The Pharmacological Implications of Flavopiridol: An Updated Overview

Flavopiridol is a flavone synthesized from the natural product rohitukine, which is derived from an Indian medicinal plant, namely Dysoxylum binectariferum Hiern. A deeper understanding of the biological mechanisms by which such molecules act may allow scientists to develop effective therapeutic strategies against a variety of life-threatening diseases, such as cancer, viruses, fungal infections, parasites, and neurodegenerative diseases. Mechanistic insight of flavopiridol reveals its potential for kinase inhibitory activity of CDKs (cyclin-dependent kinases) and other kinases, leading to the inhibition of various processes, including cell cycle progression, apoptosis, tumor proliferation, angiogenesis, tumor metastasis, and the inflammation process. The synthetic derivatives of flavopiridol have overcome a few demerits of its parent compound. Moreover, these derivatives have much improved CDK-inhibitory activity and therapeutic abilities for treating severe human diseases. It appears that flavopiridol has potential as a candidate for the formulation of an integrated strategy to combat and alleviate human diseases. This review article aims to unravel the potential therapeutic effectiveness of flavopiridol and its possible mechanism of action.


Introduction
Plants, the only producers in ecosystems, have influenced humankind and other life systems.Novel and effective therapeutic agents can be developed using bioactive products from plants that can be applicable to various dreadful illnesses, such as cancer and viral, fungal, and neurological diseases [1].This review emphasizes the therapeutic benefits associated with a compound named flavopiridol (FP).Basically, FP is a flavone and semi-synthetic derivative of the naturally occurring product rohitukine [2].Rohitukine is a chromone alkaloid found in four different plants: Dysoxylum binectariferum Hiern.(stem bark), Amoora rohituka (Roxb.)Wight & Arn.-(stem and leaves), Schumanniophyton problematicum, and Schumanniophyton magnificum [3,4].Rohitukine is widely used as a medicine on the Indian subcontinent.It has been found in preclinical studies that FP is a potent anti-cancer medication for leukemia, lymphoma, prostate carcinoma, breast cancer, bladder cancer, and liver cancer [5][6][7][8][9].Flavopiridol performs its anti-cancer function by inhibiting multiple cyclin-dependent kinases (CDKs) [10].CDK protein kinases belong to the serine/threonine protein kinase family.They regulate cell cycle progression and transcription and are a potential target for developing chemopreventive therapies [11,12].Several CDKs, including CDK1, 2, 4, and 6, directly participate in cell progression, whereas CDK7, 8, and 9 assist in transcription [2,13].
The enzymatic activities of CDK1, 2, and 4 are suppressed by flavopiridol, which leads to cell cycle arrests in the G1 and G2 phases, preventing them from entering the synthesis (S) and metaphase (M) phases, respectively [14][15][16].In addition, it inhibits CDK9, a part of the positive transcription elongation factor b (P-TEFb) that leads to the suppression of phosphorylation at the C-terminus of RNA polymerase II, inhibiting transcription [17].Among other CDK inhibitors, FP was the first to be studied in human trials [18][19][20].In 1994, clinical studies first evaluated it as a combined therapeutic regimen for acute myeloid and chronic lymphocytic leukemia [21].Despite the good results in preclinical studies, some unwanted toxicities (i.e., secretory diarrhea, hypotension, and pro-inflammatory syndrome) were found at higher doses of FP in clinical trials [22].Numerous derivatives of FP were synthesized to overcome this issue by chemical modification to improve its CDK activity, biodistribution, and therapeutic potential [23][24][25].
Flavopiridol has been extensively explored for its pharmacological therapeutic implications over the last few decades.Flavopiridol modulates several signaling pathways associated with tumor proliferation and metastasis, viral replication, fungal infection, and inflammation-associated diseases [26][27][28][29][30].Even though such bioactive semi-synthetic compounds are available, their use in treating cancer and viral, fungal, and inflammationassociated diseases is yet to be determined.Thus, understanding how a therapeutic agent such as FP acts is essential.This is imperative so that the research community can better comprehend the different signaling mechanisms underlying the onset of disease and develop innovative treatment approaches.A review is provided here, summarizing the many therapeutic applications of FP, as well as the possible molecular mechanisms of its actions.

Chemistry of Flavopiridol
Flavopiridol is also known as alvocidib, L86-8275, HL275, and NSC649890.The chemical name of FP is [4,31]-1-benzopyran-4-one] [32].This flavone is synthetically produced from the natural anti-rheumatic flavonoid rohitukine.Flavopiridol is a yellow crystalline solid that can be identified through different spectroscopic and chromatographic techniques.Its molecular formula is C 21 H 20 ClNO 5 ; its melting temperature ranges from 186 • C to 190 • C, and its molecular weight is 401.84 g mol −1 .Flavopiridol has minimal solubility in water; however, it is soluble in organic solvents, including ethanol, dimethyl sulfoxide, and dimethyl formamide [33].The synthesis of FP occurs in seven steps of chemical reactions.A detailed version of its synthesis has been reported previously [34].The crystal structure of FP was determined in complex with the cyclin-dependent kinase2 (CDK2) enzyme, which confers the flavone nucleus of the former bound to the ATP binding site pocket of the latter [35].It shows CDK-inhibitory activity by containing the D-ring, also known as the 3-hydroxy-1-methylpiperidinyl ring, as shown in Figure 1.In contrast, this ring is absent in two structurally relevant natural flavonoids, quercetin and genistein, that show poor CDK-inhibitory activity [34,36,37].

