Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9.

RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.

Retinoic acid enhances HIV-1 reverse transcription and transcription in macrophages via mTOR-modulated mechanisms.

The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4+ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4+ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.

Transcriptional regulation and therapeutic potential of cyclin-dependent kinase 9 (CDK9) in sarcoma.

Sarcomas include various subtypes comprising two significant groups - soft tissue and bone sarcomas. Although the survival rate for some sarcoma subtypes has improved over time, the current methods of treatment remain efficaciously limited, as recurrent, and metastatic diseases remain a major obstacle. There is a need for better options and therapeutic strategies in treating sarcoma. Cyclin dependent kinase 9 (CDK9) is a transcriptional kinase and has emerged as a promising target for treating various cancers. The aberrant expression and activation of CDK9 have been observed in several sarcoma subtypes, including rhabdomyosarcoma, synovial sarcoma, osteosarcoma, Ewing sarcoma, and chordoma. Enhanced CDK9 expression has also been correlated with poorer prognosis in sarcoma patients. As a master regulator of transcription, CDK9 promotes transcription elongation by phosphorylation and releasing RNA polymerase II (RNAPII) from its promoter proximal pause. Release of RNAPII from this pause induces transcription of critical genes in the tumor cell. Overexpression and activation of CDK9 have been observed to lead to the expression of oncogenes, including MYC and MCL-1, that aid sarcoma development and progression. Inhibition of CDK9 in sarcoma has been proven to reduce these oncogenes' expression and decrease proliferation and growth in different sarcoma cells. Currently, there are several CDK9 inhibitors in preclinical and clinical investigations. This review aims to highlight the recent discovery and results on the transcriptional role and therapeutic potential of CDK9 in sarcoma.

The CDK9 inhibitor enitociclib overcomes resistance to BTK inhibition and CAR-T therapy in mantle cell lymphoma.

Inhibitors of Bruton's tyrosine kinase (BTKi) and chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 are paradigm-shifting advances in treating patients with aggressive mantle cell lymphoma (MCL). However, clinical relapses following BTKi and CD19-directed CAR-T treatments are a fast-growing medical challenge. Development of novel therapies to overcome BTKi resistance (BTKi-R) and BTKi-CAR-T dual resistance (Dual-R) are urgently needed. Our single-cell RNA sequencing data revealed major transcriptomic reprogramming, with great enrichment of MYC-targets evolving as resistance to these therapies developed. Interestingly, cyclin-dependent kinase 9 (CDK9), a critical component of the positive transcription elongation factor-b complex, was among the top upregulated genes in Dual-R vs. BTKi-R samples. We therefore hypothesized that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. Enitociclib (formerly VIP152) is a selective CDK9 inhibitor whose potency against MCL has not been assessed. In this study, we found that enitociclib was highly potent in targeting lymphoma cells, with the half-maximal inhibitory concentration (IC50) ranging from 32 to 172 nM in MCL and diffuse large B-cell lymphoma cell lines. It inhibited CDK9 phosphorylation and downstream events including de novo synthesis of the short-lived proteins c-MYC, MCL-1, and cyclin D1, and induced apoptosis in a caspase-3-dependent manner. Enitociclib potently inhibited in vivo tumor growth of cell line-derived and patient-derived xenografts having therapeutic resistance. Our data demonstrate the potency of enitociclib in overcoming therapeutic resistance in MCL models and provide evidence in favor of its clinical investigation.

Discovery of novel CDK9 inhibitor with tridentate ligand: Design, synthesis and biological evaluation.

Cyclin-dependent kinase 9 (CDK9) plays a role in transcriptional regulation, which had become an attractive target for discovery of antitumor agent. In this work, beyond traditional CDK9 inhibitor with bidentate ligands in ATP binding domain, a series of novel CDK9 inhibitor with tridentate ligand were designed and synthesized. Surprisingly, this unique tridentate ligand structure endows better CDK9 inhibition selectivity compared to other CDK subtypes, and the lead candidate compound Z4-7a showed effective proliferation inhibition in HCT116 cells with acceptable pharmacokinetic properties. Research on the mechanism indicated that Z4-7a could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression. In brief, introduction of tridentate ligand might work as a promising strategy for the development of novel selective CDK9 inhibitor.

Focus on current and emerging treatment options for glioma: A comprehensive review.

