CDK9 inhibition as an effective therapy for small cell lung cancer.

Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

Transcriptional elongation control of hypoxic response.

The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.

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.

Design, synthesis, and biological evaluation of dinaciclib and CAN508 hybrids as CDK inhibitors.

The scaffolds of two known CDK inhibitors (CAN508 and dinaciclib) were the starting point for synthesizing two series of pyarazolo[1,5-a]pyrimidines to obtain potent inhibitors with proper selectivity. The study presented four promising compounds; 10d, 10e, 16a, and 16c based on cytotoxic studies. Compound 16a revealed superior activity in the preliminary anticancer screening with GI % = 79.02-99.13 against 15 cancer cell lines at 10 µM from NCI full panel 60 cancer cell lines and was then selected for further investigation. Furthermore, the four compounds revealed good safety profile toward the normal cell lines WI-38. These four compounds were subjected to CDK inhibitory activity against four different isoforms. All of them showed potent inhibition against CDK5/P25 and CDK9/CYCLINT. Compound 10d revealed the best activity against CDK5/P25 (IC50 = 0.063 µM) with proper selectivity index against CDK1 and CDK2. Compound 16c exhibited the highest inhibitory activity against CDK9/CYCLINT (IC50 = 0.074 µM) with good selectivity index against other isoforms. Finally, docking simulations were performed for compounds 10e and 16c accompanied by molecular dynamic simulations to understand their behavior in the active site of the two CDKs with respect to both CAN508 and dinaciclib.

Discovery of novel co-degradation CK1α and CDK7/9 PROTACs with p53 activation for treating acute myeloid leukemia.

Reactivating p53 activity to restore its anticancer function is an attractive cancer treatment strategy. In this study, we designed and synthesized a series of novel PROTACs to reactivate p53 via the co-degradation of CK1α and CDK7/9 proteins. Bioactivity studies showed that the selected PROTAC 13i exhibited potency antiproliferative activity in MV4-11 (IC50 = 0.096 ± 0.012 µM) and MOLM-13 (IC50 = 0.072 ± 0.014 µM) cells, and induced apoptosis of MV4-11 cells. Western-blot analysis showed that PROTAC 13i triple CK1α and CDK7/9 protein degradation resulted in the significantly increased expression of p53. At the same time, the transcriptional repression due to the degradation significantly reduced downstream gene expression of MYC, MDM2, BCL-2 and MCL-1, and reduced the inflammatory cytokine levels of TNF-α, IL-1ß and IL-6 in PMBCs. These results indicate the beneficial impact of simultaneous CK1α and CDK7/9 degradation for acute myeloid leukemia therapy.

CDK9 inhibition activates innate immune response through viral mimicry.

Cancer cells frequently exhibit hyperactivation of transcription, which can lead to increased sensitivity to compounds targeting the transcriptional kinases, in particular CDK9. However, mechanistic details of CDK9 inhibition-induced cancer cell-selective anti-proliferative effects remain largely unknown. Here, we discover that CDK9 inhibition activates the innate immune response through viral mimicry in cancer cells. In MYC over-expressing prostate cancer cells, CDK9 inhibition leads to the gross accumulation of mis-spliced RNA. Double-stranded RNA (dsRNA)-activated kinase can recognize these mis-spliced RNAs, and we show that the activity of this kinase is required for the CDK9 inhibitor-induced anti-proliferative effects. Using time-resolved transcriptional profiling (SLAM-seq), targeted proteomics, and ChIP-seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of cytokines related to the innate immune response. In particular, CDK9 inhibition activates NFκB-driven cytokine signaling at the transcriptional and secretome levels. The transcriptional signature induced by CDK9 inhibition identifies prostate cancers with a high level of genome instability. We propose that it is possible to induce similar effects in patients using CDK9 inhibition, which, we show, causes DNA damage in vitro. In the future, it is important to establish whether CDK9 inhibitors can potentiate the effects of immunotherapy against late-stage prostate cancer, a currently lethal disease.

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.

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.

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.

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.

Long noncoding RNA small nucleolar RNA host gene 5 facilitates neuropathic pain in spinal nerve injury by promoting SCN9A expression via CDK9.