Apoptosis Activation and Cell Cycle Arrest
An important criterion that distinguishes tumor cells from healthy cells and promotes tumor formation is the loss of apoptotic functions [38].Apoptosis is a programmed cell death (PCD) that involves condensation and disintegration of less compact chromatin, cleavage of DNA while forming ladders, and membrane blebbing associated with phosphatidylserine exposure [39].It is a strictly organized process regulated mainly by two types of apoptotic proteins: pro-apoptotic proteins and anti-apoptotic proteins [31].Currently, anti-cancer therapies focus on inhibiting the development and proliferation of tumor cells by targeting apoptotic pathways.In the quest to develop effective anti-cancer therapies, numerous anti-cancer compounds are being used, including FP. Flavopiridol shows anti-cancer activities mainly by directly inhibiting CDK (i.e., CDK1, 2, 4, 6, and 7) through ATP-competitive inhibitions and indirectly minimizing the levels of cyclins (i.e., cyclin D1 and cyclin D3) or CDK inhibitors (p21 and p27).Reduced levels of cyclin D (cyclin D1, cyclin A, and cyclin E) further reduce CDK enzymatic activity, leading to impaired phosphorylation of the pRb, p107, and p120 proteins (Figure 2) [17,40].

Apoptosis Activation and Cell Cycle Arrest
An important criterion that distinguishes tumor cells from healthy cells and promotes tumor formation is the loss of apoptotic functions [38].Apoptosis is a programmed cell death (PCD) that involves condensation and disintegration of less compact chromatin, cleavage of DNA while forming ladders, and membrane blebbing associated with phosphatidylserine exposure [39].It is a strictly organized process regulated mainly by two types of apoptotic proteins: pro-apoptotic proteins and anti-apoptotic proteins [31].Currently, anti-cancer therapies focus on inhibiting the development and proliferation of tumor cells by targeting apoptotic pathways.In the quest to develop effective anti-cancer therapies, numerous anti-cancer compounds are being used, including FP. Flavopiridol shows anti-cancer activities mainly by directly inhibiting CDK (i.e., CDK1, 2, 4, 6, and 7) through ATP-competitive inhibitions and indirectly minimizing the levels of cyclins (i.e., cyclin D1 and cyclin D3) or CDK inhibitors (p21 and p27).Reduced levels of cyclin D (cyclin D1, cyclin A, and cyclin E) further reduce CDK enzymatic activity, leading to impaired phosphorylation of the pRb, p107, and p120 proteins (Figure 2) [17,40].
These hypophosphorylated proteins inhibit various transcription factors (MDM-2, Myc, c-Jun, E2F) mainly responsible for growth arrest and promoting apoptosis [40].Likewise, FP decreased the levels of p21 and p27, led to the growth arrest at the G1/S phase of the cell cycle, activated caspase 9, increased the ratio of bax/bcl2, and induced apoptosis [41,42].Smith et al., (2008) have reported that a nanomolar concentration of FP was sufficient to arrest the cell cycle in the G1 and G2 phases, and it decreased expression of cyclin D1, increased expression of p21, and promoted apoptosis by activation of caspase 3 or 7, thereby inhibiting rhabdoid tumor growth [43].In a study using non-small-cell lung cancer cell lines (NCI-H661 and A549) and an osteosarcoma cell line (U2OS), the pro-apoptotic effect of FP was linked with cdk2/cyclin A inhibition and inactivation of the E2F-1 transcription factor during the late S-phase of the cell cycle, resulting in tumor cell apoptosis [44].A recent study reported that FP, with CKAP2L (cytoskeleton-associated protein 2-like) depletion, led to the suppression of tumor growth and apoptosis activation in esophageal squamous cell carcinoma [45].Several studies reported that active proliferating cancerous cells are more sensitive to FP after 24 h of treatment than resting cancerous cells [5,32,40].Later, it was observed that FP has equal potential to activate cell death in both proliferating and non-proliferating cancer cells after 24 h of treatment [4,46].Since all solid tumors contain resting cancer cells within hypoxic zones due to a lack of oxygen, FP is highly attractive in this context.This feature of FP gains explicit attention for treating chronic lymphocytic leukemia, where most of the lymphocytes exist in the quiescent stage [4].In a majority of studies to date, FP-induced tumor cell death has been determined to be independent of p53, decreasing the expression of several anti-apoptotic proteins (i.e., Bcl-X L , Bcl-2, XIAP, survivin, and Mcl-1) as shown in Figure 3 [47][48][49][50][51].These hypophosphorylated proteins inhibit various transcription factors (MDM-2, Myc, c-Jun, E2F) mainly responsible for growth arrest and promoting apoptosis [40].Likewise, FP decreased the levels of p21 and p27, led to the growth arrest at the G1/S phase of the cell cycle, activated caspase 9, increased the ratio of bax/bcl2, and induced apoptosis [41,42].Smith et al., (2008) have reported that a nanomolar concentration of FP was sufficient to arrest the cell cycle in the G1 and G2 phases, and it decreased expression of cyclin D1, increased expression of p21, and promoted apoptosis by activation of caspase 3 or 7, thereby inhibiting rhabdoid tumor growth [43].In a study using non-small-cell lung cancer cell lines (NCI-H661 and A549) and an osteosarcoma cell line (U2OS), the pro-apoptotic effect of FP was linked with cdk2/cyclin A inhibition and inactivation of the E2F-1 transcription factor during the late S-phase of the cell cycle, resulting in tumor cell apop-  Survivin is an apoptosis inhibitor in its phosphorylated form and is responsible for abnormal progression of cell cycles, as well as dodging apoptosis.However, FP suppresses the phosphorylation of survivin at threonine 34, resulting in loss of survivin activity and favoring the activation of cell death in MCF-7 (human breast cancer cells) and HeLa (human cervical cancer cells) [52].In a study using atypical thyroid cancer cells, Pinto et al., (2020) observed that FP inhibits cell proliferation, downregulates Mcl-1, and favors induction of cell cycle arrest [53].In multiple myeloma cells, FP was found to downregulate the levels of anti-apoptotic proteins (Mcl-1) and the induction of apoptosis.However, overexpressed Mcl-1 multiple myeloma cell lines restrict FP-induced apoptosis [49].Further, FP mediates apoptosis in tumor cells via caspase-dependent and -independent mechanisms (Figure 3).Caspase-dependent tumor cell death in breast cancer cells (MDA-MB-435), Burkitt's lymphoma cell line (GA-10), and human myeloid leukemic cells (U937 and HL-60) has been mediated by FP through the induction of classical mitochondrial proteins, including activation of caspases (caspase 3, 8, and 9), DNA laddering, cleavage of PARP (poly ADP-ribose polymerase) and Bid proteins, elevated levels of Bax, decreased expression of Bcl2, and the release of cytochrome C (cytC) (Figure 3) [54][55][56][57].In lung or esophageal cancer cells, FP, combined with depsipeptide, mediates apoptosis by Survivin is an apoptosis inhibitor in its phosphorylated form and is responsible for abnormal progression of cell cycles, as well as dodging apoptosis.However, FP suppresses the phosphorylation of survivin at threonine 34, resulting in loss of survivin activity and favoring the activation of cell death in MCF-7 (human breast cancer cells) and HeLa (human cervical cancer cells) [52].In a study using atypical thyroid cancer cells, Pinto et al., (2020) observed that FP inhibits cell proliferation, downregulates Mcl-1, and favors induction of cell cycle arrest [53].In multiple myeloma cells, FP was found to downregulate the levels of anti-apoptotic proteins (Mcl-1) and the induction of apoptosis.However, overexpressed Mcl-1 multiple myeloma cell lines restrict FP-induced apoptosis [49].Further, FP mediates apoptosis in tumor cells via caspase-dependent and -independent mechanisms (Figure 3).Caspase-dependent tumor cell death in breast cancer cells (MDA-MB-435), Burkitt's lymphoma cell line (GA-10), and human myeloid leukemic cells (U937 and HL-60) has been mediated by FP through the induction of classical mitochondrial proteins, including activation of caspases (caspase 3, 8, and 9), DNA laddering, cleavage of PARP (poly ADP-ribose polymerase) and Bid proteins, elevated levels of Bax, decreased expression of Bcl2, and the release of cytochrome C (cytC) (Figure 3) [54][55][56][57].In lung or esophageal cancer cells, FP, combined with depsipeptide, mediates apoptosis by activating caspase 9 and the mitochondrial release of cytC [58].Both in vitro and in vivo studies on human cholangiocarcinoma cell lines showed that FP potentiates cell cycle abortion and enhances caspase-dependent cell death [59].Some researchers reported that FP in combination with CPT11 (a DNA topoisomerase inhibitor) promotes the cleavage of apoptosis inhibitors and activation of cell death in colon cancer cells (Hct116) [60].In contrast, caspase-and Bcl2-independent cell death occurred in glioma and lung carcinoma tumors by FP via the secretion of mitochondrial apoptosis-inducing factor (AIF) [61,62].In addition, a study reported that FP promotes cell death in murine glioma cells (GL261) through the induction of caspase-dependent and -independent cell death by the secretion of cytC and AIF from the mitochondria, respectively [63].Moreover, FP may suppress the activities of the transcription factor NF-κB and make tumor cells more vulnerable to chemotherapy and radiation therapy.Through the phosphorylation of the Akt protein, the NF-κB transcription factor indirectly regulates a variety of cellular events, including cell proliferation, migration, and apoptosis.Studies have demonstrated that FP potentiates apoptosis in human leukemic cell lines in conjunction with proteasome inhibitors and PI3K (phosphatidylinositol-3-kinase) inhibitors by inhibiting the expression of NF-κB transcription factor [50,64].Furthermore, FP could also perform synergistic effects with cytostatic drugs to induce tumor cell apoptosis throughout the cell cycle by induction of caspase-dependent and -independent mechanisms [65].Using MKN-74 (human gastric tumor cells) and MCF-7, Motwani et al., (1999) investigated the synergistic effect of FP with paclitaxel to enhance the caspase activity and found that PARP cleavage led to the activation of cell death [66].Interestingly, FP in synergy with gemcitabine potentiates the activation of caspase-dependent cell death in human gastric, colon, and pancreatic tumors by suppression of the transcriptional activation of the ribonucleotide reductase M2 subunit [67].In another experimental study, it was demonstrated that FP enhances mitomycin-C-induced apoptosis in MKN-74 (gastric tumor cells) and MDA-MB-468 (breast cancer tumor cells) by inhibiting the activity of protein kinase C [68].Likewise, irinotecan-induced apoptosis in advanced hepatocellular carcinoma is potentiated by FP [69].As a collective, these shreds of evidence suggest that FP individually or in synergy with chemopreventive drugs enhances the induction of tumor cell apoptosis via caspase-dependent or -independent mitochondrial pathways by the downregulation of anti-apoptotic proteins, inactivation of cell cycle promoting proteins, and inhibition of tumor cell proliferation.