This comprehensive review delves into the current updates and challenges associated with the management of low-grade gliomas (LGG), the predominant primary tumors in the central nervous system. With a general incidence rate of 5.81 per 100000, gliomas pose a significant global concern, necessitating advancements in treatment techniques to reduce mortality and morbidity. This review places a particular focus on immunotherapies, discussing promising agents such as Zotiraciclib and Lerapolturev. Zotiraciclib, a CDK9 inhibitor, has demonstrated efficacy in glioblastoma treatment in preclinical and clinical studies, showing its potential as a therapeutic breakthrough. Lerapolturev, a viral immunotherapy, induces inflammation in glioblastoma and displays positive outcomes in both adult and pediatric patients. Exploration of immunotherapy extends to Pembrolizumab, Nivolumab, and Entrectinib, revealing the challenges and variabilities in patient responses. Despite promising preclinical data, the monoclonal antibody Depatuxizumab has proven ineffective in glioblastoma treatment, emphasizing the critical need to understand resistance mechanisms. The review also covers the success of radiation therapy in pediatric LGG, with evolving techniques, such as proton therapy, showing potential improvements in patient quality of life. Surgical treatment is discussed in the context of achieving a balance between preserving the patient's quality of life and attaining gross total resection, with the extent of surgical resection significantly influencing the survival outcomes. In addition to advancements in cancer vaccine development, this review highlights the evolving landscape of LGG treatment, emphasizing a shift toward personalized and targeted therapies. Ongoing research is essential for refining strategies and enhancing outcomes in the management of LGG.

CDK inhibition results in pharmacologic BRCAness increasing sensitivity to olaparib in BRCA1-WT and olaparib resistant in Triple Negative Breast Cancer.

One in three Triple Negative Breast Cancer (TNBC) is Homologous Recombination Deficient (HRD) and susceptible to respond to PARP inhibitor (PARPi), however, resistance resulting from functional HR restoration is frequent. Thus, pharmacologic approaches that induce HRD are of interest. We investigated the effectiveness of CDK-inhibition to induce HRD and increase PARPi sensitivity of TNBC cell lines and PDX models. Two CDK-inhibitors (CDKi), the broad range dinaciclib and the CDK12-specific SR-4835, strongly reduced the expression of key HR genes and impaired HR functionality, as illustrated by BRCA1 and RAD51 nuclear foci obliteration. Consequently, both CDKis showed synergism with olaparib, as well as with cisplatin and gemcitabine, in a range of TNBC cell lines and particularly in olaparib-resistant models. In vivo assays on PDX validated the efficacy of dinaciclib which increased the sensitivity to olaparib of 5/6 models, including two olaparib-resistant and one BRCA1-WT model. However, no olaparib response improvement was observed in vivo with SR-4835. These data support that the implementation of CDK-inhibitors could be effective to sensitize TNBC to olaparib as well as possibly to cisplatin or gemcitabine.

TAT38 and TAT38 mimics potently inhibit adipogenesis by repressing C/EBPα, PPARγ, Pi-PPARγ, and SREBP1 expression.

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20-57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20-35 TACTNCYCAKCCFQVC), core-domain (a.a. 36-46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47-57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4-11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.

Cartilage stem/progenitor cells-derived exosomes facilitate knee cartilage repair in a subacute osteoarthritis rat model.

Cartilage defects in the knee are often associated with the progression of degenerative osteoarthritis (OA), and cartilage repair is a useful strategy for managing this disease. However, cartilage repair is challenging because of the unique environment within the tissue. Recently, stem cell-based therapies have shed new light on this issue. In this study, we prepared exosomes (EXOs) from cartilage stem/progenitor cells (CSPCs) and found that treatment with EXOs increased the viability, migration, and proliferation of cultured primary chondrocytes. In a subacute OA rat model, the application of EXOs facilitated cartilage regeneration as evidenced by histological staining. Exosomal protein analysis together with bioinformatics suggested that cyclin-dependent kinase 9 (CDK9) is a key factor for chondrocyte growth and migration. Functional studies confirmed this prediction, that is, inhibiting CDK9 reduced the beneficial effects induced by EXOs in primary chondrocytes; while overexpression of CDK9 recapitulated the EXOs-induced phenotypes. RNA-Seq data showed that a set of genes involved in cell growth and migration were up-regulated by EXOs in chondrocytes. These changes could be partially reproduced by CDK9 overexpression. Overall, our data suggest that EXOs derived from primary CSPCs hold great therapeutic potential for treating cartilage defect-associated disorders such as degenerative OA, and that CDK9 is a key factor in this process.

DNMT1-mediated epigenetic suppression of FBXO32 expression promoting cyclin dependent kinase 9 (CDK9) survival and esophageal cancer cell growth.

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.

LncRNA lncMGR regulates skeletal muscle development and regeneration by recruiting CDK9 and sponging miRNAs.

