T1 Complex Research Articles

Computer simulation‐guided template selection for a molecularly imprinted polymer for selective trifluralin adsorption

AbstractTo meet selective adsorption toward trifluralin, a novel molecularly imprinted polymer (MIP) was fabricated by the dummy template molecular imprinting technology. First, computational simulation was performed to select a suitable dummy template, 3,5‐dinitro‐4‐methylbenzoic acid (T1), based on the maximum basis set superposition error (BSSE)‐corrected binding interaction energy (ΔE) of the monomer N‐vinylpyrrolidone (NVP)‐T1 complex and its structural overlap with trifluralin. Then, the MIP was prepared via the bulk polymerization. The adsorption experiments showed the MIP exhibited a trifluralin adsorption capacity of 5.1 mg g−1, an imprinting factor (IF) of 2.2, and short adsorption equilibrium time of 5 min. The adsorption of trifluralin conformed to the Freundlich adsorption (R2 = 0.985) and pseudo‐second‐order model (R2 = 0.999). In addition, the MIP exhibited selectivity to trifluralin over other adsorbents, including structural analogs (pendimethalin and oryzalin), pesticide (carbendazim), and nitrocompounds (nitrofurantoin, furazolidone, and furaltadone), with the selectivity factor (β) in the range of 1.2–3.0, respectively. In trifluralin/oryzalin mixture, the IF toward oryzalin was still as high as 1.9. The removal rate of the MIP to trifluralin in environmental water samples ranged from 90.08% to 99.04%. This study provides theoretical and experimental insights for the preparation of MIP using dummy templates.

Structure-guided design and cloning of peptide inhibitors targeting CDK9/cyclin T1 protein-protein interaction.

CDK9 (cyclin-dependent kinase 9) plays a significant role in numerous pathological conditions, such as HIV-1 infection and cancer. The interaction between CDK9 and cyclin T1 is crucial for maintaining the kinase's active state. Therefore, targeting this protein-protein interaction offers a promising strategy for inhibiting CDK9. In this study, we aimed to design and characterize a library of mutant peptides based on the binding region of cyclin T1 to CDK9. Using Osprey software, a total of 7,776 mutant peptides were generated. After conducting a comprehensive analysis, three peptides, namely, mp3 (RAADVEGQRKRRE), mp20 (RAATVEGQRKRRE), and mp29 (RAADVEGQDKRRE), were identified as promising inhibitors that possess the ability to bind to CDK9 with high affinity and exhibit low free binding energy. These peptides exhibited favorable safety profiles and displayed promising dynamic behaviors. Notably, our findings revealed that the mp3 and mp29 peptides interacted with a conserved sequence in CDK9 (residues 60-66). In addition, by designing the structure of potential peptides in the plasmid vector pET28a (+), we have been able to pave the way for facilitating the process of their recombinant production in an Escherichia coli expression system in future studies. Predictions indicated good solubility upon overexpression, further supporting their potential for downstream applications. While these results demonstrate the promise of the designed peptides as blockers of CDK9 with high affinity, additional experimental studies are required to validate their biological activity and assess their selectivity. Such investigations will provide valuable insights into their therapeutic potential and pave the way for the future development of peptide-based inhibitors targeting the CDK9-cyclin T1 complex.

Discovery of HyT-Based Degraders of CDK9-Cyclin T1 Complex.

Direct modulation of the non-kinase functions of cyclin and CDK-cyclin complexes poses challenges. We utilize hydrophobic tag (HyT) based small-molecule degraders induced degradation of cyclin T1 and its corresponding kinase partner CDK9. LL-CDK9-12 demonstrated the most potent and selective degradation ability, with DC50 values of 0.362 μM against CDK9 and 0.680 μM against cyclin T1. In prostate cancer cells, LL-CDK9-12 showed enhanced anti-proliferative activity than its parental molecule SNS032 and LL-K9-3, the previous reported CDK9-cyclin T1 degrader. Moreover, LL-CDK9-12 suppressed the downstream signaling of CDK9 and AR efficiently. Altogether, LL-CDK9-12 was an effective dual degrader of CDK9-cyclin T1 and helped study the unknown function of CDK9-cyclin T1. These results suggest that HyT-based degraders could be used as a strategy to induce the degradation of protein complexes, providing insights for the design of protein complexes' degraders.