This study aims to explore the functions and mechanisms of long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) in chronic constriction injury (CCI)-induced neuropathic pain (NP). An NP rat model was established using the CCI method and the NP severity was evaluated by paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). The expression of SNHG5, CDK9, and SCN9A was quantified in rat dorsal root ganglion, in addition to the detections of apoptosis, pathological changes, neuron number, and the co-localization of Nav1.7 and cleaved caspase-3 with NeuN. In ND7/23 cells, the apoptosis and lactate dehydrogenase concentration were assessed, as well as the relationship between SNHG5, CDK9, and SCN9A. In the dorsal root ganglion of CCI-treated rats, SNHG5 and SCN9A were upregulated and downregulation of SNHG5 suppressed SCN9A expression, increased the PWT and PWL, blocked neuroinflammation and neuronal apoptosis, and alleviated NP. Mechanistically, SNHG5 recruited CDK9 to enhance SCN9A-encoded Nav1.7 expression and promoted peripheral neuronal apoptosis and injury. In addition, SCN9A overexpression nullified the alleviative effects of SNHG5 deficiency on NP and neuron loss in CCI rats. In conclusion, SNHG5 promotes SCN9A-encoded Nav1.7 expression by recruiting CDK9, thereby facilitating neuron loss and NP after spinal nerve injury, which may offer a promising target for the management of NP.

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.

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.

Design, synthesis, and biological evaluation of novel 4,4'-bipyridine derivatives acting as CDK9-Cyclin T1 protein-protein interaction inhibitors against triple-negative breast cancer.

Cyclin-dependent kinase 9 (CDK9) is directly related to tumor development in triple-negative breast cancer (TNBC) patients. Increased CDK9 is significantly associated with poor patient prognosis, while inhibiting CDK9-Cyclin T1 protein-protein interaction has recently been demonstrated as a new approach to TNBC treatment. Herein, we synthesized a novel class of 4,4'-bipyridine derivatives as potential CDK9-Cyclin T1 PPI inhibitors against TNBC. The represented compound B19 was found to be an excellent and selective CDK9-Cyclin T1 PPI inhibitor with good potency against TNBC cell lines while exhibiting lower toxicity in normal human cell lines than the positive compound I-CDK9. Notably, compound B19 showed good pharmacokinetic properties and excellent antitumor activity against TNBC (4T1) allografts in mice with a therapeutic index of more than 42 (TGI4T1(12.5 mg/kg,i.p.) = 63.1% vs. LD50 = 537 mg/kg). Moreover, the administration of B19 in combination with the PARP inhibitor Olaparib results in a significant increase of the antitumor activity in MDA-MB-231 cells relative to that of either single agent. To our knowledge, B19 is the first reported non-metal organic compound that acts as a selective CDK9-Cyclin T1 PPI inhibitor with in vivo antitumor activity, and it may be alone and in combination with PARP inhibitor Olaparib for TNBC therapy.

Degradation of CDK9 by Ubiquitin E3 Ligase STUB1 Regulates P-TEFb Level and Its Functions for Global Target Gene Expression within Mammalian Cells.

Positive transcription elongation factor b (P-TEFb) regulates expression of diverse sets of genes within mammalian cells that have implications in several human disease pathogeneses. However, mechanisms of functional regulation of P-TEFb complex through regulation of its stability are poorly known. In this study, we show an important role of C-terminus of Hsc70-interacting protein (CHIP aka STUB1) in regulation of overall level of CDK9 and thus P-TEFb complex within mammalian cells. STUB1 acts as a ubiquitin E3 ligase for proteasomal degradation of CDK9 involving N-terminal lysine 3 (K3) residue. Whereas, overexpression of STUB1 enhances, its knockdown reduces overall CDK9 degradation kinetics within mammalian cells. Interestingly, owing to the same region of binding within CDK9, CyclinT1 protects CDK9 from STUB1-mediated degradation. Factors that cooperatively bind with CyclinT1 to form functional complex also protects CDK9 from degradation by STUB1. Knockdown of STUB1 enhances CDK9 expression and thus P-TEFb complex formation that leads to global increase in RNA polymerase II CTD phosphorylation and transcriptional activation of diverse P-TEFb target genes. Thus, we describe an important functional role of STUB1 in regulation of transcription through modulation of overall level of P-TEFb complex formation within mammalian cells.

Design and optimization of selective and potent CDK9 inhibitors with flavonoid scaffold for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a prevalent hematological tumor associated with a high morbidity and mortality rate. CDK9, functioning as a pivotal transcriptional regulator, facilitates transcriptional elongation through phosphorylation of RNA polymerase II, which further governs the protein levels of Mcl-1 and c-Myc. Therefore, CDK9 has been considered as a promising therapeutic target for AML treatment. Here, we present the design, synthesis, and evaluation of CDK9 inhibitors bearing a flavonoid scaffold. Among them, compound 21a emerged as a highly selective CDK9 inhibitor (IC50 = 6.7 nM), exhibiting over 80-fold selectivity towards most other CDK family members and high kinase selectivity. In Mv4-11 cells, 21a effectively hindered cell proliferation (IC50 = 60 nM) and induced apoptosis by down-regulating Mcl-1 and c-Myc. Notably, 21a demonstrated significant inhibition of tumor growth in the Mv4-11 xenograft tumor model. These findings indicate that compound 21a holds promise as a potential candidate for treating AML.

Identification of a diketopiperazine-based O-GlcNAc transferase inhibitor sensitizing hepatocellular carcinoma to CDK9 inhibition.

O-GlcNAcylation (O-linked ß-N-acetylglucosaminylation) is an important post-translational and metabolic process in cells that is implicated in a wide range of physiological processes. O-GlcNAc transferase (OGT) is ubiquitously present in cells and is the only enzyme that catalyses the transfer of O-GlcNAc to nucleocytoplasmic proteins. Aberrant glycosylation by OGT has been linked to a variety of diseases including cancer, neurodegenerative disorders and diabetes. Previously, we and others demonstrated that O-GlcNAcylation is notably elevated in hepatocellular carcinoma (HCC). The overexpression of O-GlcNAcylation promotes cancer progression and metastasis. Here, we report the identification of HLY838, a novel diketopiperazine-based OGT inhibitor with the ability to induce a global decrease in cellular O-GlcNAc. HLY838 enhances the in vitro and in vivo anti-HCC activity of CDK9 inhibitor by downregulating c-Myc and downstream E2F1 expression. Mechanistically, c-Myc is regulated by the CDK9 at the transcript level, and stabilized by OGT at the protein level. This work therefore demonstrates that HLY838 potentiates the antitumor responses of CDK9 inhibitor, providing an experimental rationale for developing OGT inhibitor as a sensitizing agent in cancer therapeutics.

A patent review of selective CDK9 inhibitors in treating cancer.

INTRODUCTION: The dysregulation of CDK9 protein is greatly related to the proliferation and differentiation of various cancers due to its key role in the regulation of RNA transcription. Moreover, CDK9 inhibition can markedly downregulate the anti-apoptotic protein Mcl-1 which is essential for the survival of tumors. Thus, targeting CDK9 is considered to be a promising strategy for antitumor drug development, and the development of selective CDK9 inhibitors has gained increasing attention. AREAS COVERED: This review focuses on the development of selective CDK9 inhibitors reported in patent publications during the period 2020-2022, which were searched from SciFinder and Cortellis Drug Discovery Intelligence. EXPERT OPINION: Given that pan-CDK9 inhibitors may lead to serious side effects due to poor selectivity, the investigation of selective CDK9 inhibitors has attracted widespread attention. CDK9 inhibitors make some advance in treating solid tumors and possess the therapeutic potential in EGFR-mutant lung cancer. CDK9 inhibitors with short half-life and intravenous administration might result in transient target engagement and contribute to a better safety profile in vivo. However, more efforts are urgently needed to accelerate the development of CDK9 inhibitors, including the research on new binding modes between ligand and receptor or new protein binding sites.

Bifunctional degraders of cyclin dependent kinase 9 (CDK9): Probing the relationship between linker length, properties, and selective protein degradation.

Cyclin-dependent kinase 9 (CDK9) is a promising therapeutic target in multiple cancer types, including acute myeloid leukemia (AML). Protein degraders, also known as proteolysis targeting chimeras (PROTACs), have emerged as tools for the selective degradation of cancer targets, including CDK9, complementing the activity of traditional small-molecule inhibitors. These compounds typically incorporate previously reported inhibitors and a known E3 ligase ligand to induce ubiquitination and subsequent degradation of the target protein. Although many protein degraders have been reported in the literature, the properties of the linker necessary for efficient degradation still require special attention. In this study, a series of protein degraders was developed, employing the clinically tested CDK inhibitor AT7519. The purpose of this study was to examine the effect that linker composition, specifically chain length, would have on potency. In addition to establishing a baseline of activity for various linker compositions, two distinct homologous series, a fully alkyl series and an amide-containing series, were prepared, demonstrating the dependence of degrader potency in these series on linker length and the correlation with predicted physicochemical properties.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9.

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (-)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.