CDK9 inhibition blocks the initiation of PINK1-PRKN-mediated mitophagy by regulating the SIRT1-FOXO3-BNIP3 axis and enhances the therapeutic effects involving mitochondrial dysfunction in hepatocellular carcinoma.

Mitophagy is a type of selective macroautophagy/autophagy that degrades dysfunctional or excessive mitochondria. Regulation of this process is critical for maintaining cellular homeostasis and has been closely implicated in acquired drug resistance. However, the regulatory mechanisms and influences of mitophagy in cancer are still unclear. Here, we reported that inhibition of CDK9 blocked PINK1-PRKN-mediated mitophagy in HCC (hepatocellular carcinoma) by interrupting mitophagy initiation. We demonstrated that CDK9 inhibitors promoted dephosphorylation of SIRT1 and promoted FOXO3 protein degradation, which was regulated by its acetylation, leading to the transcriptional repression of FOXO3-driven BNIP3 and impairing the BNIP3-mediated stability of the PINK1 protein. Lysosomal degradation inhibitors could not rescue mitophagy flux blocked by CDK9 inhibitors. Thus, CDK9 inhibitors inactivated the SIRT1-FOXO3-BNIP3 axis and PINK1-PRKN pathway to subsequently block mitophagy initiation. Moreover, CDK9 inhibitors facilitated mitochondrial dysfunction. The dual effects of CDK9 inhibitors resulted in the destruction of mitochondrial homeostasis and cell death in HCC. Importantly, a novel CDK9 inhibitor, oroxylin A (OA), from Scutellaria baicalensis was investigated, and it showed strong therapeutic potential against HCC and a striking capacity to overcome drug resistance by downregulating PINK1-PRKN-mediated mitophagy. Additionally, because of the moderate and controlled inhibition of CDK9, OA not led to extreme repression of general transcription and appeared to overcome the inconsistent anti-HCC efficacy and high normal tissue toxicity that was associated with existing CDK9 inhibitors. All of the findings reveal that mitophagy disruption is a promising strategy for HCC treatment and OA is a potential candidate for the development of mitophagy inhibitors.Abbreviations: BNIP3: BCL2 interacting protein 3; CCCP: carbonyl cyanide p-trichloromethoxy-phenylhydrazone; CDK9: cyclin dependent kinase 9; CHX: cycloheximide; CQ, chloroquine; DFP: deferiprone; DOX: doxorubicin; EBSS: Earle's balanced salt solution; E64d: aloxistatin; FOXO3: forkhead box O3; HCC: hepatocellular carcinoma; HepG2/ADR: adriamycin-resistant HepG2 cells; MMP: mitochondrial membrane potential; mito-Keima: mitochondria-targeted and pH-sensitive fluorescent protein; MitoSOX: mitochondrial reactive oxygen species; OA: oroxylin A; PB: phosphate buffer; PDX: patient-derived tumor xenograft; PINK1: PTEN induced kinase 1; POLR2A: RNA polymerase II subunit A; p-POLR2A-S2: Ser2 phosphorylation of RNA polymerase II subunit A; PRKN: parkin RBR E3 ubiquitin protein ligase; SIRT1: sirtuin 1.

Structure-Guided Discovery of Potent and Selective DYRK1A Inhibitors.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.

Cyclin-dependent kinase 9 (CDK9) predicts recurrence in Middle Eastern epithelial ovarian cancer.

BACKGROUND: Cyclin-dependent kinase 9 (CDK9) has been shown to play an important role in tumorigenesis of several malignancies. However, the expression of CDK9 in ovarian cancer from Middle Eastern ethnicity remains unknown. METHODS: A tissue microarray of 441 epithelial ovarian cancer (EOC) samples was used to study the expression of CDK9 immunohistochemically and their clinico-pathological associations were determined. Cox proportional hazards regression model was used for univariate and multivariate analysis of recurrence-free survival. RESULTS: CDK9 over-expression was noted in 56.2 % (248/441) of EOCs and was associated with adverse clinico-pathological parameters such as distant metastasis (p < 0.0001), stage IV tumors (p < 0.0001), tumor recurrence (p = 0.0105) and high Ki-67 index (p < 0.0001). Importantly, CDK9 over-expression was an independent predictor of poor recurrence-free survival (Hazard ratio = 1.51; 95 % confidence interval = 1.15-1.98; p = 0.0030). We also found that CDK9 outperforms Ki-67 as a predictor of tumor recurrence in EOC. CONCLUSIONS: Our results show that CDK9 expression correlates with markers of advanced disease in Middle Eastern EOC and is also a prognostic marker. CDK9 overexpression also identifies a subset of patients with highest likelihood of recurrence across the patient cohort. These patients may benefit from additional alternative therapies targeting CKD9.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Identification of novel CDK 9 inhibitors based on virtual screening, molecular dynamics simulation, and biological evaluation.

