that miR-874 plays an important role in breast cancer by inhibiting proliferation and inducing apoptosis and cell cycle arrest. In this study, CDK9 was a direct target of miR-874, which negatively regulates its proteins levels 17 . A study found that the proto-oncogene Myb expression in oestrogen receptor-positive breast cancer is downregulated by CDK9. In this study, the role of CDK9 was reinforced by using CDK9 inhibitors, e.g. SNS-032, CDKi and CAN-508. These data demonstrated an increase in tumour cell apoptosis and preventing cell growth 18 .

CDK9 clinical applications
Since CDK9 was identified as a promising therapeutic opportunity in cancer, its inhibition has become a main strategy for large pharmaceutical companies, and a number of chemical motifs have been developed. These inhibitors commonly function as ATP competitive inhibitors and have low molecular weight and drug-like properties. Several of these molecules have progressed into clinical trials as anti-proliferative agents for the treatment of various types of cancer 12,[19][20][21][22][23][24][25][26][27] . The first generation of inhibitors evaluated in clinical trials were pan-CDK inhibitors, which inhibit CDK9 as well as other CDK isoforms and other kinases ( Figure 1 and Table  1). Flavopiridol 1 was the first CDK inhibitor to enter clinical trials. Although it inhibits other CDK isoforms, including CDK4, CDK5 and CDK7, its primary anti-tumour mechanism is now understood to be through transcriptional regulation via CDK9/P-TEFb 37 . Its evaluation in phase II clinical trials for treatment of leukaemia showed up to 58% complete response, but with a high incidence of adverse effects, with up to 87% high risk 19,24,38,39 . Dinaciclib 2 is a potent pyrazolopyridine inhibitor of CDK9, CDK2, CDK5, CDK7 and CDK1 with low nanomolar IC 50 values. In in vitro studies, it inhibits Rb phosphorylation and blocks incorporation of thymidine DNA 40 . In three phase II clinical trials for the treatment of breast cancer, lung cancer and acute leukaemia, there was no complete response to treatment and there was a high incidence rate of adverse effects in up to 95% of the patients [41][42][43] . SNS-032 3 is a thiazole carboxiamide derivative with a potent CDK9 inhibitory effect (IC 50 ¼4 nM) and some activity against CDK2 and CDK7. In acute myeloid leukaemia cell lines, 3 inhibits RNA polymerase II phosphorylation, supresses Mcl-1 and XIAP and induces apoptosis 44 . It has been evaluated in phase I studies in subjects with leukaemia, but its clinical results were limited due to grade 3 and 4 toxicities, mainly myelosuppression 32,45 . RGB286638 4 is a pan-CDK inhibitors with a low nanomolar IC 50 against several CDKs. Its CDK: cyclin-dependent kinase; IC 50 : half maximal inhibitory concentration. Ã no specific IC 50 value was disclosed, but it is claimed to be 50 fold greater than the CDK9 IC 50 . ᶧno specific IC 50 value was disclosed but it claimed to be 10 fold greater than CDK9 IC 50 . evaluation in human trials demonstrated the lack of a complete response to the treatment, and 23% of the patients exhibited adverse effects 22 . Zotiraciclib (TG02 5) is a macrocyclic compound bearing phenyl-2-aminopyrimidine. It a multi-target CDK inhibitor including CDK9. In a phase I study, when combined with temozolomide for treatment of recurrent malignant gliomas, it showed a tolerable toxicity profile; investigations have progressed into a phase II trial (NCT02942264) 46 . Notably, the majority of the aforementioned inhibitors (1)(2)(3)(4)(5) showed limited success with regard to treatment with high