Recent Progress in CDK4/6 Inhibitors and PROTACs

Cell division in eukaryotes is a highly regulated process that is critical to the life of a cell. Dysregulated cell proliferation, often driven by anomalies in cell Cyclin-dependent kinase (CDK) activation, is a key pathological mechanism in cancer. Recently, selective CDK4/6 inhibitors have shown clinical success, particularly in treating advanced-stage estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. This review provides an in-depth analysis of the action mechanism and recent advancements in CDK4/6 inhibitors, categorizing them based on their structural characteristics and origins. Furthermore, it explores proteolysis targeting chimers (PROTACs) targeting CDK4/6. We hope that this review could be of benefit for further research on CDK4/6 inhibitors and PROTACs.


Introduction
The regulation of eukaryotic cell division involves complex mechanisms, with cyclins and cyclin-dependent kinase (CDK) complexes playing a pivotal role [1].Since the seminal discovery of cell cycle regulation by Tim Hunt, Paul Nurse, and Leland H. Hartwellwork that garnered a Nobel Prize-20 CDKs have been identified.CDK1-7 and CDK14-18 are primarily associated with cell cycle regulation, while CDK7-13, CDK19, and CDK20 are involved in transcription [2][3][4][5].Owing to their critical roles in cell cycle progression, cellular transcription, and apoptotic pathways, CDKs have emerged as significant targets in anticancer drug development.
The advent of third-generation CDK inhibitors marked a notable improvement in selectivity, activity, and toxicity.Selective CDK4/6 inhibitors, in particular, have achieved remarkable success in clinical applications, notably in advanced-stage ER-positive breast cancer treatments.Four drugs from this class have been approved by the FDA, with three for cancer and one for myeloprotection [7][8][9][10].Current research is focused on enhancing the selectivity of CDK inhibitors and addressing drug resistance.There are many excellent reviews on pan-inhibitors, and this review concentrates on selective CDK4/6 inhibitors [11][12][13][14][15].
The advent of third-generation CDK inhibitors marked a notable improvement in selectivity, activity, and toxicity.Selective CDK4/6 inhibitors, in particular, have achieved remarkable success in clinical applications, notably in advanced-stage ER-positive breast cancer treatments.Four drugs from this class have been approved by the FDA, with three for cancer and one for myeloprotection [7][8][9][10].Current research is focused on enhancing the selectivity of CDK inhibitors and addressing drug resistance.There are many excellent reviews on pan-inhibitors, and this review concentrates on selective CDK4/6 inhibitors [11][12][13][14][15].

The Biological Rationale for Targeting CDK4/6
The classical and best-documented function of CDK4/6 in cell proliferation is that cyclin D1-CDK4/6 phosphorylates the retinoblastoma protein (RB1) and RB-like proteins (RBL1 and RBL2), impacting the G1-S phase transition (Figure 2).The unphosphorylated RB1 interacts with E2F transcription family members, blocking their activity and repressing transcription essential for S phase entry.Phosphorylated RB1, on the other hand, releases E2F, which promotes transcription of cyclin E that associates with CDK2 to further phosphorylate RB1, resulting in the facilitation of the S phase entry.This process is intricately regulated by external signals and mitogenic signaling, with aberrations in the CDK4/6-RB-E2F axis observed in various cancers [16,17].
However, there are some debates surrounding this model.For instance, RB1 is monophosphorylated during the G1 phase and becomes inactivated in the late G1 phase by cyclin E-CDK2, which hyperphosphorylates RB1 on multiple residues.Further, the phosphorylation of RB1 by cyclin D-CDK4/6 is crucial for normal cell-cycle progression, highlighting the need for more research in this area to elucidate the biological function of the CDK4/6-RB axis [18].