Long non-coding RNAs (lncRNAs) play an essential role in vertebrate myogenesis and muscle diseases. However, the dynamic expression patterns, biological functions, and mechanisms of lncRNAs in skeletal muscle development and regeneration remain largely unknown. In this study, a novel lncRNA (named lncMGR) was differentially expressed during breast muscle development in fast- and slow-growing chickens. Functionally, lncMGR promoted myoblast differentiation, inhibited myoblast proliferation in vitro, and promoted myofiber hypertrophy and injury repair in vivo. Mechanistically, lncMGR increased the mRNA and protein expression of skeletal muscle myosin heavy chain 1 A (MYH1A) via both transcriptional and post-transcriptional regulation. Nuclear lncMGR recruited cyclin-dependent kinase 9 (CDK9) to the core transcriptional activation region of the MYH1A gene to activate MYH1A transcription. Cytoplasmic lncMGR served as a competitive endogenous RNA (ceRNA) to competitively absorb miR-2131-5p away from MYH1A and subsequently protected the MYH1A from miR-2131-5p-mediated degradation. Besides miR-2131-5p, cytoplasmic lncMGR could also sponge miR-143-3p to reconcile the antagonist between the miR-2131-5p/MYH1A-mediated inhibition effects and miR-143-3p-mediated promotion effects on myoblast proliferation, thereby inhibiting myoblast proliferation. Collectively, lncMGR could recruit CDK9 and sponge multiple miRNAs to regulate skeletal muscle development and regeneration, and could be a therapeutic target for muscle diseases.

Targeting CDK9 in Cancer: An Integrated Approach of Combining In Silico Screening with Experimental Validation for Novel Degraders.

The persistent threat of cancer remains a significant hurdle for global health, prompting the exploration of innovative approaches in the quest for successful therapeutic interventions. Cyclin-dependent kinase 9 (CDK9), a central player in transcription regulation and cell cycle progression, has emerged as a promising target to combat cancer. Its pivotal role in oncogenic pathways and the pressing need for novel cancer treatments has propelled CDK9 into the spotlight of drug discovery efforts. This article presents a comprehensive study that connects a multidisciplinary approach, combining computational methodologies, experimental validation, and the transformative Proteolysis-Targeting Chimera (PROTAC) technology. By uniting these diverse techniques, we aim to identify, characterize, and optimize a new class of degraders targeting CDK9. We explore these compounds for targeted protein degradation, offering a novel and potentially effective approach to cancer therapy. This cohesive strategy utilizes the combination of computational predictions and experimental insights, with the goal of advancing the development of effective anticancer therapeutics, targeting CDK9.

RNF20 contributes to epigenetic immunosuppression through CDK9-dependent LSD1 stabilization.

Cyclin-dependent kinase 9 (CDK9) plays a critical role in transcription initiation and is essential for maintaining gene silencing at heterochromatic loci. Inhibition of CDK9 increases sensitivity to immunotherapy, but the underlying mechanism remains unclear. We now report that RNF20 stabilizes LSD1 via K29-mediated ubiquitination, which is dependent on CDK9-mediated phosphorylation. This CDK9- and RNF20-dependent LSD1 stabilization is necessary for the demethylation of histone H3K4, then subsequent repression of endogenous retrovirus, and an interferon response, leading to epigenetic immunosuppression. Moreover, we found that loss of RNF20 sensitizes cancer cells to the immune checkpoint inhibitor anti-PD-1 in vivo and that this effect can be rescued by the expression of ectopic LSD1. Our findings are supported by the observation that RNF20 levels correlate with LSD1 levels in human breast cancer specimens. This study sheds light on the role of RNF20 in CDK9-dependent LSD1 stabilization, which is crucial for epigenetic silencing and immunosuppression. Our findings explore the potential importance of targeting the CDK9-RNF20-LSD1 axis in the development of new cancer therapies.

PBRM1 loss is associated with increased sensitivity to MCL1 and CDK9 inhibition in clear cell renal cancer.

