Correction: de Jesus et al. Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-κB Cascade: In Vitro and In Silico Investigations. Molecules 2022, 27, 1078.

There was an error in the original publication [...].

Feed-forward regulatory loop driven by IRF4 and NF-κB in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive hematological malignancy derived from mature CD4+ T-lymphocytes. Here, we demonstrate the transcriptional regulatory network driven by 2 oncogenic transcription factors, IRF4 and NF-κB, in ATL cells. Gene expression profiling of primary ATL samples demonstrated that the IRF4 gene was more highly expressed in ATL cells than in normal T cells. Chromatin immunoprecipitation sequencing analysis revealed that IRF4-bound regions were more frequently found in super-enhancers than in typical enhancers. NF-κB was found to co-occupy IRF4-bound regulatory elements and formed a coherent feed-forward loop to coordinately regulate genes involved in T-cell functions and development. Importantly, IRF4 and NF-κB regulated several cancer genes associated with super-enhancers in ATL cells, including MYC, CCR4, and BIRC3. Genetic inhibition of BIRC3 induced growth inhibition in ATL cells, implicating its role as a critical effector molecule downstream of the IRF4-NF-κB transcriptional network.

Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-kB Cascade: In Vitro and In Silico Investigations.

Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1-3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = -9.2 kcal/mol) and the IKKα activation loop (BE = -9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.

Enhancer profiling identifies critical cancer genes and characterizes cell identity in adult T-cell leukemia.

A number of studies have recently demonstrated that super-enhancers, which are large cluster of enhancers typically marked by a high level of acetylation of histone H3 lysine 27 and mediator bindings, are frequently associated with genes that control and define cell identity during normal development. Super-enhancers are also often enriched at cancer genes in various malignancies. The identification of such enhancers would pinpoint critical factors that directly contribute to pathogenesis. In this study, we performed enhancer profiling using primary leukemia samples from adult T-cell leukemia/lymphoma (ATL), which is a genetically heterogeneous intractable cancer. Super-enhancers were enriched at genes involved in the T-cell activation pathway, including IL2RA/CD25, CD30, and FYN, in both ATL and normal mature T cells, which reflected the origin of the leukemic cells. Super-enhancers were found at several known cancer gene loci, including CCR4, PIK3R1, and TP73, in multiple ATL samples, but not in normal mature T cells, which implicated those genes in ATL pathogenesis. A small-molecule CDK7 inhibitor, THZ1, efficiently inhibited cell growth, induced apoptosis, and downregulated the expression of super-enhancer-associated genes in ATL cells. Furthermore, enhancer profiling combined with gene expression analysis identified a previously uncharacterized gene, TIAM2, that was associated with super-enhancers in all ATL samples, but not in normal T cells. Knockdown of TIAM2 induced apoptosis in ATL cell lines, whereas overexpression of this gene promoted cell growth. Our study provides a novel strategy for identifying critical cancer genes.

HIV Tat/P-TEFb Interaction: A Potential Target for Novel Anti-HIV Therapies.

Transcription is a crucial step in the life cycle of the human immunodeficiency virus type 1 (HIV 1) and is primarily involved in the maintenance of viral latency. Both viral and cellular transcription factors, including transcriptional activators, suppressor proteins and epigenetic factors, are involved in HIV transcription from the proviral DNA integrated within the host cell genome. Among them, the virus-encoded transcriptional activator Tat is the master regulator of HIV transcription. Interestingly, unlike other known transcriptional activators, Tat primarily activates transcriptional elongation and initiation by interacting with the cellular positive transcriptional elongation factor b (P-TEFb). In this review, we describe the molecular mechanism underlying how Tat activates viral transcription through interaction with P-TEFb. We propose a novel therapeutic strategy against HIV replication through blocking Tat action.

Quantification of the HIV transcriptional activator complex in live cells by image-based protein-protein interaction analysis.

The virus-encoded Tat protein is essential for HIV transcription in infected cells. The interaction of Tat with the cellular transcription elongation factor P-TEFb (positive transcriptional elongation factor b) containing cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9) is critical for its activity. In this study, we use the Fluoppi (fluorescent-based technology detecting protein-protein interaction) system, which enables the quantification of interactions between biomolecules, such as proteins, in live cells. Quantitative measurement of the molecular interactions among Tat, CycT1 and CDK9 has showed that any third molecule enhances the binding between the other two molecules. These findings suggest that each component of the Tat:P-TEFb complex stabilizes the overall complex, thereby supporting the efficient transcriptional elongation during viral RNA synthesis. These interactions may serve as appropriate targets for novel anti-HIV therapy.