rates of adverse effects. These outcomes may be due to the lack of selectivity for CDK9. The second generation of inhibitors evaluated in clinical trials are selective CDK9 inhibitors. In 2017, researchers at Bayer identified the aminotriazine derivative atuveciclib (BAY-1143572, 6) as the first highly selective CDK9 inhibitor. In a biochemical assay, it is highly potent against CDK9 (IC 50 ¼6 nM) and selective over other CDK isoforms (>150 fold) 26 . In an adult T-cell leukaemia/lymphoma model, it inhibits RNA polymerase II phosphorylation and induces apoptosis through Myc and Mcl-1 depletion 47 . It is currently being evaluated in phase I clinical trials for treatment of advance acute leukaemia (NCT02345382) 48 . The same research group has also identified a second selective and potent CDK9 inhibitor, BAY-1251152 7, an aminopyridine derivative structurally related to 6. This compound presents similar biological results (CDK9 IC 50 ¼4 nM and selectivity >50 fold over other CDKs). It is currently being evaluated in a phase I clinical trial for treatment of acute leukaemia (NCT02635672) 36,49 . A novel and potent aminopyridine derivative, AZD4573 8, has been identified by researchers at AstraZeneca. It shows an CDK9 IC 50 value of 3 nM and selectivity over other CDKs and kinases claimed to be >10 fold. In a haematological tumour model, it induces apoptosis through suppression of Mcl-1 expression 50 . It is currently being evaluated in a phase I clinical trial in patients with refractory haematological malignancies (NCT03263637) 51 .

The new CDK9 patent literature
Patents were collected from world intellectual property organisation (WIPO), Espacenet and Scifinder databases using "CDK9" and "cancer" as keywords and combining the results. Patents that did not cover small molecule inhibitors or not related to cancer as well as duplicated documents were manually excluded. The selected patent applications were classified according to structural similarity of their subject compounds in to seven structure classes. Patent documents in languages other than English were translated to understand the inventions' claims.

2-Aminopyridines/pyrimidines
In 2018, GenFleet Therapeutics filed a patent on 5-chloro-2-aminopyridines as potent and selective CDK9 inhibitors (patent in Chinese) for treatment of haematological malignancies ( Table 2). Compounds 9 and 10 exhibit CDK9 inhibition IC 50 values of 0.93 and 1.27 nM, respectively. In addition, they show selectivity at least 1000 fold over other CDK isoforms and high selectivity over a panel of 468 kinases 52 . In cell-based assays, 9 and 10 show good selectivity for cancer cells: they exhibit strong cytotoxicity against leukaemia and lymphoma cell lines (average IC 50 values of 31 nM) with no effect on normal cell lines (CHL and CHO). In acute myeloid leukaemia (AML) cell lines (HL-60, OCI-AML-3 and MV4-11) and the acute promyeloid leukaemia cell line NB-4, compound 9 presents concentration-dependent inhibition of CDK9 phosphorylation through downregulation of RNA polymerase II, Mcl-1 and c-Myc. In addition, it induces caspase-3 protein cleavage by poly (ADP-ribose) polymerase (PARP) and blocks the cells in G0-G1 phase in the above-mentioned four cell lines. In the MV4-11 mouse model, 9 reduces the tumour weight by 98.7% with a high dose (20-30 mg/kg), but this high dose shows a negative effect on Balb/c mouse body weight. However, with the low dose of 10 mg/kg, the tumour weight is significantly reduced, the body weight of the mice remains stable during the treatment and the compound is well tolerated 53 .