MCL1 is a member of the BCL2 family of apoptosis regulators, which play a critical role in promoting cancer survival and drug resistance. We previously described PRT1419, a potent, MCL1 inhibitor with anti-tumor efficacy in various solid and hematologic malignancies. To identify novel biomarkers that predict sensitivity to MCL1 inhibition, we conducted a gene essentiality analysis using gene dependency data generated from CRISPR/Cas9 cell viability screens. We observed that clear cell renal cancer (ccRCC) cell lines with damaging PBRM1 mutations displayed a strong dependency on MCL1. PBRM1 (BAF180), is a chromatin-targeting subunit of mammalian pBAF complexes. PBRM1 is frequently altered in various cancers particularly ccRCC with ~40% of tumors harboring damaging PBRM1 alterations. We observed potent inhibition of tumor growth and induction of apoptosis by PRT1419 in various preclinical models of PBRM1-mutant ccRCC but not PBRM1-WT. Depletion of PBRM1 in PBRM1-WT ccRCC cell lines induced sensitivity to PRT1419. Mechanistically, PBRM1 depletion coincided with increased expression of pro-apoptotic factors, priming cells for caspase-mediated apoptosis following MCL1 inhibition. Increased MCL1 activity has been described as a resistance mechanism to Sunitinib and Everolimus, two approved agents for ccRCC. PRT1419 synergized with both agents to potently inhibit tumor growth in PBRM1-loss ccRCC. PRT2527, a potent CDK9 inhibitor which depletes MCL1, was similarly efficacious in monotherapy and in combination with Sunitinib in PBRM1-loss cells. Taken together, these findings suggest PBRM1 loss is associated with MCL1i sensitivity in ccRCC and provide rationale for the evaluation of PRT1419 and PRT2527 for the treatment for PBRM1-deficient ccRCC.

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb.

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9, and is not involved with releasing positive transcription elongation factor b from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

Small molecule inhibitors of transcriptional cyclin-dependent kinases impose HIV-1 latency, presenting "block and lock" treatment strategies.

Current antiretroviral therapy for HIV-1 infection does not represent a cure for infection as viral rebound inevitably occurs following discontinuation of treatment. The "block and lock" therapeutic strategy is intended to enforce proviral latency and durably suppress viremic reemergence in the absence of other intervention. The transcription-associated cyclin-dependent protein kinases (tCDKs) are required for expression from the 5´ HIV-1 long-terminal repeat, but the therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency has not been characterized. Here, we expanded previous observations to directly compare the effect of highly selective small molecule inhibitors of CDK7 (YKL-5-124), CDK9 (LDC000067), and CDK8/19 (Senexin A), and found each of these prevented HIV-1 provirus expression at concentrations that did not cause cell toxicity. Inhibition of CDK7 caused cell cycle arrest, whereas CDK9 and CDK8/19 inhibitors did not, and could be continuously administered to establish proviral latency. Upon discontinuation of drug administration, HIV immediately rebounded in cells that had been treated with the CDK9 inhibitor, while proviral latency persisted for several days in cells that had been treated with CDK8/19 inhibitors. These results identify the mediator kinases CDK8/CDK19 as potential "block and lock" targets for therapeutic suppression of HIV-1 provirus expression.

MC180295 is a highly potent and selective CDK9 inhibitor with preclinical in vitro and in vivo efficacy in cancer.

BACKGROUND: Inhibition of cyclin-dependent kinase 9 (CDK9), a novel epigenetic target in cancer, can reactivate epigenetically silenced genes in cancer by dephosphorylating the SWI/SNF chromatin remodeler BRG1. Here, we characterized the anti-tumor efficacy of MC180295, a newly developed CDK9 inhibitor. METHODS: In this study, we explored the pharmacokinetics of MC180295 in mice and rats, and tested the anti-tumor efficacy of MC180295, and its enantiomers, in multiple cancer cell lines and mouse models. We also combined CDK9 inhibition with a DNA methyltransferase (DNMT) inhibitor, decitabine, in multiple mouse models, and tested MC180295 dependence on T cells. Drug toxicity was measured by checking body weights and complete blood counts. RESULTS: MC180295 had high specificity for CDK9 and high potency against multiple neoplastic cell lines (median IC50 of 171 nM in 46 cell lines representing 6 different malignancies), with the highest potency seen in AML cell lines derived from patients with MLL translocations. MC180295 is a racemic mixture of two enantiomers, MC180379 and MC180380, with MC180380 showing higher potency in a live-cell epigenetic assay. Both MC180295 and MC180380 showed efficacy in in vivo AML and colon cancer xenograft models, and significant synergy with decitabine in both cancer models. Lastly, we found that CDK9 inhibition-mediated anti-tumoral effects were partially dependent on CD8 + T cells in vivo, indicating a significant immune component to the response. CONCLUSIONS: MC180380, an inhibitor of cyclin-dependent kinase 9 (CDK9), is an efficacious anti-cancer agent worth advancing further toward clinical use.

Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.

GFH009 is a potent, highly selective, small molecule that targets and inhibits the activity of the CDK9/cyclin T1 regulatory complex of P-TEFb. This study aimed to develop and validate a highly selective and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for precise quantification of GFH009 in rat plasma. This method was subsequently employed for conducting toxicokinetic studies of GFH009 in rats. Plasma was prepared using a simple protein precipitation method by acetonitrile. Chromatographic separation of the analytes was achieved on a BEH C18 analytical column with a rapid 3.0 min run time and a flow rate of 0.5 ml/min. The calibration curves for plasma samples exhibited excellent linearity over a wide concentration range of 1.0-1,000 ng/ml for GFH009. Intra- and inter-day accuracies were within 92.7-105.7%, and precisions were no more than 6.7%. Furthermore, the analyte demonstrated stability under four different storage conditions, with variations of <15.0%. This study pioneers a methodological innovation by introducing a highly reliable, specific and sensitive analytical method for GFH009 in rat plasma. The successful application of this method in toxicokinetic studies further underscores its significance, offering valuable insights for the methodology of clinical pharmacokinetic research.

CDK7 and CDK9 inhibition interferes with transcription, translation, and stemness, and induces cytotoxicity in GBM irrespective of temozolomide sensitivity.

BACKGROUND: Glioblastoma (GBM) is refractory to current treatment modalities while side effects of treatments result in neurotoxicity and cognitive impairment. Here we test the hypothesis that inhibiting CDK7 or CDK9 would effectively combat GBM with reduced neurotoxicity. METHODS: We examined the effect of a CDK7 inhibitor, THZ1, and multiple CDK9 inhibitors (SNS032, AZD4573, NVP2, and JSH150) on GBM cell lines, patient-derived temozolomide (TMZ)-resistant and responsive primary tumor cells and glioma stem cells (GSCs). Biochemical changes were assessed by western blotting, immunofluorescence, multispectral imaging, and RT-PCR. In vivo, efficacy was assessed in orthotopic and subcutaneous xenograft models. RESULTS: CDK7 and CDK9 inhibitors suppressed the viability of TMZ-responsive and resistant GBM cells and GSCs at low nanomolar concentrations, with limited cytotoxic effects in vivo. The inhibitors abrogated RNA Pol II and p70S6K phosphorylation and nascent protein synthesis. Furthermore, the self-renewal of GSCs was significantly reduced with a corresponding reduction in Sox2 and Sox9 levels. Analysis of TCGA data showed increased expression of CDK7, CDK9, SOX2, SOX9, and RPS6KB1 in GBM; supporting this, multispectral imaging of a TMA revealed increased levels of CDK9, Sox2, Sox9, phospho-S6, and phospho-p70S6K in GBM compared to normal brains. RNA-Seq results suggested that inhibitors suppressed tumor-promoting genes while inducing tumor-suppressive genes. Furthermore, the studies conducted on subcutaneous and orthotopic GBM tumor xenograft models showed that administration of CDK9 inhibitors markedly suppressed tumor growth in vivo. CONCLUSIONS: Our results suggest that CDK7 and CDK9 targeted therapies may be effective against TMZ-sensitive and resistant GBM.

Integrated computational approaches for designing potent pyrimidine-based CDK9 inhibitors: 3D-QSAR, docking, and molecular dynamics simulations.

CDK9 is an emerging target for the development of anticancer drugs. The development of CDK9 inhibitors with significant potency had consistently posed a formidable challenge. In the current research, a number of computational methodologies, such as, 3D-QSAR, molecular docking, fingerprint analysis, molecular dynamic (MD) simulations followed by MMGB/PBSA and ADMET studies were used systemically to uncover the binding mechanism of pyrimidine derivatives against CDK9. The CoMFA and CoMSIA models having high q2 (0.53, 0.54) and r2 values (0.96, 0.93) respectively indicating that model could accurately predict the bioactivities of CDK9 inhibitors. Using the R-group exploration technique implemented by the Spark™ by Cresset group, the structural requirements revealed by the contour maps of model were utilized strategically to create an in-house library of 100 new CDK9 inhibitors. Additionally, the compounds from the in-house library were mapped into 3D-QSAR model which predicted pIC50 values comparable to the experimental values. A comparison between 3D-QSAR generated contours and molecular docking conformation of ligands was performed to elucidate the essentials of CDK9 inhibitor design. MD simulations (100 ns) were performed on the selected docked complexes A21, A14 and D98 which contributed in validating the binding interactions. According to the findings of binding free energy analysis (MMGB/PBSA), It was observed that residues CYS106 and GLU107 had a considerable tendency to facilitate ligand-protein interactions via H-bond interactions. The aforementioned findings have the potential to enhance researchers comprehension of the mechanism underlying CDK9 inhibition and may be utilized in the development of innovative and efficacious CDK9 inhibitors.