Anti-Metastatic Effect
Tumor metastasis is one of the fatal characteristics of tumor cells through which they disseminate beyond their original location to surrounding and distant tissues or organs [70].It occurs in a sequential manner where tumor cells first evade anchorage-dependent cell death (anoikis) by detaching from the extracellular matrix (ECM); second, tumor cells invade across the ECM to gain access to blood circulation through the circulatory system and lymphatic system; third, tumor cells extravasate and arrest at distant sites of the human body; fourth, tumor cells colonize and form micro-metastases by warding off immune attack; and finally, the cells instigate neovascularization to promote tumor growth and establish macro-metastases [71].An estimated 90% of cancer mortality occurs due to tumor metastasis [38].Detecting and inhibiting malignant tumor metastasis is a significant challenge that must always be addressed in the modern practice of cancer treatment [70,72].In order to combat this problem, scientists have found several natural and chemical compounds that inhibit tumor metastasis [73][74][75].Anti-metastatic agents generally include angiogenesis inhibitors, integrin antagonists, VEGF inhibitors, anti-MMP agents, and receptor tyrosine kinase inhibitors [76,77].The use of FP as an anti-metastatic agent has been documented for more than three decades in cancer research because it possesses various activities, such as decreased VEGF secretion, anti-angiogenesis activity, anti-invasion activity, and decreased secretion of MMP enzymes (Figure 4) [54,78,79].Using ovarian carcinoma (OCa-I) as an in vivo tumor model, Mason et al., (2004) determined a significant decrease in metastatic nodules in the lungs after FP treatment [80].Another group of researchers found that FP-treated human osteosarcoma cells showed anti-metastatic activity, as the inhibition of R-cadherin and induction of P-cadherin expression led to the reduced migration and invasion of cancerous cells [81].Similarly, it was evidenced that FP decreased expression of galectin-3 and increased expression of E-cadherin in KRAS mutant lung adenocarcinoma cells, which reflects its anti-metastatic effects (Figure 4) [82].Additionally, in vivo mice experiments confirmed that FP can significantly reduce the ability of human osteosarcoma cells (SJSA-1 and 143B) to metastasize into the lungs [81].Further, several studies reported that the synergistic interaction between FP and other chemopreventive agents increased their anti-invasive and anti-metastatic activities against metastatic cancer.A recent study investigated FP in conjunction with quisinostat (a histone deacetylase inhibitor), and found that the combination is a potential regimen for treating cutaneous and uveal metastatic melanoma, irrespective of NRAS and KRAS mutations [83].Likewise, another study reported that a synergistic combination of FP with carfilzomib led to the activation of G2/M cell cycle arrest, decreased levels of X-linked inhibitor of apoptosis (XIAP), and augmented apoptosis.This drug combination exposed to mice with human adrenocortical cancer xenografts provoked a significant decrease in cancerous growth due to increased cleaved caspase and decreased expression of XIAP [84].Holkova et al., (2011) noted that a bortezomib (proteasome inhibitor) and FP combination is a potential therapeutic option against recurrent or refractory B-cell neoplasms [85].Similarly, researchers have found that gemcitabine and irinotecan, in combination with FP, possess potentiated anti-metastatic effects that facilitate the treatment of metastatic cancers [86].In an in vivo breast cancer mice model, FP in combination with sorafenib (Raf inhibitor) had anti-metastatic activity, causing a significant reduction in the lung metastatic tumor.This is possibly due to the decreased Mcl-1 expression and Rb signaling in the cancer cell [87].In summary, it was concluded that FP itself or combined with other anti-cancerous drugs, has potent tumor metastasis inhibitory activity due to its ability to suppress the exodus and infiltration of tumor cells through numerous mechanisms.

