Current Pharmaceutical Design - Volume 23, Issue 28, 2017

Background: The viral transactivator Tat protein is a key modulator of HIV-1 replication, as it regulates transcriptional elongation from the integrated proviral genome. Tat recruits the human transcription elongation factor b, and other host proteins, such as the super elongation complex, to activate the cellular RNA polymerase II, normally stalled shortly after transcription initiation at the HIV promoter. By means of a complex set of interactions with host cellular factors, Tat determines the fate of viral activity within the infected cell. The virus will either actively replicate to promote dissemination in blood and tissues, or become dormant mostly in memory CD4+ T cells, as part of a small but long-living latent reservoir, the main obstacle for HIV eradication. Objective: In this review, we summarize recent advances in the understanding of the multi-step mechanism that regulates Tat-mediated HIV-1 transcription and RNA polymerase II release, to promote viral transcription elongation. Early events of the human transcription elongation factor b release from the inhibitory 7SK small nuclear ribonucleoprotein complex and its recruitment to the HIV promoter will be discussed. Specific roles of the super elongation complex subunits during transcription elongation, and insight on recently identified cellular factors and mechanisms regulating HIV latency will be detailed. Conclusion: Understanding the complexity of HIV transcriptional regulation by host factors may open the door for development of novel strategies to eradicate the resilient latent reservoir.

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.

The general mechanism involved in Tat activation of RNA Polymerase II (RNAP II) elongation of the integrated HIV-1 was elucidated over 20 years ago. This mechanism involves Tat binding to the TAR RNA element that forms at the 5'; end of viral transcripts and recruiting a general RNAP II elongation factor termed as PTEFb. This elongation factor consists of CDK9 and Cyclin T1, and when recruited by Tat to TAR RNA, CDK9 was proposed to phosphorylate the carboxyl terminal domain of RNAP II and thereby activate elongation. Research in the past two decades has shown that the mechanism of Tat action is considerably more complicated than this simple model. In metabolically active cells, CDK9 and Cyclin T1 are now known to be largely sequestered in a RNA-protein complex termed the 7SK RNP. CDK9 and Cyclin T1 are released from the 7SK RNP by mechanisms not yet fully elucidated and along with Tat, bind to TAR RNA and orchestrate the assembly of a Super Elongation Complex (SEC) containing several additional proteins. CDK9 in the SEC then phosphorylates multiple substrates in the RNAP II complex to activate elongation. Importantly for therapeutic strategies, CDK9 and Cyclin T1 functions are down-regulated in resting CD4+ T cells that harbor latent HIV-1, and their up-regulation is required for reactivation of latent virus. Current strategies for a functional cure of HIV-1 infection therefore are likely to require development of latency reversal agents that up-regulate CDK9 and Cyclin T1 function in resting CD4+ T cells.

Background: Infection by the human immunodeficiency virus has become a treatable disease, which could not be cured because the virus persists in the face of an efficacious drug treatment. Current efforts for the rescue of replication-competent virus from cellular reservoirs are limited to drugs targeting transcriptional reactivation of the dormant virus and clinical trials so far are disappointing. One explanation could be that posttranscriptional pathways are not optimal thus impeding full reactivation of the virus, which is required for purging the cellular reservoirs. Objective: This review is focused on the post-transcriptional pathways of viral RNA processing. In this review, the complex regulation of viral RNAs in the nucleus, their export to the cytoplasm and engagement in translation and packaging are discussed in the context of reactivation and latency. In addition, post-transcriptional regulation of viral and cellular gene expression, by RNA interference, is considered with respect to HIV-1 reactivation from latency. Conclusion: Complete rescue of virus from the cellular reservoir is likely to require a combination of drugs targeting transcriptional and post-transcriptional pathways carefully tailored to HIV-1.

Following seminal discoveries by Rosen and co-workers in 1985, the HIV-1 TAR has emerged as one of the most extensively studied regulatory elements of the HIV-1 genome. Located adjacent to the long terminal repeat promoter, this cis-acting motif, in conjunction with the viral Tat protein, plays a critical role in viral genomic RNA synthesis via modification of the transcription complex. As such, the Tat/TAR axis has been the subject of intense efforts aimed at developing therapeutic interventions, directed against both the protein and nucleic acid components. While these efforts have to date been largely unsuccessful, current strategies to develop a functional cure for HIV have spawned renewed interest in targeting the Tat/TAR complex as a means of impairing virus reactivation and ultimately reducing the size of the latent reservoir pool. At the same time, advances in high throughput technologies, coupled with an increased understanding of RNA biology and function, have led to the identification of novel agents with enhanced potency and selectivity against a variety of cis-acting regulatory RNAs. In this review, recent approaches utilized to identify small molecules, peptides and evolved proteins with respect to targeting HIV-1 TAR are discussed.