A novel IKKα inhibitor, noraristeromycin, blocks the chronic inflammation associated with collagen-induced arthritis in mice.

OBJECTIVES: To evaluate the therapeutic efficacy of a novel inhibitor for IκB kinase alpha (IKKα), noraristeromycin (NAM), for murine experimental model of rheumatoid arthritis, collagen- induced arthritis (CIA). METHODS: NAM has been chemically synthesized as reported earlier. CIA was induced in DBA/1JNCrlj mice by intradermal inoculation of bovine type II collagen (col II) together with Freund Complete Adjuvant. Following the Day 21 booster injection of col II with Freund Incomplete Adjuvant, the animals were monitored for the development of arthritis and clinically evaluated. NAM was administered orally at different doses prior to induction (prophylactic protocol) or after the emergence of definitive arthritis (therapeutic protocol). RESULTS: Here we demonstrate the experimental evidence that oral administration of NAM could completely prevent the occurrence of experimental arthritis in CIA mouse model at 0.3 mg/kg with ED50 value of approximately 0.1 mg/kg twice daily. Moreover, twice daily oral therapeutic dosage of 1 mg/kg of NAM significantly inhibited the paw swelling and disease progression even after the occurrence of experimental CIA. In addition, NAM exhibited an excellent pharmacokinetics in mice and oral administration of NAM could suppress the production of TNFα elicited by lipopolysaccharide (LPS) in a dose-dependent manner. CONCLUSIONS: These results indicated that IKKα inhibition is an effective novel therapy for the treatment of chronic inflammatory processes such as those associated with RA and other related conditions.

Identification of a novel CDK9 inhibitor targeting the intramolecular hidden cavity of CDK9 induced by Tat binding.

HIV-1 transcription is specifically augmented by a transcriptional activator complex composed of Tat, an HIV-1-encoded activator, and the host transcription elongation factor P-TEFb, which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1. Several observations suggest that P-TEFb is an attractive anti-HIV-1 drug target. However, the long-term cytotoxicity of CDK9 inhibitors hinders their widespread use in HIV-1 therapy. Thus, novel and safe inhibitors are sorely needed. By performing molecular dynamics simulations of the 3D structure of Tat/P-TEFb, we previously identified a unique cavity structure of CDK9, the CDK9 hidden cavity, that is specifically induced by Tat binding. Here, we attempted to identify compounds that fit this cavity and inhibit CDK9 activity by in silico screening. We identified compounds that could inhibit CDK9 activity. One of such compound, 127, showed the strongest inhibitory activity against CDK9. Interestingly, it also inhibited CDK6 to a similar extent. We inspected the amino acid sequence and structural properties of the CDK9 hidden cavity to determine whether it is conserved in other CDKs, such as CDK6. The Ile61, comprising the center of the CDK9 hidden cavity, appears to be crucial for its kinase activity, thus indicating that the identification of the CDK9 hidden cavity may provide vital information for the development of novel CDK9 inhibitors.

In Vitro Antiviral Activity of Mentha cordifolia Plant Extract in HIV-1 Latently Infected Cells Using an Established Human Cell Line.

Emergence of drug resistance demands new therapeutic strategies against the human immunodeficiency virus (HIV). Currently, there is an increasing research focus on targeting gene expression-the crucial step wherein new viruses and new viral strains are amplified. Moreover, natural products are also being considered as potential candidates for new antivirals. We screened the extract obtained from a Philippine medicinal plant, Mentha cordifolia (Mc). In this study, we demonstrated that Mc ammonium sulfate extract has antiretroviral activity against HIV. HIV-1 latently infected cells (OM10.1) were pretreated with Mc extract and activated with TNFα. In treated cells, viral replication was inhibited in both cell culture supernatant and whole cell lysates. The level of viral production, as measured by the viral p24 protein concentration, was very much inhibited under noncytotoxic concentrations to the similar level without addition of TNFα. Luciferase assays, however, showed that Mc does not inhibit the HIV-1 long terminal repeat-driven gene expression. IκBα degradation and p65 nuclear translocation was also not affected as visualized through Western blot and immunofluorescence. These observations demonstrated that Mc possessed an antiviral component against HIV-1 and warrant further work to explore its target of action at a later step of gene expression. Our study introduces a potential source of a lead compound that targets steps in the HIV life cycle.

A-kinase-interacting protein 1 (AKIP1) acts as a molecular determinant of PKA in NF-kappaB signaling.

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

Application of fluorescent-based technology detecting protein-protein interactions to monitor the binding of hepatitis B virus X protein to DNA-damage-binding protein 1.