Anti-Angiogenesis
Angiogenesis is the process of developing new blood vessels from existing ones.It occurs during menstruation, wound healing, and under various pathological conditions, including tumor growth, arthritic synovium, and proliferative retinopathy [88].In tumor cells, angiogenesis is mainly responsible for neovascularization through the activation of endothelial cells by tumor-derived cytokines like vascular endothelial growth factor (VEGF), which encourages tumor cells to survive and expand [89].The most potent angiogenic growth factor, VEGF, is generally released during hypoxic conditions to help tumor cells adapt to this microenvironment, driven by hypoxia-induced factor-1 (HIF-1α)

Anti-Angiogenesis
Angiogenesis is the process of developing new blood vessels from existing ones.It occurs during menstruation, wound healing, and under various pathological conditions, including tumor growth, arthritic synovium, and proliferative retinopathy [88].In tumor cells, angiogenesis is mainly responsible for neovascularization through the activation of endothelial cells by tumor-derived cytokines like vascular endothelial growth factor (VEGF), which encourages tumor cells to survive and expand [89].The most potent angiogenic growth factor, VEGF, is generally released during hypoxic conditions to help tumor cells adapt to this microenvironment, driven by hypoxia-induced factor-1 (HIF-1α) [90].Inhibition of cancer cell proliferation can be achieved by targeting angiogenic growth factors, oncogenes, membrane receptors, proteases, and signaling transduction factors involved in the neovascularization of tumor cells.Flavopiridol decreased the expression of VEGF mRNA and its consecutive protein in human monocytes under hypoxic environments by reducing VEGF mRNA stability (Figure 5) [91].Another group of researchers found that FP inhibited the Src transcription factor and downregulated the expression of VEGF [92].Further, it was evidenced that FP could also decrease c-fos-induced VEGFD expression by the inhibition of fos promoter activation and PKC [93].In the studies on GI-LI-N, LAN-5, and ACN (human neuroblastoma cells), researchers demonstrated that FP inhibits the expression of VEGF, which is induced by HIF-1α, picolinic acid, and desferrioxamine [94].Also, Newcomb et al., (2005) found that the inhibition of angiogenesis by FP in human glioma cell lines (U87MG and T98G) via downregulation of VEGF expression through a decrease in HIF-1α levels occurred even when a proteasome inhibitor was present [78].Another vital component of the angiogenesis process is matrix metalloproteinase (MMP) enzymes, which are secreted by tumor cells to facilitate cancer cell infiltration and migration through destruction of extracellular matrix components (Figure 5).It was found that FP impaired the release of MMP2 and MMP9 enzymes in breast tumor cells, which eventually caused the suppression of tumor invasion and migration, as observed in a Matrigel invasion assay [54].Likewise, FP-treated human glioma cells showed decreased secretion of MMP2, were unable to induce the proteolysis of the extracellular matrix, and favored the inhibition of tumor invasion [78].Takada et al., (2004) noted that FP-mediated inhibition of MMP9 secretion occurred through impaired NF-κB enzymatic activity induced by NF-κB kinase, IκBα kinase, tumor necrosis factor (TNF) receptor 1 (also referred as TNFR1), TNF-receptor-associated factor-2 (TRAF2), and TNF receptor-associated death domain (TRADD) (Figure 5) [79].Another study observed the anti-angiogenic function of FP in the human colon carcinoma cell line (RKO).It showed a 70% decrease in blood vessel formation using the mouse xenograft Matrigel invasion model [95].Since human RKO cell lines are devoid of αvβ3and αvβ5-integrins, this suggests that the FP-mediated anti-angiogenic effect is independent of integrin inhibition.In contrast, Yang et al., (2014) found a synergistic combination of FP with paclitaxel that showed anti-angiogenic effects by decreasing αvβ3 integrin expression in endothelial cells and increasing expression of αvβ3 integrin in ovarian carcinoma cell lines (SKOV3).It was assessed that both in vitro and in vivo anti-angiogenic activity was evaluated using endothelial cell tube formation and tumor microvessel density, respectively [96].Similarly, a combination of FP with docetaxel in transgenic Gγ/T-15 mice induced apoptosis and decreased angiogenesis, leading to the inhibition of primary and metastatic prostate cancer [97].Further, authors reported that FP is a better anti-angiogenic molecule than the known angiogenesis inhibitor TNP-470, as observed in a Matrigel invasion model of human colon carcinoma xenografts [98].
Interestingly, Mcferrin et al., (2010) reported another possible mechanism for FPmediated anti-angiogenic effects.Researchers observed that FP inhibits the activity of CDK9 and vGPCR (Kaposi sarcoma-associated herpesvirus G-protein coupled receptor) in human umbilical vein endothelial cells (HUVECs), which further decreases the secretion of VEGF-A and VEGF-C, leading to a decrease in cell migration and new blood vessel formation [99].All the pieces of evidence indicated that FP possesses anti-angiogenic effects through numerous mechanisms such as decreasing hypoxic-induced VEGF secretion through decreased HIF-1α expression, decreasing MMP enzyme secretion by inactivation of NF-κB, and inhibition of vGPCR that further inhibits VEGF secretion (Figure 5).Altogether, FP possesses anti-apoptotic and anti-angiogenic activities.Hence, it could be applied to the treatment of different cancers in the future.2010) reported another possible mechanism for FP-mediated anti-angiogenic effects.Researchers observed that FP inhibits the activity of CDK9 and vGPCR (Kaposi sarcoma-associated herpesvirus G-protein coupled receptor) in human umbilical vein endothelial cells (HUVECs), which further decreases the secretion of VEGF-A and VEGF-C, leading to a decrease in cell migration and new blood vessel formation [99].All the pieces of evidence indicated that FP possesses anti-angiogenic effects through numerous mechanisms such as decreasing hypoxic-induced VEGF secretion through decreased HIF-1α expression, decreasing MMP enzyme secretion by inactivation of NF-κB, and inhibition of vGPCR that further inhibits VEGF secretion (Figure 5).Altogether, FP possesses anti-apoptotic and anti-angiogenic activities.Hence, it could be applied to the treatment of different cancers in the future.

Anti-Inflammatory Effects
Inflammatory diseases occur when the immune system fails to eliminate organisms or foreign substances, resulting in excessive and prolonged inflammation.This event contributes to the progression and pathogenesis of a broad range of severe inflammationassociated diseases such as cancer, asthma, atherosclerosis, neurological illnesses, and arthritis [100,101].A study reported the dose-dependent inhibition of inflammatory

Anti-Inflammatory Effects
Inflammatory diseases occur when the immune system fails to eliminate organisms or foreign substances, resulting in excessive and prolonged inflammation.This event contributes to the progression and pathogenesis of a broad range of severe inflammationassociated diseases such as cancer, asthma, atherosclerosis, neurological illnesses, and arthritis [100,101].A study reported the dose-dependent inhibition of inflammatory processes in murine collagen-induced arthritis conditions by FP [102].One of the marked characteristics of inflammation is massive leukocyte infiltration from the bloodstream into the tissue site via the ability of leukocytes to interact with endothelial cells.FP has the potential to show anti-inflammatory effects by inhibiting the leukocyte extravasation and leukocyte-endothelial cell interaction through the suppression of endothelial cell activation [103].In support of this fact, it was observed that FP blocked neutrophil infiltration and reduced the cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1) expressions in a concanavalin A-induced murine hepatitis model [103].Additionally, it was proved that FP-mediated suppression of macrophage infiltration occurs when given through the intrathecal route [104].Further, it was found that FP abrogates ICAM-1 expression as well as NF-κB mediated gene transcription by impairment of CDK9 activity, not by suppressing the canonical NF-κB activation pathway (i.e., abrogation of IκBα kinase and p65 subunit phosphorylation) [103].Despite being part of P-TEFb (positive transcriptional elongation factor b), CDK9 has been reported to bind with the cytoplasmic part of glycoprotein (gp130), the receptor for IL-6, a pro-inflammatory cytokine [103].To strengthen the observation of the previous study, it was observed that FP inhibited IL-6/STAT3 (signal transducer and activation of transcription 3) signaling in hepatocarcinoma (hepG2 cell line) via a JAK/STAT signaling pathway, which is a crucial inflammatory pathway to increase neutrophil survival [105].Further, it was evidenced that FP suppressed IFN-γ mediated nitric oxide (NO) formation within vascular endothelial cells by reducing expression levels of inducible NO synthase (iNOS), which leads to the inactivation of STAT1 and its downstream molecule IFN-γ responsive factor (IRF1) in JAK/STAT signaling, suggesting an anti-inflammatory effect (Figure 6) [106].It is imperative to recognize that NO and TNF-α are crucial inflammatory molecules and excess production of them is associated with the pathophysiology of many inflammatory illnesses [107].Interestingly, Haque et al., (2011) reported that FP decreased the production of pro-inflammatory mediators, including TNF-α and NO, in lipopolysaccharide (LPS)-activated cells [108].In addition to NF-κB and IκBα kinase, it also inhibits the series of MAPK (mitogen-activated protein kinases), including p38, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases, and stress-induced protein kinases, in the presence of LPS [108].Further, it was found that FP-treated inhibition of TNF-α and NO in the presence of lipopolysaccharide occurred by abrogation of MAPK and NF-κB stimulation via the MyD88-(Myeloid differentiation factor 88) dependent TLR2 (Toll-like receptor 2) ligand, which may explain the importance of FP to mitigate the inflammatory response (Figure 6) [108].Basically, the four major signaling pathways exist, including JAK/STAT, MAPK, PI3K, and NF-κB, which regulate inflammatory signals and promote the survival of leukocytes [101].FP can inhibit the Akt phosphorylation that suppresses the NF-κB induction and controls different biological events, including migration, proliferation, inflammation, and apoptosis (Figure 6) [57].Dose-and timedependent abrogation of TNF-α-induced NF-κB by FP was reported in different cell lines, including human kidney cells (A293), human myeloid leukemic cells (HL60), and human T-lymphocyte cells (Jurkat) [79].In addition to TNF-α, FP also suppressed NF-κB induced by other molecules, such as inflammatory agents, tumor promoters, and carcinogens [79].This inhibition occurs through the suppression of Akt phosphorylation, and a canonical NF-κB stimulation cascade results in the suppression of TNF-α-induced NF-κB regulated genes such as MMP9, cyclooxygenase-2, and cyclin D1, indicating the immunomodulatory and anti-inflammatory role of FP as illustrated in Figure 6 [79].In another study, FP was found to have anti-arthritic effects by inhibiting the production of inflammatory mediators (i.e., iNOS) and extracellular-matrix-degrading genes (i.e., MMP1, 3, 9, and 13), protecting human cartilage explants from the adverse effects of pro-inflammatory cytokines (i.e., IL-1β, TNF-α, and LPS) while maintaining cell survival and function [109].The in vitro and in vivo studies on the bone remodeling model indicate that FP suppresses RANKL (receptor activator of nuclear kappa B ligand) signaling pathways by suppressing the non-canonical NF-κB activation cascade [110].Information on RANKL suggests that it is a transmembrane protein of the TNF superfamily and is closely linked to acute and chronic inflammation, which strengthens the role of FP as an anti-inflammatory agent [111,112].
Chang and his colleagues reported the abrogation of inflammatory mediators in FP alone or in combination with PLGA (poly (lactic-co-glycolic acid)) nanoparticle-treated astrocytes through significant inhibition of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) and induction of an anti-inflammatory cytokine (i.e., IL-10) [104,113].Similar results were obtained in a fungal keratitis mouse model, wherein FP inhibited the inflammation induced in fungal keratitis through the production of the IL-1β, IL-6, and TNF-α cytokines while promoting the production of the IL-10 cytokine [29].All the evidence indicated herein points towards FP's ability to inhibit inflammation through a multitude of mechanisms, as discussed above, making it a potential anti-inflammatory agent for future clinical research.
as an anti-inflammatory agent [111,112].Chang and his colleagues reported the abrogation of inflammatory mediators in FP alone or in combination with PLGA (poly (lactic-coglycolic acid)) nanoparticle-treated astrocytes through significant inhibition of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) and induction of an anti-inflammatory cytokine (i.e., IL-10) [104,113].Similar results were obtained in a fungal keratitis mouse model, wherein FP inhibited the inflammation induced in fungal keratitis through the production of the IL-1β, IL-6, and TNF-α cytokines while promoting the production of the IL-10 cytokine [29].All the evidence indicated herein points towards FP's ability to inhibit inflammation through a multitude of mechanisms, as discussed above, making it a potential anti-inflammatory agent for future clinical research.