Background: Despite efficient suppression of HIV-1 replication, current antiviral drugs are not able to eradicate HIV-1 infection. Permanent HIV-1 suppression or complete eradication requires novel biological approaches and therapeutic strategies. Our previous studies showed that HIV-1 transcription is regulated by host cell protein phosphatase-1. We also showed that HIV-1 transcription is sensitive to the reduction of intracellular iron that affects cell cycle-dependent kinase 2. We developed protein phosphatase 1-targeting small molecules that inhibited HIV-1 transcription. We also found an additional class of protein phosphatase-1-targeting molecules that activated HIV-1 transcription and reported HIV-1 inhibitory iron chelators and novel curcumin analogs that inhibit HIV-1. Here, we review HIV-1 transcription and replication with focus on its regulation by protein phosphatase 1 and cell cycle dependent kinase 2 and describe novel small molecules that can serve as future leads for anti-HIV drug development. Results: Our review describes in a non-exhaustive manner studies in which HIV-1 transcription and replication are targeted with small molecules. Previously, published studies show that HIV-1 can be inhibited with protein phosphatase-1-targeting and iron chelating compounds and curcumin analogs. These results are significant in light of the current efforts to eradicate HIV-1 through permanent inhibition. Also, HIV-1 activating compounds can be useful for “kick and kill” therapy in which the virus is reactivated prior to its inhibition by the combination antiretroviral therapy. Conclusion: The studies described in our review point to protein phosphatase-1 as a new drug target, intracellular iron as subject for iron chelation and novel curcumin analogs that can be developed for novel HIV-1 transcription- targeting therapeutics.

Background: HIV-1 can be preserved in long-lived resting CD4+ T- and myeloid cells, forming a viral reservoir in tissues of the infected individuals. Infected patients primarily receive cART, which, to date, is the most efficient treatment against HIV/AIDS. However, the major problem in the eradication of HIV-1 from patients is the lack of therapeutic approaches to recognize the latent HIV-1 provirus and to eliminate latently infected cells. Results: In the current review, we describe the effect of HIV-1 transcriptional inhibitors CR8#13 and F07#13 using a series of in vitro and in vivo assays. We found that both of these compounds regulate p-TEFb in infected cells, and terminate transcription at two sites, either at the LTR or early gag regions. The resulting short transcripts are termed TAR and TAR-gag, respectively. These nascent RNAs are capable of binding to SWI/SNF components, including mSin3A/HDAC-1 complex and potentially serve as a scaffolding RNA. Both TAR and TAR-gag are detected as large complexes from treated infected cells when using chromatography. Both transcripts are non-coding in T-cells and monocytes, and potentially recruit suppressive factors along with RNAbinding proteins to the DNA resulting in Transcriptional Gene Silencing (TGS). Finally, these compounds suppress activated virus when using a latent humanized mouse model. Conclusion: Collectively, these data implicate transcription inhibitors as regulators of the viral promoter through short non-coding RNAs and chromatin remodeling factors. These RNAs give specificity toward either viral DNA and/or nascent mRNA when functioning as TGS.

An ability to store cells (if they cannot be used fresh) reduces cell wastage, thereby increasing the supply of transplantable material. Cell storage is also valuable in scientific research, allowing material to be archived and experiments to be repeated from the same tissue source and facilitating research collaboration by allowing stored samples to be distributed. Cryopreservation is currently considered the most promising and successful, long-term biological conservation method. Its use has led to optimizing survival, improving protocols and stem cell and neuroglia viability, thereby assuring its future use in neuroregeneration and restoration regarding cell therapy. The success of conservation processes in ensuring cell viability depends on aspects such as the characteristics, cells' cryobiological behavior, isolation methodologies, cell freezing, the use and choice of cryoprotectants and such aspects' influence on intra- and extra-cellular dynamics. This review deals with cells' cryobiological behavior, cryopreservation and cryoprotectants, emphasizing on stem cell and neuroglial populations as therapeutic target regarding nervous system diseases.