In 2013, researchers at Changzhou Le Sun Pharmaceuticals disclosed 2-aminopyrimidines (11-13) for treatment of proliferative disorders (Table 3). Compound 11 (CDKI-73) inhibits CDK9 as well as other CDK isoforms, including CDK1, CDK2 and CDK5. It also shows a low nanomolar inhibitory effect towards Aroura A, Aroura B and GSK3b. In MTT proliferative assay, it shows a cytotoxicity IC 50 of 30 nM in MCF-7 and HCT-116 cell lines. In primary CLL cells, CDKI-73 shows a LD 50 of 80 nM using an apoptosis assay with no effect on normal B cells and T cells. In pharmacokinetic measurements in mice, it exhibits 56% oral bioavailability following a 10 mg/kg oral dose 54 . In combination with fludarabine, it shows strong synergistic effect on 48 h cytotoxicity assay using CLL cells. Also, the combination markedly represses Bcl-2, Mcl-1, XIAP, CCND1 and CCNDD2 gene expression. Furthermore, the synergistic combination retains the pro-survival, CDK40L-expression co-culture condition, which is known condition to induce resistance to fludarabine 55 . In 2019, Si et al. at Ancureall Pharmaceuticals filed a patent on 2-aminopyrimidines (patent in Chinese). A total of 172 compounds were prepared and assayed against CDK9 (the most interesting examples 14-18 are shown in Figure 2). No specific IC 50 values were disclosed but it claimed to be <10 nM against CDK9 in biochemical assay and <1 nM against cell growth in cell-based assay using 44 CDK9-expressing tumour cell lines, including human acute leukaemia MOM13 cells. In in vivo animal studies, compound 14 effectively inhibits tumour growth by 91.3% at dose of 50 mg/kg with no effect on the body weight of the mice 56 .
Bayer filed 10 patent applications during 2013-2018 covering 2-aminopyridines/pyrimidines as selective CDK9 inhibitors for cancer therapy. In three publications, Lucking et al. presented disubstituted 5-fluoropyrimidine compounds bearing a sulfondiimine and sulphone group (representative examples are shown in Figure  3 and Table 4). These compounds show CDK9 IC 50 values between 4 and 51 nM and CDK2 IC 50 between 190 and 2300 nM. In cytotoxicity assay, these examples exhibit IC 50 between 35 and 1270 nM in eight cell lines. In pharmacokinetic assays, these derivatives exhibit thermodynamic aqueous solubility between 1.2 and 259 mg/L at pH 6.5. In a Caco-2 permeability test, these examples show an efflux ratio between 1.1 and 6.9 60,62,63 . In follow-up publications, the same group claimed macrocyclic 2-aminopyridine/ pyrimidines with improved potency and selectivity towards CDK9. A total of 32 derivatives were prepared and assayed against CDK9 and CDK2. The most potent and selective examples are shown in Table 4. These examples have reported single digit nanomolar CDK9 IC 50 values in in vitro assay and selectivity exceeds 1000 fold over CDK2. These examples exhibit low nanomolar IC 50 values in proliferation assay using eight cancer cell lines 57,58 . In separate publications, the same group disclosed five 5-flouoro-N-(pyridyl-2yl) pyridine-2-amine derivatives (Table 4) as potent CDK9 inhibitors and selectivity no greater than 29 fold over CDK2. In cytotoxicity assay in seven cell lines, these examples show low nanomolar IC 50 values 59,61,64-66 . An agent from these patents has progressed into clinical trials (BAY-1251152, 7) for treatment of leukaemia, as described in the "CDK9 clinical applications" section (see Table 1).
Wang et al. at Aucentra Therapeutics filed a patent on 2-aminopyrimidines bearing an imidazopyridine group. A total of 181 compounds were prepared and assessed against CDK9 and other CDK isoforms. Compounds 33 and 34 are representative examples from this series; they inhibit CDK9 with K i values of 6 and 8 nM, respectively, and selectivity greater than 40 fold over other CDKs (Table 5). In a cell viability assay, they exhibit anti-proliferative activity with GI 50 in the sub-micromolar range in 12 cancer cell lines including prostate cancer, breast cancer, ovarian cancer and leukaemia cell lines 67 .

Other heteroaryl compounds
In 2014, researchers from ViroStatics have filed a patent on 4-aminopyrimidines as CDK9 inhibitors and assessed their antiviral and anti-proliferative effects (patent in Japanese). The patent includes 148 compounds with the general formula 39 ( Figure 5), most of which show CDK9 IC 50 value <10 mM and supress the viability and proliferation of tumour cells with IC 50 of 0.4 mM 72 .