Efficacy of Flavopiridol as a Therapeutic Agent
The emergence of pathogens that cause life-threatening diseases remains a problem for their treatment and effective disease management and is a significant threat to global public health.Prevention of new infections and their progression to active disease is critical to reducing the burden of the diseases and death caused by them.To address this problem, research is constantly sought for better alternatives to antimicrobial agents due to drug resistance among pathogenic microbes [114].There is potential for bioactive

Efficacy of Flavopiridol as a Therapeutic Agent
The emergence of pathogens that cause life-threatening diseases remains a problem for their treatment and effective disease management and is a significant threat to global public health.Prevention of new infections and their progression to active disease is critical to reducing the burden of the diseases and death caused by them.To address this problem, research is constantly sought for better alternatives to antimicrobial agents due to drug resistance among pathogenic microbes [114].There is potential for bioactive compounds derived from natural sources to be used to combat drug resistance issues and reduce antibiotic off-target effects [115].In addition to the anti-cancerous, anti-metastasis, anti-angiogenic, and anti-inflammatory functions of FP, it has also been extensively used as a treatment for different diseases such as viral infections, parasitic infections, and fungal infections caused by several harmful pathogens, as shown in Table 1.It has been observed that it abrogates the replication of different viruses, such as human immunodeficiency virus (HIV), herpes simplex virus (HSV1 and 2), human cytomegalovirus (HCMV), human adenovirus (hAdV5 and hAdV53), and influenza virus A (HINI, H3N2, H7N9) by suppression of mRNA transcription through P-TEFb/CDK9 complex inhibition [26][27][28]116,117]. Similarly, it inhibits the growth of different parasites, including Leishmania mexicana, Toxoplasma gondii, and Plasmodium falciparum [29,118].Interestingly, it has been noted that FP inhibits fungal growth, biofilm formation, and the adhesion ability of Aspergillus fumigatus, decreases inflammation, and induces autophagy [29].Aside from these, it has several other pharmacological benefits, described in Table 1, and the underlying molecular mechanisms have been proposed.In summary, it was concluded that FP has antimicrobial and other therapeutic benefits that could be used to overcome drug resistance and unnecessary outcomes.