Epigenetic oncogenesis, biomarkers and emerging chemotherapeutics for breast cancer

Breast cancer remains one of the leading causes of cancer-related deaths globally and the most prominent among females, yet with limited effective therapeutic options. Most of the current medications are challenged by various factors including low efficacy, incessant resistance, immune evasion and frequent recurrence of the disease. Further understanding of the prognosis and identification of plausible therapeutic channels thus requires multimodal approaches. In this review, epigenetics studies of several pathways to BC oncogenesis via the inducement of oncogenic changes on relevant markers have been overviewed. Similarly, the counter-epigenetic mechanisms to reverse such changes as effective therapeutic strategies were surveyed. The epigenetic oncogenesis occurs through several pathways, notably, DNMT-mediated hypermethylation of DNA, dysregulated expression for ERα, HER2/ERBB and PR, histone modification, overexpression of transcription factors including the CDK9-cyclin T1 complex and suppression of tumour suppressor genes. Scientifically, the regulatory reversal of the mechanisms constitutes effective epigenetic approaches for mitigating BC initiation, progression and metastasis. These were exhibited at various experimental levels by classical chemotherapeutic agents including some repurposable drugs, endocrine inhibitors, monoclonal antibodies and miRNAs, natural products, metal complexes and nanoparticles. Dozens of the potential candidates are currently in clinical trials while others are still at preclinical experimental stages showing promising anti-BC efficacy. The review presents a model for a wider understanding of epigenetic oncogenic pathways to BC and reveals plausible channels for reversing the unpleasant changes through epigenetic modifications. It advances the science of therapeutic designs for ameliorating the global burden of BC upon further translational studies.

Discovery of Small-Molecule Degraders of the CDK9-Cyclin T1 Complex for Targeting Transcriptional Addiction in Prostate Cancer.

Aberrant hyperactivation of cyclins results in carcinogenesis and therapy resistance in cancers. Direct degradation of the specific cyclin or cyclin-dependent kinase (CDK)-cyclin complex by small-molecule degraders remains a great challenge. Here, we applied the first application of hydrophobic tagging to induce degradation of CDK9-cyclin T1 heterodimer, which is required to keep productive transcription of oncogenes in cancers. LL-K9-3 was identified as a potent small-molecule degrader of CDK9-cyclin T1. Quantitative and time-resolved proteome profiling exhibited LL-K9-3 induced selective and synchronous degradation of CDK9 and cyclin T1. The expressions of androgen receptor (AR) and cMyc were reduced by LL-K9-3 in 22RV1 cells. LL-K9-3 exhibited enhanced anti-proliferative and pro-apoptotic effects compared with its parental CDK9 inhibitor SNS032 and suppressed downstream signaling of CDK9 and AR more effectively than SNS032. Moreover, LL-K9-3 inhibited AR and Myc-driven oncogenic transcriptional programs and exerted stronger inhibitory effects on several intrinsic target genes of AR than the monomeric CDK9 PROTAC (Thal-SNS032).

Application of low‐dose CT to the creation of 3D‐printed kidney and perinephric tissue models for laparoscopic nephrectomy

PurposeThe aim of this study was to explore the feasibility of 3D printing of kidney and perinephric fat based on low‐dose CT technology.Patients and MethodsA total of 184 patients with stage T1 complex renal tumors who underwent laparoscopic nephrectomy were prospectively enrolled and divided into three groups: group A (conventional dose kidney and perinephric fat 3D printing group, n = 62), group B (low‐dose kidney and perinephric fat 3D printing, n = 64), and group C (conventional dose merely kidney 3D printing group, n = 58). The effective dose (ED), signal‐to‐noise ratio (SNR), and contrast‐to‐noise ratio (CNR) were determined. The 3D printing quality was evaluated using a 4‐point scale, and interobserver agreement was assessed using the intraclass correlation coefficient (ICC).ResultsThe ED of group B was lower than that of group A, with a decrease of 55.1%. The subjective scores of 3D printing quality in all groups were 3 or 4 points. The interobserver agreement among the three observers in 3D printing quality was good (ICC = 0.84–0.92). The perioperative indexes showed that operation time (OT), warm ischemia time (WIT), estimated blood loss (EBL), and laparoscopic partial nephrectomy (LPN) conversion to laparoscopic radical nephrectomy (LRN) in groups A or B were significantly less than those in group C. LPN was more frequent in group A and group B than in group C (all p < 0.017). There were no significant differences in perioperative indexes between group A and group B (all p > 0.017).ConclusionLow‐dose CT technology can be effectively applied to 3D printing of kidney and perinephric fat and reduce the patient's radiation dose without compromising 3D printing quality. 3D printing of kidney and perinephric fat can significantly increase the success rate of LPN and decrease OT, WIT, and EBL.

The CDK9–cyclin T1 complex mediates saturated fatty acid–induced vascular calcification by inducing expression of the transcription factor CHOP

Vascular calcification (or mineralization) is a common complication of chronic kidney disease (CKD) and is closely associated with increased mortality and morbidity rates. We recently reported that activation of the activating transcription factor 4 (ATF4) pathway through the saturated fatty acid (SFA)-induced endoplasmic reticulum (ER) stress response plays a causative role in CKD-associated vascular calcification. Here, using mouse models of CKD, we 1) studied the contribution of the proapoptotic transcription factor CCAAT enhancer–binding protein homologous protein (CHOP) to CKD-dependent medial calcification, and 2) we identified an additional regulator of ER stress–mediated CHOP expression. Transgenic mice having smooth muscle cell (SMC)–specific CHOP expression developed severe vascular apoptosis and medial calcification under CKD. Screening of a protein kinase inhibitor library identified 16 compounds, including seven cyclin-dependent kinase (CDK) inhibitors, that significantly suppressed CHOP induction during ER stress. Moreover, selective CDK9 inhibitors and CRISPR/Cas9-mediated CDK9 reduction blocked SFA-mediated induction of CHOP expression, whereas inhibitors of other CDK isoforms did not. Cyclin T1 knockout inhibited SFA-mediated induction of CHOP and mineralization, whereas deletion of cyclin T2 and cyclin K promoted CHOP expression levels and mineralization. Of note, the CDK9–cyclin T1 complex directly phosphorylated and activated ATF4. These results demonstrate that the CDK9–cyclin T1 and CDK9–cyclin T2/K complexes have opposing roles in CHOP expression and CKD-induced vascular calcification. They further reveal that the CDK9–cyclin T1 complex mediates vascular calcification through CHOP induction and phosphorylation-mediated ATF4 activation.

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and safeguard transcriptome integrity1. Pol II accumulation downstream of the cleavage and polyadenylation signal (CPS) can facilitate recruitment of factors involved in mRNA 3’-end formation and termination2, but how this sequence is initiated remains unclear. In a chemical-genetic screen, we identified human protein phosphatase 1 (PP1) isoforms as substrates of positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex3. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis24. Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain (CTD) can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after Cdk9 inhibition in vivo. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, concomitant with accumulation of Pol II phosphorylated at CTD repeat residue Ser25. A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq)6, and is genetically suppressed by ablation of Cdk9 target sites in Spt5. These results suggest that the transition from elongation to termination by Pol II coincides with Dis2-dependent reversal of Cdk9 signaling—a switch analogous to a Cdk1-PP1 circuit that controls mitotic progression4.

HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

BackgroundHIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality.ResultsMass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR—Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E—poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue.ConclusionPhosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.

Effects of herbal mixture extracts on obesity in rats fed a high-fat diet

The aim of this study was to investigate and compare the effects of three herbal mixture extracts on obesity induced by high-fat diet (HFD) in rats. The prescriptions—Pericarpium citri reticulatae and Fructus crataegi—were used as matrix components and mixed with Ampelopsis grossedentata, Salvia miltiorrhiza, and epigallocatechin-3-gallate (EGCG) to form T1, T2, and T3 complexes, respectively. Results revealed that HFD feeding significantly increased body weight gain, fat deposition, plasma lipid profiles, hepatic lipid accumulation, and hepatic vacuoles formation, but decreased plasma levels of adiponectin in rats. Only the T1 complex showed the tendency, although not significantly so, for decreased HFD-induced body weight gain. T1 and T3 complexes significantly reduced HFD-induced fat deposition, and plasma levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol. Only the T1 complex significantly increased HFD-reduced adiponectin levels in plasma, but decreased HFD-increased triglyceride content in liver tissues. All complexes effectively inhibited HFD-induced vacuoles formation. The content of dihydromyricetin, salvianolic acid B, and EGCG in T1, T2, and T3 complexes was 18.25 ± 0.07%, 22.20 ± 0.10%, and 18.86 ± 0.04%, respectively. In summary, we demonstrated that herbal mixture extracts, especially T1 complex, exhibit antiobesity activity in HFD-fed rats.

Identification of novel inhibitors against Cyclin Dependent Kinase 9/Cyclin T1 complex as: Anti cancer agent.

Cell cycle consists of different types of phases, transition from G1, S, G2, M. Inhibition of associated CDKs like CDK9/Cyclin T1 complex, which are indirectly involved in the Cell cycle progression in the form of transcription elongation, reduces diverse diseases such as Cardiac Hypertrophy, Alzheimer’s, Cancer, AIDS and Inflammation. Glide tool of the Schrodinger software has been used for performing Structure Based Virtual Screening and Docking against Drug Bank and MDPI database. The best hits were identified which go and bind in the active site of the target where ATP binds for the activity. The ADMET, MM-GBSA and DFT analysis is also done for the same. Compound 4-{4-[4-(3-aminopropoxy)phenyl]-1H-pyrazol-5-yl}-6-chlorobenzene-1,3-diol (DB08045) was found to be more potent, novel and selective as an inhibitor. Hopefully compound (DB08045) could be used as an anti-cancer agent for the treatment of life-threatening diseases.

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 (Mg1 2+). 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 Mg1 2+ ion, coupled with the Mg2 2+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg1 2+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg1 2+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg1 2+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

In silico design of small molecule inhibitors of CDK9/cyclin T1 interaction

In order to design a small molecule which potentially may interfere with CDK9/cyclin T1 complex formation and therefore influence its physiological role, a computational study of dynamics and druggability of CDK9 binding surface was conducted. Druggability estimates and pocket opening analyses indicated binding regions of cyclin T1 residues, Phe 146 and Lys 6, as starting points for the design of small molecules with the potential to inhibit the CDK9/cyclin T1 association. A pharmacophore model was created, based on these two residues and used to select potential inhibitor structures. Binding energies of the inhibitors were estimated with MM-GBSA. A good correlation of MM-GBSA energies and FTMap druggability predictions was observed. Amongst studied compounds a derivative of 2-amino-8-hydroxyquinoline was identified as the best potential candidate to inhibit CDK9/cyclin T1 interactions.

The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

Computational study and peptide inhibitors design for the CDK9 – cyclin T1 complex

Cyclin dependent kinase 9 (CDK9) is a protein that belongs to the cyclin-dependent kinases family, and its main role is in the regulation of the cell transcription processes. Since the increased activity of CDK9 is connected with the development of pathological processes such as tumor growth and survival and HIV-1 replication, inhibition of the CDK9 could be of particular interest for treating such diseases. The activation of CDK9 is initiated by the formation of CDK9/cyclin T1 complex, therefore disruption of its formation could be a promising strategy for the design of CDK9 inhibitors. In order to assist in the design of potential inhibitors of CDK9/cyclin T1 complex formation, a computational study of the CDK9/cyclin T1 interface was conducted. Ten peptides were designed using the information from the analysis of the complex, hot spot residues and fragment based design. The designed peptides were docked to CDK9 structures obtained by molecular dynamics simulations of CDK9/cyclin T1 complex and the CDK9 alone and their binding affinities were evaluated using molecular mechanics Poisson Boltzman surface area (MM-PBSA) method and steered molecular dynamics (SMD). Designed peptide sequences LQTLGF and ESIILQ, both derived from the surface of cyclin T1, as well as the peptide sequence PRWPE, derived from fragment based design, showed the most favorable binding properties and were selected for our further studies.

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising cyclin-dependent kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.