AbbVie has been the main contributor to CDK9 patent literature in the last decade; they have mainly focussed on anti-proliferative activity. A wide array of scaffolds have been studied, including pyrrolopyridines/pyrimidines, tetracyclic systems and pyridines. In 2014, Lai 73 .
In 2017, researchers at AstraZeneca filed a patent on pyridine and pyrimidine amide derivatives as inhibitors of CDK9, with potential uses in hyper-proliferative diseases. Around 83 compounds were prepared and assayed in a CDK9 assay with two ATP concentrations (at Km and a high concentration of 5 mM). Several of the compounds show a single digit nanomolar CDK9 IC 50 at   Figure 6 and Table 7. An agent from this series is   Figure 3. ᶧAverage of eight cell lines.
currently being evaluated clinical trials (AZD4573, 8) for treatment of haematological malignancies, as described in the "CDK9 clinical applications" section (see Table 1). The Lead Discovery Centre claimed the 2-aminotriazine derivative 47 as a potent and selective CDK9 inhibitor for treatment of cancer ( Figure 7). Compound 47 exhibits CDK9 inhibitory activity with an IC 50 of 1 mM and selectivity greater than 30 fold against other CDKs, including CDK2, CDK1, CDK4, CDK6 and CDK7, and greater than 50 fold across a kinase panel. In cell-based assay, it shows anti-proliferative activity with IC 50 in sub-micromolar range against various cell lines 75 .
Bayer filed a patent on 2-aminotriazines as CDK9 inhibitors. A total of 91 compounds were prepared and assessed against CDK9 and CDK2 (representative examples are shown in Table 8). These compounds show CDK9 IC 50 values between 2 and 30 nM and selectivity greater that 100 fold over CDK2. In cytotoxicity assays, these examples exhibit IC 50 in sub-micromolar range in six cell lines. In pharmacokinetic assays, these derivatives exhibit good thermodynamic aqueous solubility up to 1200 mg/L at pH 6.5 76 . An agent from this patent has progressed into clinical trials (atuveciclib, BAY-1143572, 8) for treatment of leukaemia, as described in the "CDK9 clinical applications" section (see Table 1).

Pyrrol[2,3-b]pyridines
AbbVie published seven patent applications from 2014 to 2016 covering more than 13,600 derivatives of the pyrrolo[2,3-b]pyridine scaffold as CDK9 inhibitors targeting cancer. Examples of the most interesting compounds are illustrated in (general structures 51-53, Figure 9 and Table 9). They inhibit CDK9 with IC 50 values     Figure 10). No specific biological values were disclosed, but the most potent inhibitors claimed to have >70% inhibition at 500 nM in in vitro CDK9 assay and >70% inhibition at 10 mM in a proliferation assay against five cell lines, including the prostate cancer cell lines PC-3 81 .  Table 10) show two-digit nanomolar IC 50 values against CDK9 and an average EC 50 of 0.11 mM in a cytotoxicity assay against A431 and H929 cell lines 83 .