Derivatives of Flavopiridol
As already stated, FP has CDK-inhibitory activity that abrogates the continuation of the cell cycle by arresting the G1 and G2/M phases.This CDK-inhibitory activity is mainly responsible for anti-cancerous, anti-inflammatory, and other pharmacological applications.However, the significant issues hampering FP's transition from research to the clinic are non-selective kinase inhibitory activity, low target specificity, moderate target affinity, and bioavailability in biological systems.To overcome all these problems, a wide range of FP derivatives have been synthesized by chemical modifications aiming for better biological and pharmacological functionalities.The structure-activity relationship of FP was mentioned in Figure 1.It has been observed that replacement of the chromone moiety of FP by 4-hydroxy benzofuranone improves the kinase inhibitory activity (CDK1 and CDK2) and selectivity against CDK4 and also enhances the anti-cancerous activity [130].Thio-and oxo-FP analogs increase the selectivity of FP for CDK1 by discriminating from other kinases.In addition, these FP analogs also possess anti-proliferative activity against different human cancer cell lines [131].Another piece of research indicates that the Dring of FP is critical for CDK-inhibitory activity, and olefin analogs of FP have shown selective kinase inhibitory activity against CDK4, as well as tumor growth inhibition in MCF-7 cells [34].Interestingly, P-276-00 is another derivate of FP that shows selective CDK1, CDK4, and CDK9 inhibitory activity compared to other CDKs.Additionally, it also has anti-tumor activities against 14 human cancer cell lines [132].Further, it was found that the 2-fluorophenyl analog of FP shows 40-fold more selective inhibition to P-TEFb than other CDKs, leading to the inhibition of HIV-1 Tat transactivation and replication; therefore, it could be used as an anti-HIV1 therapeutic [133].Voruciclib and IIIM-290 are oral bioavailable FP analogs with selective CDK9 inhibitory activity.Voruciclib has shown anti-tumor activity when combined with other anti-cancer drugs to treat different cancers [24].Another derivative, IIIM-290, displayed caspase-dependent apoptosis in pancreatic cancer and exhibited anti-tumor activity against a wide range of cancer cell lines [23].Surprisingly, Ibrahim et al., (2020) have reported that thioether-benzimidazoles analogs of FP by chemical modification at the C-ring led to the inhibition of CDK9 and CDK10.This FP derivative is the only compound that has shown CDK10 inhibitory activity, and it also possesses anti-cancer effects by inhibiting tumor growth in seven cancer cell lines [25].The CDK-inhibitory activity and anti-cancerous effects of other clinically active FP derivatives are summarized in Tables 2 and 3, respectively.

Adverse Effects of Flavopiridol
Flavopiridol has several therapeutic benefits, including anti-cancerous, anti-inflammatory, anti-viral, anti-fungal, and anti-parasitic functions, which have already been described in the previous sections.Despite all these advantages of FP, there is still some dirt in the picture.The significant toxicity concerns associated with using FP are secretory diarrhea and pro-inflammatory syndrome (i.e., tiredness, fever, local tumor pain, and intonation of acute phase reactants) associated with hypotension [19].Flavopiridol's effects on in vitro epithelial cell models were assessed to explain its unanticipated toxic effects.Flavopiridol caused chloride secretion in these models, implying that therapeutic antidiarrheal medicines should be used to prevent secretory diarrhea [2].The maximum tolerated dose (MTD) was increased from 50 mg m −2 /24 h to 78 mg m −2 /24 h for three days following prophylactic treatment for diarrhea with cholestyramine and loperamide [2].As an attempt to understand the pro-inflammatory syndrome, different cytokines were examined in the plasma prior to and following FP administration.It was observed that plasma IL-6 levels significantly increased dose-dependently [19,136].In the phase I clinical study, it was observed that the MTD of FP was 37.5 mg m −2 /24 h, and dose-limiting side effects were observed at 53 mg m −2 /24 h, including fatigue, vomiting, neutropenia, diarrhea, and nausea [2,19].In addition, non-dose-limiting toxicities, such as anorexia and local tumor pain, were observed.Similarly, in another clinical study, dose-limiting side effects were observed at 40 mg m −2 /24 h MTD for three days with vomiting and nausea [2].In a phase II dose escalation study of FP, substantial side effects were observed, including anorexia, fatigue, nausea, tumor pain, dyspnea, diarrhea, and cough [137,138].In another toxicological study, Wirger et al., (2005) reported that at 1 mg/kg/day of FP over three weeks, different side effects were observed, including leukopenia, lesions in the spleen and bone marrow, and gastrointestinal toxicity [7].Interestingly, in one study, it was found that FP showed drug resistance, though the mechanism is unknown.Later, it was revealed that FP mediates endoplasmic reticulum (ER) stress-induced autophagy, which is responsible for its resistance [139].In a recent study, it was found that a majority of FP-treated human prostate cancer cells (DU145) died.However, a small sub-population survived and emerged as FP-resistant cancer cells (DU145 FP ) [140].DU145 FP cancer cells have shown different characteristics, including slow growth, mitochondrial depolarization, induced abundant anti-apoptotic proteins, and less sensitivity to docetaxel and cisplatin [140].These FPresistant cancer cells induce mitochondrial lesions that modulate cell metabolism, motility, and advanced neoplastic growth potential [140].

Clinical Trials: Results and Experiences
Several clinical trials have demonstrated FP's effectiveness against many cancers, including leukemia, lymphoma, and breast, prostate, pancreatic, stomach, lung, liver, head and neck tumors [141,142].It has been shown that FP is safe and effective in patients suffering from different cancers when administered intravenously per day for several weeks [141,142].Two significant drawbacks to this drug being used alone as an anticancer agent in clinical trials are low plasma concentrations due to its binding with serum proteins and unwanted side effects due to its high doses [142].However, in some cases, this phytochemical can exert anti-cancer activity alone or in combination with some established anti-cancer medications, including vorinostat and gemcitabine hydrochloride in adult solid tumors, cisplatin in ovarian epithelial cancer, bortezomib in B-cell neoplasms, doxorubicin hydrochloride in sarcomas, venetoclax in leukemia, paclitaxel in esophageal cancer, and docetaxel in breast and pancreatic cancer [141,142].It has been reported that over 60 clinical trials, varying in clinical phases, have been performed with FP; these trials are listed in detail in Table 4.

Conclusions and Future Perspectives
The current research on FP has demonstrated its effectiveness in numerous clinical areas.It has been suggested that FP can modulate multiple molecular signaling pathways associated with the development of different cancers, and inflammatory, neurological, viral, fungal, and parasitic diseases.However, future research should explore other therapeutic functions of FP using different omics approaches such as system biology, genomics, epigenomics, transcriptomics, proteomics, and metabolomics.It would be possible to overcome the evolution of cellular resistance by synthesizing FP derivatives to improve the therapeutic efficacy of drugs for targeted diseases.It is possible to increase the effectiveness of the FP manifold by using synergistic approaches with other natural and synthetic anti-cancer medications or drug molecules.Furthermore, the application of FP with engineered nanoparticles, such as polymeric nanoparticles, liposomes, magnetic nanoparticles, exosomes, and metallic nanoparticles, enhanced the drug loading capacity, half-life in biological systems, sustained release, and selective biodistribution.Additionally, the pharmacokinetic and pharmacodynamic profiles were ameliorated over traditional formulations, preventing tumor metastasis and enhancing cancer therapy.Yang et al. (2009) reported that liposomal formulations of FP administered to mice led to reduced systemic clearance, improved plasma concentrations, and increased elimination phase half-life relative to FP alone [143,144].Moreover, the clinical efficacy of FP with different nanoformulations against different cancers and other targeted diseases must be studied.