The Biological Rationale for Targeting CDK4/6
The classical and best-documented function of CDK4/6 in cell proliferation is that cyclin D1-CDK4/6 phosphorylates the retinoblastoma protein (RB1) and RB-like proteins (RBL1 and RBL2), impacting the G1-S phase transition (Figure 2).The unphosphorylated RB1 interacts with E2F transcription family members, blocking their activity and repressing transcription essential for S phase entry.Phosphorylated RB1, on the other hand, releases E2F, which promotes transcription of cyclin E that associates with CDK2 to further phosphorylate RB1, resulting in the facilitation of the S phase entry.This process is intricately regulated by external signals and mitogenic signaling, with aberrations in the CDK4/6-RB-E2F axis observed in various cancers [16,17].
However, there are some debates surrounding this model.For instance, RB1 is monophosphorylated during the G1 phase and becomes inactivated in the late G1 phase by cyclin E-CDK2, which hyperphosphorylates RB1 on multiple residues.Further, the phosphorylation of RB1 by cyclin D-CDK4/6 is crucial for normal cell-cycle progression, highlighting the need for more research in this area to elucidate the biological function of the CDK4/6-RB axis [18].
CDK4/6 also influences cell cycle progression through kinase-independent mechanisms.For instance, the Cyclin-dependent kinase inhibitor 1/kinase inhibitory protein (CIP/KIP) protein family, including p21 CIP1 , p27 kIP1 , and p57 kIP2 , binds cyclin E-CDK2 and suppresses its activity (Figure 3).Upregulation of cyclin D and the formation of cyclin D-CDK4/6 complexes, which competitively bind CIP/KIP, leads to redistribution of CIP/KIP, thus activating cyclin E-CDK2 and promoting the G1-S transition.Beyond facilitating the G1-S transition, Cyclin D-CDK4/6 promotes tumor progression through various pathways.For example, it phosphorylates and stabilizes transcription factor FOXM1, which promotes cell-cycle progression and protects cancer cells from entering senescence [19].Cyclin D-CDK4 also phosphorylates SMAD3 and inhibits its transcriptional activity, which disables the anti-proliferative ability of growth factor beta (TGF-β) [20].Cyclin D-CDK4/6 phosphorylates and inactivates tuberous sclerosis complex (TSC2), a negative regulator of rapamycin complex 1 (mTORC1), which subsequently activates mTORC1.CDK6 binds the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and the promoter of proviral integration of molony murine leukemia virus 1 (PIM1) pro-oncogenic kinase, stimulating their expression.Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased expression of FLT3 and PIM1, which induced cell cycle arrest and apoptosis [21].The research on Cyclin D-CDK4/6 promoting tumor progression has also been well reviewed [22].These diverse  Beyond facilitating the G1-S transition, Cyclin D-CDK4/6 promotes tumor progression through various pathways.For example, it phosphorylates and stabilizes transcription factor FOXM1, which promotes cell-cycle progression and protects cancer cells from entering senescence [19].Cyclin D-CDK4 also phosphorylates SMAD3 and inhibits its transcriptional activity, which disables the anti-proliferative ability of growth factor beta (TGF-β) [20].Cyclin D-CDK4/6 phosphorylates and inactivates tuberous sclerosis complex (TSC2), a negative regulator of rapamycin complex 1 (mTORC1), which subsequently activates mTORC1.CDK6 binds the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and the promoter of proviral integration of molony murine leukemia virus 1 (PIM1) pro-oncogenic kinase, stimulating their expression.Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased expression of FLT3 and PIM1, which induced cell cycle arrest and apoptosis [21].The research on Cyclin D-CDK4/6 promoting tumor progression has also been well reviewed [22].These diverse Beyond facilitating the G1-S transition, Cyclin D-CDK4/6 promotes tumor progression through various pathways.For example, it phosphorylates and stabilizes transcription factor FOXM1, which promotes cell-cycle progression and protects cancer cells from entering senescence [19].Cyclin D-CDK4 also phosphorylates SMAD3 and inhibits its transcriptional activity, which disables the anti-proliferative ability of growth factor beta (TGF-β) [20].Cyclin D-CDK4/6 phosphorylates and inactivates tuberous sclerosis complex (TSC2), a negative regulator of rapamycin complex 1 (mTORC1), which subsequently activates mTORC1.CDK6 binds the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and the promoter of proviral integration of molony murine leukemia virus 1 (PIM1) pro-oncogenic kinase, stimulating their expression.Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased expression of FLT3 and PIM1, which induced cell cycle arrest and apoptosis [21].The research on Cyclin D-CDK4/6 promoting tumor progression has also been well reviewed [22].These diverse roles of CDK4/6 in tumor progression underscore their potential as targets in cancer therapy.

The Overview of CDK4/6 Sites
All CDKs possess a dual-leaf structure, with the N-terminal comprising β-sheet elements and the C-terminal formed by α-helices.The N-terminal leaf contains G-ring inhibitory components, while the C-terminal leaf is characterized by activating fragments and phosphorylation sites (serine or threonine, referred to as the T-loop).CDK4 is located on chromosome 12q14.1,consisting of a narrow region approximately 3.2 kbp in length.CDK6, mapped to human chromosome 7q21.2,encodes a cytoplasmic protein comprising 326 amino acids and weighing about 37 kDa.The structural and functional similarities between CDK4 and CDK6, with 71% homology in amino acids, facilitate the design of compounds as CDK4/6 inhibitors [23].
The cocrystallization study of Ribociclib and CDK6 (PDB ID: 5l2t) suggests that the 2-aminopyrimidine moiety of Ribociclib forms hydrogen bonds with VAL101, and the pyrrole ring interacts with PHE98, potentially underpinning its inhibitory effect (Figure 4).Additionally, Ribociclib's piperazine ring, extending outside the protein cavity, seems to play a role in modulating hydrosolubility and selectivity against other CDKs.roles of CDK4/6 in tumor progression underscore their potential as targets in can therapy.

The Overview of CDK4/6 Sites
All CDKs possess a dual-leaf structure, with the N-terminal comprising β-s elements and the C-terminal formed by α-helices.The N-terminal leaf contains Ginhibitory components, while the C-terminal leaf is characterized by activating f ments and phosphorylation sites (serine or threonine, referred to as the T-loop).CDK located on chromosome 12q14.1,consisting of a narrow region approximately 3.2 kb length.CDK6, mapped to human chromosome 7q21.2,encodes a cytoplasmic pro comprising 326 amino acids and weighing about 37 kDa.The structural and functio similarities between CDK4 and CDK6, with 71% homology in amino acids, facilitate design of compounds as CDK4/6 inhibitors [23].
The cocrystallization study of Ribociclib and CDK6 (PDB ID: 5l2t) suggests that 2-aminopyrimidine moiety of Ribociclib forms hydrogen bonds with VAL101, and pyrrole ring interacts with PHE98, potentially underpinning its inhibitory effect (Fig 4).Additionally, Ribociclib's piperazine ring, extending outside the protein cavity, se to play a role in modulating hydrosolubility and selectivity against other CDKs.

Approved CDK4/6 Inhibitors for Marketing
The third generation of CDK inhibitors, characterized by enhanced selectivity duced side effects, and improved pharmacokinetic properties, has shown significant tential in cancer therapy, particularly in breast cancer.Palbociclib (Ibrance/PD-0332 Compound 8, Figure 5), developed by Pfizer, was the first FDA-approved CDK4/6 in itor, marking a milestone in the development of these drugs.It exhibits IC50 va against CDK4/6 of 11 and 9 nM, respectively [7].Palbociclib received accelerated proval in the US in February 2015 for first-line treatment of advanced or metast ER-positive, HER2-negative breast cancer in postmenopausal women.It has shown cacy in reducing the proliferation of ER-positive breast cancer cell lines in vitro blocking Rb phosphorylation, causing G1 phase arrest [24].Studies also indicate combined therapy with palbociclib and antiestrogen agents (e.g., letrozole and ful trant) leads to a more significant reduction in phosphorylated Rb levels, E2F and Fox levels, and downstream target gene expression.In the xenotransplantation mode

Approved CDK4/6 Inhibitors for Marketing
The third generation of CDK inhibitors, characterized by enhanced selectivity, reduced side effects, and improved pharmacokinetic properties, has shown significant potential in cancer therapy, particularly in breast cancer.Palbociclib (Ibrance/PD-0332991, Compound 8, Figure 5), developed by Pfizer, was the first FDA-approved CDK4/6 inhibitor, marking a milestone in the development of these drugs.It exhibits IC 50 values against CDK4/6 of 11 and 9 nM, respectively [7].Palbociclib received accelerated approval in the US in February 2015 for first-line treatment of advanced or metastatic ER-positive, HER2-negative breast cancer in postmenopausal women.It has shown efficacy in reducing the proliferation of ER-positive breast cancer cell lines in vitro by blocking Rb phosphorylation, causing G1 phase arrest [24].Studies also indicate that combined therapy with palbociclib and antiestrogen agents (e.g., letrozole and fulvestrant) leads to a more significant reduction in phosphorylated Rb levels, E2F and FoxM1 levels, and downstream target gene expression.In the xenotransplantation model of ER-positive breast cancer derived from patients, palbociclib plus anti-estrogen letrozole (compared with any drug alone) has a greater inhibitory effect on Rb phosphorylation, downstream signal transduction, and tumor growth [25].
Ribociclib (Compound 9, Figure 5), developed by Novartis, is another oral, smallmolecule inhibitor of CDK4/6, approved in the USA in March 2017 [8].It displays IC 50 values against CDK4/6 of 10 nM and 39 nM, respectively.Patient-derived xenograft models of ER-positive breast cancer have demonstrated the enhanced efficacy of ribociclib in combination with antiestrogen agents (letrozole or fulvestrant) and, in some cases, further improvement when combined with a phosphatidylinositol 3-kinase (PI3K) inhibitor [26].The antitumor effects of ribociclib have also been demonstrated in vitro in leukemia cells.
Abemaciclib (Verzenio/LY2835219, Compound 10, Figure 5), an oral inhibitor of CDK4/6 developed by Eli Lilly, was approved in the USA on 28 September 2017 [9].It is indicated in combination with fulvestrant for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, in combination with fulvestrant in women with disease progression following endocrine therapy, and as a monotherapy in adult patients with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting [27][28][29].It shows IC 50 values against CDK4 of 2 nM.
Trilaciclib (G1T28, Compound 11, Figure 5), developed by G1 Therapeutics (formerly G-Zero Therapeutics), is a transient inhibitor of CDK4/6 with IC 50 values of 1 nM and 4 nM.Trilaciclib induces a transient, reversible G1 cell cycle arrest of proliferating hematopoietic stem and progenitor cells (HSPCs) in bone marrow, protecting them from chemotherapy (myeloprotection) [10].On 12 February 2021, trilaciclib received its first approval in the USA to decrease the incidence of chemotherapy-induced myelosuppression in adult patients when administered prior to a platinum/etoposide-containing regimen or topotecan-containing regimen for extensive-stage small cell lung cancer (ES-SCLC) [30,31].
Dalpiciclib (SHR6390, Compound 12, Figure 5), approved by the National Drug Administration (NMPA) on 31 December 2021, shows IC 50 values against CDK4/6 of 12.4 and 9.9 nM [32].It exhibits effective antiproliferative activity against various human RB-positive tumor cells, specifically inducing G1 phase arrest and cell aging while reducing the level of Ser780-phosphorylated RB protein.
Birociclib (XZP-3287, Compound 13, Figure 5), developed by Xuanzhu Biotechnology Co., Ltd., received the NMPA approval in 2022.It is indicated for locally advanced or metastatic adult breast cancer patients with hormone receptor (HR) positive and human epidermal growth factor receptor 2 (Her2) negative who have received two or more endocrine treatments and one chemotherapy in the metastatic setting and disease progression [33].
Molecules 2023, 28, x FOR PEER REVIEW 5 of 23 ER-positive breast cancer derived from patients, palbociclib plus anti-estrogen letrozole (compared with any drug alone) has a greater inhibitory effect on Rb phosphorylation, downstream signal transduction, and tumor growth [25].Ribociclib (Compound 9, Figure 5), developed by Novartis, is another oral, small-molecule inhibitor of CDK4/6, approved in the USA in March 2017 [8].It displays IC50 values against CDK4/6 of 10 nM and 39 nM, respectively.Patient-derived xenograft models of ER-positive breast cancer have demonstrated the enhanced efficacy of ribociclib in combination with antiestrogen agents (letrozole or fulvestrant) and, in some cases, further improvement when combined with a phosphatidylinositol 3-kinase (PI3K) inhibitor [26].The antitumor effects of ribociclib have also been demonstrated in vitro in leukemia cells.
Abemaciclib (Verzenio/LY2835219, Compound 10, Figure 5), an oral inhibitor of CDK4/6 developed by Eli Lilly, was approved in the USA on 28 September 2017 [9].It is indicated in combination with fulvestrant for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, in combination with fulvestrant in women with disease progression following endocrine therapy, and as a monotherapy in adult patients with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting [27][28][29].It shows IC50 values against CDK4 of 2 nM.
Trilaciclib (G1T28, Compound 11, Figure 5), developed by G1 Therapeutics (formerly G-Zero Therapeutics), is a transient inhibitor of CDK4/6 with IC50 values of 1 nM and 4 nM.Trilaciclib induces a transient, reversible G1 cell cycle arrest of proliferating hematopoietic stem and progenitor cells (HSPCs) in bone marrow, protecting them from chemotherapy (myeloprotection) [10].On 12 February 2021, trilaciclib received its first approval in the USA to decrease the incidence of chemotherapy-induced myelosuppression in adult patients when administered prior to a platinum/etoposide-containing regimen or topotecan-containing regimen for extensive-stage small cell lung cancer (ES-SCLC) [30,31].
Dalpiciclib (SHR6390, Compound 12, Figure 5), approved by the National Drug Administration (NMPA) on 31 December 2021, shows IC50 values against CDK4/6 of 12.4 and 9.9 nM [32].It exhibits effective antiproliferative activity against various human RB-positive tumor cells, specifically inducing G1 phase arrest and cell aging while reducing the level of Ser780-phosphorylated RB protein.
Birociclib (XZP-3287, Compound 13, Figure 5), developed by Xuanzhu Biotechnology Co., Ltd., received the NMPA approval in 2022.It is indicated for locally advanced or metastatic adult breast cancer patients with hormone receptor (HR) positive and human epidermal growth factor receptor 2 (Her2) negative who have received two or more endocrine treatments and one chemotherapy in the metastatic setting and disease progression [33].