The use of nanotechnology in neurosciences has been evolving since new treatments, diagnoses and biomolecule monitoring are needed to find safer treatments for central nervous system diseases (CNDs). Nanotechnology employs devices that interact with biological systems allowing molecular interactions with a high degree of specificity. This review considers concepts associated with nanotechnology and leading areas of neurosciences with nanotechnology research.

Adipokines are bioactive proteins that mediate proliferation, metabolism, inflammation, and angiogenesis. Adiponectin is an important adipokine that exerts multiple key functions via its anti-metabolic syndrome and anti-inflammatory properties. A number of adiponectin receptors, AdipoR1, AdipoR2 and T-cadherin, have been identified. Recent studies have suggested the involvement of adiponectin and receptors in several cancers, including prostate, breast, endometrial, brain, and colon cancer. Altered levels of adiponectin expression, or its interacting receptors, in cancers can lead to dysregulation of signaling pathways. Our current review describes the molecular mechanisms underlying the anti-tumorigenesis activity of adiponectin and the role of its receptors in prostate carcinogenesis, and provides perspectives of adiponectin-mediated signaling as a potential target for therapy.

Parkinson's disease is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra pars compacta region. An important mechanism contributing to its development is oxidative stress, induced by the imbalance between the endogenous antioxidant defenses and free radicals production. Naturally occurring bioactive compounds exhibit high antioxidant capacity that may help reducing oxidative stress and even reverse the damage induced by ROS. Fruits are particularly rich in phytochemicals with antioxidant effect, and their properties against the development of neurodegenerative diseases are of great interest. This review discusses how the fruits bioactive compounds and synthetic analogs have been assessed for their ability to regulate molecular pathways involved in neuronal survival such as MAPK, Nrf2, and NF-ΚB, thus elucidating the possible therapeutic and neuroprotective actions of these compounds.

Astrocytes are important glial cells involved in the ionic regulation of the extracellular fluid in the Central Nervous System (CNS), the formation of the blood brain barrier (BBB) and the support to neurons for the maintenance of the Krebs cycle intermediaries. Even though these cells are known to be important for the brain functioning, several of their functions and their development have not been fully elucidated. In this context, identifying the algorithms used for their analysis plays a pivotal role in the development of new strategies in the study of astrocytes. The main objective of this review is to summarize the techniques that have helped to obtain transcriptomic data in astrocytes and the new algorithms that were used to perform the analysis of experimental data, elucidating new studies in which these had been used. We also highlight the current transcriptomics approaches targeting astrocytes function as a possible target for pharmacological interventions.

Background: The blood-brain barrier restricts drug penetration to the central nervous system. Targeted nanocarriers are new potential tools to increase the brain entry of drugs. Ligands of endogenous transporters of the blood-brain barrier can be used as targeting vectors for brain delivery of nanoparticles. Objective: We tested biotin-labeled solid nanoparticles for the first time and compared to biotinylated glutathione- labeled nanoparticles in brain endothelial cells. Method: Neutravidin coated fluorescent polystyrene nanoparticles were derivatized with biotin and biotinylated glutathione. As a human in vitro blood-brain barrier model hCMEC/D3 brain endothelial cells were used. Cell viability by MTT test, uptake and transfer of the nanoparticles across the endothelial monolayers were measured. The uptake of the nanoparticles was visualized by confocal microscopy. Results: The tested nanoparticles caused no change in cell viability. The uptake of biotin- and glutathione-labeled nanoparticles by brain endothelial cells was time-dependent and significantly higher compared to non-labeled nanoparticles. The penetration of the glutathione-labeled nanoparticles across the endothelial monolayer was higher than the biotin-targeted ones. Biotin- and glutathione-targeted nanoparticles were visualized in hCMEC/D3 cells. We verified that hCMEC/D3 express mRNA for sodium-dependent multivitamin transporter (SMVT/SLC5A6) responsible for the blood-brain barrier transport of biotin. Conclusion: Biotin as a ligand increased the uptake and the transfer of nanoparticles across brain endothelial cells. Biotinylated glutathione could further increase nanoparticle permeability through endothelial monolayers supporting its use as a brain targeting vector.