Interruption of RNA processing machinery by a small compound, 1-[(4-chlorophenyl)methyl]-1H-indole-3-carboxaldehyde (oncrasin-1)

Protein kinase Ciota (PKCiota) is activated by oncogenic Ras proteins and is required for K-Ras-induced transformation and colonic carcinogenesis in vivo. However, the role of PKCiota in signal transduction and oncogenesis is not clear. We recently identified a small molecule, designated 1-[(4-chlorophenyl)methyl]-1H-indole-3-carboxaldehyde (oncrasin-1), that can selectively kill K-Ras mutant cancer cells and induce abnormal nuclear aggregation of PKCiota in sensitive cells but not in resistant cells. To determine the causes and biological consequences of PKCiota aggregates in the nucleus, we analyzed the effect of oncrasin-1 on proteins involved in DNA repair and RNA processing. Our results showed that oncrasin-1 treatment led to coaggregation of PKCiota and splicing factors into megaspliceosomes but had no obvious effects on the DNA repair molecule Rad51. Moreover, oncrasin-1 treatment suppressed the phosphorylation of the largest subunit of RNA polymerase II and the expression of intronless reporter genes in sensitive cells but not in resistant cells, suggesting that suppression of RNA transcription is a major effect of oncrasin-1 treatment. Studies with cultured cells or with recombinant proteins showed that oncrasin-1 can disrupt the interaction of PKCiota and cyclin-dependent protein kinase 9/cyclin T1 complex, which is known to phosphorylate the largest subunit of RNA polymerase II and is required for RNA transcription. Together, our results suggest that oncrasin-1 suppresses the function of RNA processing machinery and that PKCiota might be involved in the biological function of RNA processing complexes.

Inhibition of HIV-1 by DpT-Based Iron Chelators

HIV-1 transcription is activated by HIV-1 Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. Elongation of HIV-1 transcription is mediated by the interaction of Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II. Tat itself is phosphorylated by host cell cycle-dependent kinase 2 (CDK2) [1] and inhibition of CDK2 by tridentate iron chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone, (311) or ICL670 (deferasirox) inhibits HIV-1 transcription [2]. In addition to the inhibition of CDK2, 311 and ICL670 also prevent association of CDK9 with cyclin T1 [2], which could lead to inhibition of CDK9 activity and also to inhibition of HIV-1 transcription. Recently, a group of novel di-2-pyridylketone thiosemicarbazone (DpT) based tridentate iron chelators were shown to exhibit marked antiproliferative activity in vivo [3]. Here we screened DpT-based and also 2-benzoylpyridine thiosemicarbazone (BpT)-based tridentate iron chelators and identified three chelators, Dp44mT, Bp4eT and Bp4aT, that inhibited HIV-1 transcription but were not cytotoxic as determined by propidium iodide uptake, LDH release and calcein-AM uptake. The inhibition of HIV-1 transcription was observed in CEM HIV-1 LTR-GFP cells infected with Adeno-Tat and in 293T cells transiently transfected with HIV-1-LTR LacZ and Tat-expressing vectors with IC50s in the mid-nanomolar range. These new iron chelators also inhibited HIV-1 replication in CEM and THP-1 cells at 10 mM concentration. Analysis of the molecular mechanism of HIV-1 inhibition revealed that the DpT- and BpT-based iron chelators inhibited the activities of both CDK9/cyclin T1 and CDK2. The CDK9/cyclin T1 complex was disrupted in the cells treated with iron chelators, suggesting a possible mechanism for the inhibition of CDK9. In conclusion, our findings provide further evidence that iron chelators may inhibit HIV-1 transcription by deregulating CDK2 and CDK9. The projected therapeutic index of the selected DpT-based iron chelators was over 103 suggesting their potential usefulness as future anti-retroviral therapeutics.

CDK9 a Potential Target for Drug Development

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.

Cdk9/Cyclin T1 complex: A key player during the activation/differentiation process of normal lymphoid B cells

Cdk9/Cyclin T1 complex is very important in controlling specific differentiative pathways of several cell types. Limited data are available regarding the expression of Cdk9/Cyclin T1 in hematopoietic and lymphoid tissues. Cdk9/Cyclin T1 expression seems to be related to particular stages of lymphoid differentiation/activation. In this study, we observed that the expression level of Cdk9/Cyclin T1 in vivo increases in memory B cells compared to naïve B cells, and in activated B cells, compared to non-activated ones. The expression level of the Cdk9/Cyclin T1 complex does not increase in cells induced to differentiate in vitro. In addition, we showed that Cdk9 interacts with E12 and E47, specifically activated during Germinal Center (GC) reaction. Taken together this data suggests an active role for the Cdk9/Cyclin T1 complex during lymphoid differentiation through germinal center reaction.