Other bicyclic compounds
In 2013, the SNU R&D Foundation disclosed pyrrolo [2,3-d]pyrimidine-5-carboxamide derivatives for the prevention or treatment of liver cancer. The most interesting derivatives, compounds 64 and 65 (shown in Figure 12), inhibit CDK9 with 1 and 40% residual activity, respectively, at 100 nM. They also show some inhibitory activity against other CDK isoforms, including CDK1, CDK2 and CDK7. In western blotting experiments, both compounds effectively inhibit the phosphorylation of RNA polymerase II Ser2 with 1.6% residual activity at 10 nM. Compound 65 successfully downregulates Mcl-1, survivin and XIAP in SNU-354 cells (hepatocellular carcinoma cell line). Moreover, this reduction is attributed to mRNA, confirmed by real-time polymerase chain reaction (RT-PCR). Cell viability was measured by MTT assay in SNU-354 cells. The results showed that the cell viability with 64 and 65 is 36.6 and 16.8% residual activity at 50 mM, respectively. This inhibitory effect on cell growth is greater than that of the standard CDK inhibitors olomoucine and roscovitine. Compounds 64 and 65 induce apoptosis of SNU-354 cells, confirmed by a PARP cleavage experiment. In particular, 65 induces PARP cleavage after 6 h. Furthermore, both compounds increase caspase activity in a dose-dependent manner. In animal studies using a SNU-354 cancer xenograft model, 65 reduces the tumour growth by 15% at 4 mg/kg/day and 47% at 20 mg/kg/day 84 .  Figure 6.     Cell-viability IC 50 in A431 and H929 are 0.025 and 0.077 mM, respectively. In cell western blots, the IC 50 to reduce phosphorylation of RNA polymerase II is 0.17 mM.
In a H929 xenograft model in mice, the compound shows 60% total growth inhibition with a 5 mg/kg dose. 79
In a H929 xenograft model in mice, the compound shows 66% total growth inhibition with a 3.75 mg/kg dose. 80 55 Cell-viability EC 50 in H929 is 0.8 mM.
In a H929 xenograft model in mice, the compound shows 39% total growth inhibition with 7.5 mg/kg dose.   In 2015, Bondke et al. at Cancer Research Technology filed a patent on pyrazolo[1,5-a]pyrimidine-5,7-diamine scaffold as CDK inhibitors for treatment of proliferative disorders. Compound 66 inhibits CDK9 with an IC 50 of 1.1 mM, but it also strongly inhibits CDK7 other CDK isoforms, including CDK2 and CDK1 (Table 11). In a cell growth inhibition assay, compound 66 inhibits the growth of breast cancer cell line (MCF7) and colorectal cancer cell line (HTC116) with an IC 50 <1 mM. In an HCT116 tumour xenograft model, it reduces the tumour growth by 65% with 100 mg/kg daily dose 85 . In a separate publication in collaboration with Carrick Therapeutics, the same group disclosed 67 mainly as a CDK7 inhibitor, but it also strongly inhibits CDK9 (Table 11). No further biological data were provided in the above mentioned publication 86 .
Temple University filed a patent application on CDK9 inhibitors focussed on a benzothiazin-3-one core. The authors claimed compound 68 ( Figure 13) as a CDK9 inhibitor for treatment of cancer, with a CDK9 IC 50 value of 93.7 nM. In an anti-proliferative assay, it shows selective toxicity to cancer cell lines over normal human stem cells, with an average IC 50 of 0.26 mM in eight cancer cell lines, including prostate cancer, pancreatic cancer, breast cancer and leukaemia 87 .

Chromones
In 2019, researchers at China Pharmaceutical University filed a patent on a series of flavonoids as antitumour agents (patent in Chinese). The most potent derivatives, 69 and 70, are claimed to inhibit the growth of HepG2, A549, HCT116 and THP-1 cancer cell lines, with IC 50 values from 0.4 to 4.4 mM. The researchers claimed that both compounds are monospecific CDK9 inhibitors with low nanomolar potency and selectivity greater than 50 fold over other CDK isoforms (Table 12). In addition, 69 and 70 show reasonable in vitro physiochemical properties, including aqueous solubility and lipophilicity. In MV4-11 cancer cells, 69 induces apoptosis in a concentration-dependent manner; it exhibits 40% apoptosis at 1 mM. In a western blot assay in MV4-11 cell lines, 69 inhibits CDK9 activity by reducing the expression of RNA polymerase II and induces apoptosis in an Mcl-1 dependent manner. It also shows sustained induction of cleaved caspase-3 in MV4-11 cells 88 .