AIMS: Cyclin-dependent kinase 9 (CDK9) is a member of the CDK subfamily and plays a major role in the regulation of transcriptional elongation. It has attracted widespread attention as a therapeutic target for cancer. Here, we aimed to explore novel CDK 9 inhibitors by using a hybrid virtual screening strategy. MAIN METHODS: A hybrid virtual screening strategy was constructed with computer-aided drug design (CADD). First, compounds were filtered in accordance with Lipinski's rule of five and adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Second, a 3D-QSAR pharmacophore model was built and used as a 3D query to screen the obtained hit compounds. Third, the hit compounds were subjected to molecular docking studies. Fourth, molecular dynamics (MD) simulations were performed on CDK9 in complex with the final hits to examine the structural stability. Finally, CDK9 kinase biochemical assay was performed to identify the biological activity of the hit compounds. KEY FINDINGS: Seven hit compounds were screened out. These hit compounds showed drug-like properties in accordance with Lipinski's rule of five and ADMET. Complexes involving the six hit compounds bound to CDK9 exhibited good structural stability in the MD simulation. Furthermore, these six hit compounds had strong inhibitory activity against CDK9 kinase. In particular, hit 3 showed the most promising activity with the percentage of 71%. SIGNIFICANCE: The six hit compounds may be promising novel CDK9 inhibitors, and the hybrid virtual screening strategy designed in this study provides an important reference for the design and synthesis of novel CDK9 inhibitors.

CDK9 inhibitors in acute myeloid leukemia.

Current treatment for acute myeloid leukemia (AML) is less than optimal, but increased understanding of disease pathobiology and genomics has led to clinical investigation of novel targeted therapies and rational combinations. Targeting the cyclin-dependent kinase 9 (CDK9) pathway, which is dysregulated in AML, is an attractive approach. Inhibition of CDK9 leads to downregulation of cell survival genes regulated by super enhancers such as MCL-1, MYC, and cyclin D1. As CDK9 inhibitors are nonselective, predictive biomarkers that may help identify patients most likely to respond to CDK9 inhibitors are now being utilized, with the goal of improving efficacy and safety.

Identification of Atuveciclib (BAY†1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer.

Selective inhibition of exclusively transcription-regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY-958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer.

Discovery and SAR of novel pyrazolo[1,5-a]pyrimidines as inhibitors of CDK9.

The serine-threonine kinase CDK9 is a target of emerging interest for the development of anti-cancer drugs. There are multiple lines of evidence linking CDK9 activity to cancer, including the essential role this kinase plays in transcriptional regulation through phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Indeed, inhibition of CDK9 has been shown to result in a reduction of short-lived proteins such as the pro-survival protein Mcl-1 in malignant cells leading to the induction of apoptosis. In this work we report our initial studies towards the discovery of selective CDK9 inhibitors, starting from the known multi-kinase inhibitor PIK-75 which possesses potent CDK9 activity. Our series is based on a pyrazolo[1,5-a]pyrimidine nucleus and, importantly, the resultant lead compound 18b is devoid of the structural liabilities present in PIK-75 and possesses greater selectivity.

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study.

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg12+). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg12+ ion, coupled with the Mg22+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg12+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg12+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg12+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

Efficacy of cyclin-dependent-kinase 9 inhibitors in a murine model of mixed-lineage leukemia.

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1.

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Protective effect of berberine on antioxidant enzymes and positive transcription elongation factor b expression in diabetic rat liver.

The protective effect of berberine against antioxidant, antilipid peroxidation in serum and liver tissue, and positive transcription elongation factor b (P-TEFb) expression in liver tissue of type 2 diabetic rats was investigated. Overnight fasted rats were intraperitoneally injected 35 mg/kg streptozotocin. Diabetic rats were admitted after 2 weeks and given a high-carbohydrate/high-fat diet to induce hyperlipidemias. From week 16, diabetic rats were treated with 75, 150, 300 mg/kg berberine, 100mg/kg fenofibrate or 4 mg/kg rosiglitazone for another 16 weeks. P-TEFb (composed of cyclin-dependent kinase 9 and cyclin T1) mRNA and protein expression in liver tissue were detected by real time PCR and immunohistochemistry, respectively. Berberine significantly up-regulated the declined cyclin-dependent kinase 9, cyclin T1 mRNA and protein expression in diabetic rat liver. Berberine obviously decreased malondialdehyde level and increased catalase, superoxide dismutase, glutathione peroxidase, and glutathione activities in liver tissue and serum of diabetic rats. These results suggest that the effects of berberine on up-regulation of P-TEFb expression, antioxidant and antilipid peroxidation may be related to its protective potential on diabetes.

Crystal structure of HIV-1 Tat complexed with human P-TEFb.

Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat.P-TEFb complex containing HIV-1 Tat, human Cdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat.P-TEFb complex and block HIV replication.

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4+ T cells.

BACKGROUND: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. RESULTS: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. CONCLUSION: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4+ T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively.

Pkn is a novel partner of cyclin T2a in muscle differentiation.

With the aim to find novel partners of human Cyclin T2a, we performed a two-hybrid screening in yeast using the full-length cDNA of this cyclin as bait, and a human heart cDNA library as preys source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKNalpha, a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co-immunoprecipitation and in vitro pull-down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD-responsive promoter, pointed out that PKNalpha was able to enhance MyoD-dependent transcription, and that this effect was further increased when cyclin T2a was co-overexpressed. Finally, overexpression of both Cyclin T2a and PKNalpha in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation-induced differentiation. Taken together, our data strengthen the hypothesis that Cyclin T2a plays a role in muscle differentiation, and propose PKNalpha as a novel partner of Cyclin T2a in this process.