Figure 1 .
Figure 1.The chemical structure of flavopiridol.A, B, C, and D represent the different aromatic rings present in the structure.The encircled regions mark the functional groups essential to flavopiridol's functional activity.

Figure 1 .
Figure 1.The chemical structure of flavopiridol.A, B, C, and D represent the different aromatic rings present in the structure.The encircled regions mark the functional groups essential to flavopiridol's functional activity.

Figure 2 .
Figure 2. Flavopiridol controls cell cycle progression by targeting different cyclin and CDK proteins of the G1 and G2 phases.The red arrows represent the effect of flavopiridol on the expression, and the arrow directions indicate the increase or decrease in the expression of these proteins.

Figure 2 .
Figure 2. Flavopiridol controls cell cycle progression by targeting different cyclin and CDK proteins of the G1 and G2 phases.The red arrows represent the effect of flavopiridol on the expression, and the arrow directions indicate the increase or decrease in the expression of these proteins.

Figure 3 .
Figure 3.An illustration of the apoptosis induced by flavopiridol.Flavopiridol promotes the apoptosis of tumor cells by targeting different proteins involved in the intrinsic and extrinsic apoptotic pathways.The red arrows represent the effect of flavopiridol on the expression, and the arrow directions indicate the increase or decrease in the expression of the respective proteins.

Figure 3 .
Figure 3.An illustration of the apoptosis induced by flavopiridol.Flavopiridol promotes the apoptosis of tumor cells by targeting different proteins involved in the intrinsic and extrinsic apoptotic pathways.The red arrows represent the effect of flavopiridol on the expression, and the arrow directions indicate the increase or decrease in the expression of the respective proteins.

Figure 4 .
Figure 4. Schematic representation of flavopiridol targeting the regulatory molecular players of tumor metastasis.Flavopiridol mediates the inhibition of tumor metastasis by decreasing MMP proteins' secretion via inhibition of NF-κB activity, increasing expression of E-cadherin and P-cadherin, and inhibition of wnt signaling through the inhibition of GSK3β.The red arrows represent the actions performed by flavopiridol, whereas the arrow directions indicate the increase or decrease in expression.

Figure 4 .
Figure 4. Schematic representation of flavopiridol targeting the regulatory molecular players of tumor metastasis.Flavopiridol mediates the inhibition of tumor metastasis by decreasing MMP proteins' secretion via inhibition of NF-κB activity, increasing expression of E-cadherin and P-cadherin, and inhibition of wnt signaling through the inhibition of GSK3β.The red arrows represent the actions performed by flavopiridol, whereas the arrow directions indicate the increase or decrease in expression.

28 Figure 5 .
Figure 5. Modulation of molecular targets contributing to tumor angiogenesis by flavopiridol.Flavopiridol inhibits the formation of new blood vessels by targeting different signaling pathways, including decreasing VEGF secretion, suppressing MAPK pathways by inhibiting c-Jun and c-Fos transcription factors, and decreasing the secretion of MMP proteins.The red arrows here represent the actions performed by flavopiridol, whereas the arrow directions indicate the increase or decrease in expression.Interestingly, Mcferrin et al., (2010) reported another possible mechanism for FP-mediated anti-angiogenic effects.Researchers observed that FP inhibits the activity of CDK9 and vGPCR (Kaposi sarcoma-associated herpesvirus G-protein coupled receptor) in human umbilical vein endothelial cells (HUVECs), which further decreases the secretion of VEGF-A and VEGF-C, leading to a decrease in cell migration and new blood vessel formation[99].All the pieces of evidence indicated that FP possesses anti-angiogenic effects through numerous mechanisms such as decreasing hypoxic-induced VEGF secretion through decreased HIF-1α expression, decreasing MMP enzyme secretion by inactivation of NF-κB, and inhibition of vGPCR that further inhibits VEGF secretion (Figure5).Altogether, FP possesses anti-apoptotic and anti-angiogenic activities.Hence, it could be applied to the treatment of different cancers in the future.

Figure 5 .
Figure 5. Modulation of molecular targets contributing to tumor angiogenesis by flavopiridol.Flavopiridol inhibits the formation of new blood vessels by targeting different signaling pathways, including decreasing VEGF secretion, suppressing MAPK pathways by inhibiting c-Jun and c-Fos transcription factors, and decreasing the secretion of MMP proteins.The red arrows here represent the actions performed by flavopiridol, whereas the arrow directions indicate the increase or decrease in expression.

Figure 6 .
Figure 6.Schematic representation of the anti-inflammatory activity of flavopiridol expression.Flavopiridol exerts its anti-inflammatory effects by targeting proteins of different cell signaling pathways, such as NF-κB, JAK-STAT, MAPK, PI-3K, and PKC.The red arrows here represent the actions performed by flavopiridol, whereas the arrow directions indicate the increase or decrease in expression.

Figure 6 .
Figure 6.Schematic representation of the anti-inflammatory activity of flavopiridol expression.Flavopiridol exerts its anti-inflammatory effects by targeting proteins of different cell signaling pathways, such as NF-κB, JAK-STAT, MAPK, PI-3K, and PKC.The red arrows here represent the actions performed by flavopiridol, whereas the arrow directions indicate the increase or decrease in expression.

Table 1 .
Description of the therapeutic effects of flavopiridol and the proposed mechanisms of action.

Table 3 .
Summary of the anti-cancerous activity of flavopiridol-based derivatives.

Table 4 .
Various clinical trials are reported for the inhibition of tumor development and metastasis.