The hepatitis B virus X protein (HBx) and the V protein of paramyxovirus simian virus 5 (SV5-V) interact with DNA damage-binding protein 1 (DDB1), a cellular enzyme involved in DNA repair and cell cycle regulation, to stimulate viral activity. DDB1 has several cellular substrates, and the amino acid sequences of the binding sites in the viral proteins and their substrates are notably dissimilar. To determine whether HBx binds preferentially to DDB1, despite differences in the amino acid sequences, we developed a system to monitor DDB1 binding in living cells through a protein-protein visuali-zation system, designated fluorescent-based technology detecting protein-protein interactions (Fluoppi). HBx in association with DDB1 formed clear fluorescent puncta. The number of these fluorescent puncta increased with an increase in the amount of HBx. The binding of HBx to DDB1 inhibited the cellular substrate DDB1-CUL4A-associated factor 9 (DCAF9) from binding to DDB1. The inhibitor nitazoxanide prevented the viral proteins HBx and SV5-V from binding to DDB1 but did not inhibit the binding of DCAF9 or HBx(ΔNC), which constitutes the binding site of HBx. Our results demonstrate that the Fluoppi system is useful for monitoring the binding of HBx to DDB1 as well as for examining the effect of drugs on DDB1-Hbx binding.

Inhibition of inflammatory cytokine production from rheumatoid synovial fibroblasts by a novel IkappaB kinase inhibitor.

Nuclear factor-kappaB (NF-kappaB) is involved in the pathophysiology of rheumatoid arthritis (RA) and is considered to be a feasible molecular target in treating patients. In the RA joint tissues, activation of NF-kappaB is often observed together with high amounts of the proinflammatory cytokines tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta. TNFalpha and IL-1beta are known to stimulate NF-kappaB signaling and are produced as the effect of NF-kappaB signaling, thus forming a vicious cycle leading to a self-perpetuating nature of rheumatoid inflammation and expansion of such inflammatory response to other joints. Because a kinase called IkappaB kinase complex (IKK) is involved in the NF-kappaB activation cascade, we examined the effect of a novel IKK inhibitor, (7-[2-(cyclopropyl-methoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride; CHPD), on the production of inflammatory cytokines from rheumatoid synovial fibroblasts (RSF). TNFalpha stimulation induced production of inflammatory cytokines such as IL-6 and IL-8 in RSF, and the extent of IL-6 and IL-8 induction was dramatically reduced by CHPD under noncytotoxic concentrations. Likewise, expression of il-6 and il-8 genes was significantly reduced by CHPD. In addition, chromatin immunoprecipitation assays revealed that the DNA binding of NF-kappaB (p65) to il-8 promoter in RSF was induced after TNFalpha stimulation and that, upon CHPD treatment to RSF for 1 h, the NF-kappaB binding to il-8 promoter was significantly decreased. Here, we have demonstrated that an IKKbeta inhibitor, CHPD, acts as an effective inhibitor for the production of inflammatory cytokines in response to proinflammatory cytokines. These findings indicate that such a IKKbeta inhibitor could be a feasible candidate for an antirheumatic drug.

Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

Molecular docking analysis of the protein-protein interaction between RelA-associated inhibitor and tumor suppressor protein p53 and its inhibitory effect on p53 action.

RelA-associated inhibitor (RAI) was initially identified as a protein that interacts with the p65 subunit (RelA) of nuclear factor-kappaB. It was recently found to interact with the p53 tumor suppressor protein. RAI is a structural homolog of the p53-binding protein 2 and I kappaB family proteins, and is known to inhibit the DNA-binding activities of p65 and p53. In the present study, we have attempted to predict the 3-dimensional structure of RAI in complex with p53 using computational chemistry. In order to evaluate the predicted structure model, we created a series of RAI mutants in which the amino acid residues involved in the interaction with p53 were mutated, and examined their activities in blocking p53-mediated bax gene expression. Our observations support the validity of the predicted 3-dimensional model of the p53-RAI protein complex. Based on the p53-RAI complex model, we have demonstrated the biological importance of the R248 and R273 residues of p53, and the D775 and E795 residues of RAI, in the protein-protein interaction between p53 and RAI and the biological actions of these proteins. These findings will further clarify the biological actions of RAI in carcinogenesis and can be used for the development of a novel strategy in blocking the actions of RAI. The possible biological implications of RAI are also discussed.

Inhibition of human immunodeficiency virus type 1 replication by blocking IkappaB kinase with noraristeromycin.