The Council of Scientific and Industrial Research filed a patent on chromones related to rohitukine [5,7-     inhibits 37% tumour growth with a 70 mg/kg/day intraperitoneal dose without mortality. Compounds 71 and 72 show good aqueous solubility (>1500 mg/mL) 89 (Table 13).  Table 14. They exhibit very low nanomolar IC 50 against CDK9. They also inhibit other isoforms, including CDK2, CDK7, CDK12 and CDK13 which could contribute to their anti-proliferative activity 90 (Figure 14). Smith et al. at Apogee Biotechnology filed a patent for diaminothiazole derivatives as anti-proliferative and anti-inflammatory agents. Compound 80 shows a CDK9 IC 50 value of 0.32 mM. It also has some activity against GSK3b, SK1 and SK2. It shows sub-micromolar IC 50  In in vivo animal studies using a murine pancreatic cancer Pan02 xenograft model, compound 80 reduces the tumour growth by 70% at dose of 5 mg/kg/day 91 (Figure 15).

Macrocyclic compounds
In 2015, Frey et al. at AbbVie explored several tetracyclic systems as CDK9 inhibitors for cancer treatment. A total of 193 compounds were designed and prepared with general scaffold 81 presented in Figure 16. Many of these compounds inhibit CDK9 with an IC 50 between 0.021 and 1 mM and cytotoxicity IC 50 between 0.006 and 5 mM in H929 cells (Table 15) 92 .

Summary and perspective
CDK9 plays a central role in transcription through phosphorylation of RNA polymerase II. At the present time, there is strong evidence that implicates CDK9 as a cancer target. Its anticancer mechanism is believed to be through promotion of short-lived anti-apoptotic genes such as Mcl-1 and Myc. Targeting CDK9 has received great academic and industrial interest. AbbVie, Bayer and Novartis have been the main contributors to the CDK9 patent literature. 2-Aminopyridines/pyrimidines and pyrrolo [2,3-b]pyridines/pyrimidines are the main chemical motifs for CDK9 inhibitors. (Supplementary Table 1 is a list of the CDK9 patents published from 2012 to date classified by chemical type and applicant). Numerous potent and selective CDK9 inhibitors have been disclosed with single-digit nanomolar potency and selectivity greater than 30 fold over other    Figure 14.
CDK isoforms (e.g. 2-aminopyridines 9 and 10, 2-aminopyrimidines 33 and 40, 2-aminotriazine 47 and flavonoids 69 and 70). Clinical applications of early pan-CDK9 inhibitors (1-5) have yielded unambiguous positive results that have mainly been attributed to the lack the selectivity. Therefore, pharmaceutical companies have focussed on developing mono-specific CDK9 inhibitors over the last decade. Despite the challenge in targeting a single CDK isoform due to the high structural similarity of the active sites among homologous kinase isoforms, it has been possible to selectively inhibit CDK9 using a number of chemical scaffolds. Indeed, three selective CDK9 clinical agents (6)(7)(8) are currently being evaluated in human trials, but no results have been disclosed yet.
On the other hand, targeting multiple survival pathways by pan or dual inhibitors can be interesting clinically. A number of in vitro studies support the above statement, such as the reported synergism between CDK9 and BRD4 inhibition to reduce Myc expression and dual inhibition of CDK9-mediated Mcl-1 and PI3Kmediated Bcl-xL 96,97 . Moreover, there have been positive clinical outcomes with some pan-kinase inhibitors, e.g. palbociclib, initially developed as a selective CDK4/6 inhibitor. However, a recent study has shown that it is also a potent CDK9 inhibitor and engages with several lipid kinases 98 . Such evidence will argue against the prevailing view of removing all off-target effects. Additional preclinical and clinical studies are required to determine whether selectively targeting CDK9 will lead to better cancer therapy.     Figure 16.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This research project was funded by the Deanship of Scientific Research, Princess Nourah bint Abdulrahman University, through the Fast-tract Research Funding program.