Other Structures
Compound 73 (Figure 15) is a highly selective CDK4/6 inhibitor with IC50 values of 9.2 and 7.8 nM, which were found through screening the Merck sample repository and further optimization [83].In 2015, Takao Horiuchi et al. reported a series of CDK inhibitors, including Compound 74 (Figure 15), which showed CDK2/4 inhibition with IC50 values of 880 and 22 nM [84].Compounds 75 and 76 (Figure 15) exhibited inhibition activity against CDK6, with IC50 values of 115.38 nM and 726.25 nM, respectively.Moreover, they increased the levels of bax and p53 and decreased the levels of bcl-2 [85].

Natural Product Inhibiting CDK4/6
In 1997, Jun'ichi et al. reported the isolation and identification of Konbu'acidin A (77, Figure 16), which exhibits inhibition activity against CDK4 with an IC50 of 20 µg/mL [86].It is noteworthy that there are two guanidine moieties in the molecule.In 2020, Abdel Nasser B.

Other Structures
Compound 73 (Figure 15) is a highly selective CDK4/6 inhibitor with IC 50 values of 9.2 and 7.8 nM, which were found through screening the Merck sample repository and further optimization [83].In 2015, Takao Horiuchi et al. reported a series of CDK inhibitors, including Compound 74 (Figure 15), which showed CDK2/4 inhibition with IC 50 values of 880 and 22 nM [84].Compounds 75 and 76 (Figure 15) exhibited inhibition activity against CDK6, with IC 50 values of 115.38 nM and 726.25 nM, respectively.Moreover, they increased the levels of bax and p53 and decreased the levels of bcl-2 [85].

Other Structures
Compound 73 (Figure 15) is a highly selective CDK4/6 inhibitor with IC50 va 9.2 and 7.8 nM, which were found through screening the Merck sample reposito further optimization [83].In 2015, Takao Horiuchi et al. reported a series of CDK tors, including Compound 74 (Figure 15), which showed CDK2/4 inhibition wi values of 880 and 22 nM [84].Compounds 75 and 76 (Figure 15) exhibited inhibit tivity against CDK6, with IC50 values of 115.38 nM and 726.25 nM, respectively.M ver, they increased the levels of bax and p53 and decreased the levels of bcl-2 [85].

Natural Product Inhibiting CDK4/6
In 1997, Jun'ichi et al. reported the isolation and identification of Konbu'acidin Figure 16), which exhibits inhibition activity against CDK4 with an IC50 of 20 µg/mL [8 noteworthy that there are two guanidine moieties in the molecule.In 2020, Abdel Na

PROTAC Targeting CDK4/6 and Cyclins
Recently, PROTAC, which degrades CDK4/6, has been considered a strategy to overcome drug resistance.In addition, degradation of CDK4/6 also eliminates other functions of CDK4/6 besides kinase activity, which benefits cancer therapy.

CDK4/6 Inhibitors in Clinical Research
In recent years, CDK4/6 inhibitors have been developed rapidly, and some have gradually entered clinical trials.In this section, we list some drugs that have already entered clinical research.
G1T38 (Compound 109, Figure 20, Table 1), developed by G1 Therapeutics ((North Carolina, USA)), is a novel potent CDK4/6 inhibitor with good selectivity and oral bioavailability.At present, it is in a Phase-II-stage clinical study.G1T38 reduced RB phosphorylation, blocked cells in the G1 phase, and inhibited cell proliferation in various CDK4/6dependent oncogenic cell lines, including breast, melanoma, leukemia, and lymphoma cells.In addition, G1T38 accumulates in mouse xenograft tumors but in plasma, with less neutropenia.All these good pharmacokinetic and pharmacodynamic properties make G1T38 a continuous, daily oral anti-tumor agent [111].Ebvaciclib (Compound 110, Figure 20, Table 1), developed by Pfizer (New York, NY, USA), is a CDK2/4/6 inhibitor.In November 2018, the in vitro and in vivo data of Ebvaciclib were first released at the 30th AACR Annual Conference held in Dublin, Ireland.Ebvaciclib has a higher binding affinity for CDK2, 4, 5, and 6, which is 40 times higher than that of CDK1 and CDK9.In March 2018, a Phase-I/II-trial treated patients with HR-positive, HER2-negative breast cancer, metastatic triple-negative breast cancer, or advanced cisplatin-resistant epithelial ovarian cancer/fallopian tube cancer [112].
MM-D37K (Table 1) is a synthetic peptide composed of p16 INK4a (a specific inhibitor of cyclin D-CDK4 and CDK6) and cell penetrating peptide (CPP)-Antp (Penetratin).It is a non-ATP-competitive CDK4/6 inhibitor that is in Phase-II-stage clinical research.The merit of MM-D37K over the existing ATP-competitive CDK inhibitors will be explored, benefiting the development of next-generation CDK inhibitors [113].
BPI-16350 (Table 1), developed by Beida Pharmaceutical (Hangzhou, China), is in a Phase-III-stage clinical study.It is applied to treat locally advanced, recurrent, or metastatic breast cancer that has progressed HR+/HER2−, combined with Fluvastatin [114].RGT-419B (Table 1), developed by Shanghai Qilu Ruige Pharmaceutical Research and Development Co., Ltd.(Shanghai, China), is a potent CDK2/4/6 inhibitor that is in Phase I clinical research.The informed use of RGT-419B is for the unmet medical needs of patients with refractory or recurrent disease after previous treatment and patients with advanced/metastatic breast cancer to improve the survival rate and quality of life [115].FCN-437c (Table 1) is developed by Fuchuang Pharmaceuticals (Chongqing, China), a subsidiary of Fosun Pharmaceuticals.On 23 January 2019, a Phase I clinical trial was conducted in the United States.In September 2020, the Phase II clinical study was conducted in China for patients with ER+/HER2− advanced breast cancer (excluding Hong Kong, Macao, and Taiwan) [116].TY-302 (Table 1 1), developed by Amgen (FLX BIO) (California, USA), is in Phase I clinical study.FLX925 selectively acts on FLT3 and CDK4/6, and its current indication is acute myeloid leukemia (AML) [120].P-276-00 (Compound 112, Figure 20, Table 1), developed by Piramal (Mumbai, India), is in phase II clinical studies for advanced refractory neoplasms and multiple myeloma.It also has inhibitory activity against TNF-α.It also has anti-inflammatory activity, and its first indication for application is for the treatment of mucositis caused by severe radiation in patients with head and neck cancer [121].SPH4366 (

Summary and Prospect
Recently, selective CDK4/6 inhibitors have shown clinical success, particularly in treating advanced-stage estrogen receptor ER+/HER2− breast cancer.Herein, we mainly review the mechanism of action and the progress of CDK4/6 inhibitors.These compounds have been categorized based on molecule similarity and origin.In addition, the proteolysis targeting chimers (PROTACs) targeting CDK4/6 have been reviewed.
However, in the clinic, some patients develop primary or acquired drug resistance.For example, about 20% of breast cancer patients receiving CDK4/6 inhibitor treatment have no response to treatment [131].These patients already have genetic mutations in their tumor cells, allowing them to avoid the effects of CDK4/6 inhibitors and continue to proliferate in the presence of drugs.Thus far, many primary resistance mechanisms have been identified, all of which seem to involve activation of the cyclin D-CDK4/6-Rb pathway [132].
In addition, the activation of cyclin D-CDK4/6-Rb, activation of other proliferation pathways, changes in the tumor microenvironment, and regulation of tumor metabolism may also lead to the emergence of acquired drug resistance [132].Within 2 years of starting treatment with CDK4/6 inhibitors in the PALOMA-2 study, over 30% of enrolled patients developed resistance to palbociclib [133].Furthermore, 40 months later, over 70% of patients in the combination of palbociclib and letrozole group in this study experienced tumor progression during treatment.As prolonged exposure to CDK4/6 inhibitors continues, more and more patients have developed drug resistance; ultimately, all patients receiving CDK4/6 inhibitor treatment will develop acquired resistance [132].
The coming research may involve the following aspects: First, the more potent and selective inhibitors that overcome drug resistance according to the mechanism of drug resistance.Secondly, the dual or multiple targeting inhibitors may have been researched for their synergetic effects or myeloprotection.Finally, the PROTACs may have been tried for anti-resistance based on the action mechanism of PROTAC and the non-enzymatic function of CDK4/6, which benefits cancer therapy.

Figure 1 .
Figure 1.The first and second generations of CDK inhibitors.

Figure 1 .
Figure 1.The first and second generations of CDK inhibitors.

Figure 2 .
Figure 2. The classical mechanism of CDK4/6 driving the cell cycle.

Figure 3 .
Figure 3.The non-enzymatic function of CDK4/6 on the phosphorylation of RB.

Figure 3 .
Figure 3.The non-enzymatic function of CDK4/6 on the phosphorylation of RB.

Figure 3 .
Figure 3.The non-enzymatic function of CDK4/6 on the phosphorylation of RB.

Figure 4 .
Figure 4. (a) The 3D X-ray crystal structure of CDK6 with Ribociclib (PDB ID: 5l2t); (b) 3D inte tion between CDK6 and Ribociclib.Protein is prepared with discovery studio.Images are pared with Free Maestro.

Figure 4 .
Figure 4. (a) The 3D X-ray crystal structure of CDK6 with Ribociclib (PDB ID: 5l2t); (b) 3D interaction between CDK6 and Ribociclib.Protein is prepared with discovery studio.Images are prepared with Free Maestro.

Figure 14 .
Figure 14.Structures derived from high-throughput screening and natural products.

Figure 14 .
Figure 14.Structures derived from high-throughput screening and natural products.

Figure 14 .
Figure 14.Structures derived from high-throughput screening and natural products.

Figure 20 .
Figure 20.Some structures in clinical research.

Figure 20 .
Figure 20.Some structures in clinical research.
), developed by Zhengzhou Taiji Hongnuo Pharmaceutical Co., Ltd.(Zhengzhou, China), is a potent and highly selective oral CDK4/6 inhibitor.It was in Phase I clinical trials in December 2019 [117].TQB3616 (Table 1), developed by Zhengda Tianqing Pharmaceutical (Lianyungang, China), is in Phase III clinical study.It is applied to patients with breast cancer and lung cancer, including HR+/HER2− late/metastatic breast cancer and ER+/HER2+ late/metastatic breast cancer [118].BEBT-209 (Table 1), developed by Guangzhou Beibeite Pharmaceutical Co., Ltd.(Guangzhou, China), is in Phase II clinical study for patients with advanced breast cancer.Unlike the already-marketed CDK4/6 anti-tumor inhibitors, BEBT-209 improves the selectivity of CDK4 over CDK6, which is expected to reduce the hematological and immunosuppressive toxicity caused by CDK6 activity inhibition [119].FLX925 (Compound 111, Figure 20, Table

Table 1 )
, developed by Shanghai Pharmaceutical Group (Shanghai, China), is in Phase II/III clinical study.It is used for advanced solid tumor, local, or metastatic breast cancer [122].

Table 1 .
Drugs in clinical research.

Table 1 )
, developed by Shanghai Xunhe Pharmaceutical Technology Co., Ltd.(Shanghai, China), is in Phase I clinical trial.Its intended use is to treat advanced solid tumors, including colorectal cancer, breast cancer, ovarian cancer, etc. [127].HS-10342 (Table 1), developed for Hansen Pharmaceutical (Shanghai, China), is in a Phase I clinical trial.Its intended use is for the treatment of patients with advanced breast cancer with ER+/HER2− [128].QHRD110 (Table 1), developed by Changzhou Qianhong Biochemical Pharmaceutical (Changzhou, China), is in phase I clinical trials [129].NUV-422 (Table 1) is a novel inhibitor of the cyclin-dependent kinase CDK2/4/6, developed by Nuvation Bio.(New York, NY, USA).It is a Phase II clinical study for patients with HR+/HER2− advanced breast cancer [130].