Nuclear factor kappaB (NF-kappaB) is one of the critical transcription factors in inflammatory responses and replication of viruses such as human immunodeficiency virus (HIV). In fact, it has been demonstrated that various NF-kappaB inhibitors could block HIV replication. To explore more potent NF-kappaB inhibitors, we focused on carbocyclic adenine nucleosides that had been reported to have anti-inflammatory effects. We synthesized 15 carbocyclic adenine nucleoside compounds and examined their effects on the NF-kappaB-dependent gene expression using HEK293 cell line. Among these compounds, noraristeromycin (NAM) exhibited the most potent inhibitory effect on the NF-kappaB activity under the non-cytotoxic concentrations. NAM-inhibited IkappaBalpha phosphorylation and degradation upon stimulation of cells with tumour necrosis factor-alpha (TNF-alpha). In addition, NAM prevented p65 phoshorylation. These findings suggested that both IkappaB kinase-alpha (IKK-alpha) and -beta were targeted by NAM. Interestingly, in vitro kinase assay revealed that NAM inhibited the kinase activity of IKK-alpha more potently than that of IKK-beta. When we treated the cell lines, OM10.1 and Molt4/IIIB, in which HIV-1 is latently and chronically infected, we found a strong suppressive effect of NAM on HIV-1 viral replication upon stimulation with TNF-alpha.

Magnolia ovovata extract and its active component magnolol prevent skin photoaging via inhibition of nuclear factor kappaB.

Transcriptional activity of nuclear factor kappaB (NF-kappaB) is induced by environmental signals including inflammation, UV irradiation and oxidative stress. It was shown that the NF-kappaB activity greatly contributes to the skin photoaging process. Thus, it is plausible that NF-kappaB inhibitors could directly prevent skin photoaging. In this study, we found that Magnolia ovovata extract inhibited NF-kappaB-mediated gene expression and demonstrated that external swabbing with Magnolia extract preventing skin photoaging processes through keratinocyte hyperproliferation and degradation of collagen fibers in mice skin. We have identified magnolol as the solely responsible active compound in Magnolia extract. Magnolol effectively inhibited the NF-kappaB-dependent transcription, but no effect was observed with other inducible transcription factors such as activator protein-1 (AP-1) and cyclic-AMP responsive element-binding protein (CREB). In addition, magnolol was effective in inhibiting the production of basic fibroblast growth factor (bFGF) and matrix metalloprotease-1 (MMP-1) from the cells overexpressing p65, a major subunit of NF-kappaB. Although magnolol did not affect the phosphorylation and degradation of IkappaBalpha, it inhibited the nuclear translocation of the activated NF-kappaB. These findings suggest that Magnolia extract and its active component magnolol can be used to prevent the skin photoaging via inhibiting NF-kappaB by external topical application.

The Tat/P-TEFb Protein-Protein Interaction Determining Transcriptional Activation of HIV.

Human immunodeficiency virus type (HIV) transcription is crucial for its life cycle and is primarily involved in the maintenance of viral latency. HIV transcription is regulated by both viral and cellular transcription factors. Numerous epigenetic factors, as well as transcriptional suppressor proteins, play major roles in the maintenance of transcriptional silencing of viral gene expression from the proviral DNA. Once inducible transcription factors such as nuclear factor κB are activated through extracellular signaling, viral latency is terminated and transcription from the silenced proviral DNA is initiated. Transcriptional induction by cellular factors is immediately followed by high gene expression via the function of the virus-encoded transcriptional activator Tat. Interestingly, unlike other known transcriptional activators, Tat primarily activates transcriptional elongation, rather than initiation, by interacting with and activating cellular positive transcriptional elongation factor b (P-TEFb). In this review, we describe how HIV transcription is negatively and positively regulated through its life cycle and the molecular mechanism underlying how Tat activates viral transcription. We propose a novel strategy against viral replication in which regulated transcriptional processes play important roles in determining the extent of viral replication. The structural details of how Tat interacts with P-TEFb are described, which may be useful for the development of effective and specific anti-HIV therapies.

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor.

Involvement of nuclear factor-kappaB (NF-kappaB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-kappaB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-kappaB in addition to the phosphorylation of IkappaBalpha and the activation of NF-kappaB DNA binding and that various target genes of NF-kappaB including bcl-x(L), XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IkappaB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC(50) values ranging from 18 to 35 mumol/L concomitantly with inhibition of the phosphorylation of IkappaBalpha/p65 and NF-kappaB DNA-binding, down-regulation of the NF-kappaB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IkappaB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-kappaB.

Molecular dynamics simulation and experimental verification of the interaction between cyclin T1 and HIV-1 Tat proteins.

The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2' helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1' and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound.