Objective: The protein kinase C (PKC) agonist bryostatin-1 has shown significant ex-vivo potency to revert HIV-1 latency, compared with other latency reversing agents (LRA). The safety of this candidate LRA remains to be proven in treated HIV-1-infected patients. Methods: In this pilot, double-blind phase I clinical-trial (NCT 02269605), we included aviraemic HIV-1-infected patients on triple antiretroviral therapy to evaluate the effects of two different single doses of bryostatin-1 (10 or 20 μg/m²) compared with placebo. Results: Twelve patients were included, four in each arm. Bryostatin-1 was well tolerated in all participants. Two patients in the 20 μg/m² arm developed grade 1 headache and myalgia. No detectable increases of cell-associated unspliced (CA-US) HIV-1-RNA were observed in any study arm, nor differences in HIV-1 mRNA dynamics between arms (P = 0.44). The frequency of samples with low-level viraemia did not differ between arms and low-level viraemia did not correlate with CA-US HIV-1-RNA levels (P = 0.676). No changes were detected on protein kinase C (PKC) activity and in biomarkers of inflammation (sCD14⁺ and interleukin-6) in any study arm. After the single dose of bryostatin-1, plasma concentrations were under detection limits in all the patients in the 10 μg/m² arm, and below 50 pg/ml (0.05 nmol/l) in those in the 20 μg/m² arm. Conclusion: Bryostatin-1 was safe at the single doses administered. However, the drug did not show any effect on PKC activity or on the transcription of latent HIV, probably due to low plasma concentrations. This study will inform next trials aimed at assessing higher doses, multiple dosing schedules or combination studies with synergistic drugs.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers’ properties as a real tool for viral infection detection and treatment.

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.

The mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are involved in oncogenic transformation and can promote metastasis and tumor progression. In human cells, there are four MNKs isoforms (MNK1a/b and MNK2a/b), derived from two genes by alternative splicing. These kinases play an important role controlling the expression of specific proteins involved in cell cycle, cell survival and cell motility via eukaryotic initiation factor 4E (eIF4E) regulation, but also through other substrates such as heterogeneous nuclear ribonucleoprotein A1, polypyrimidine tract-binding protein-associated splicing factor and Sprouty 2. In this review, we provide an overview of the role of MNK in human cancers, describing the studies conducted to date to elucidate the mechanism involved in the action of MNKs, as well as the development of MNK inhibitors in different hematological cancers and solid tumors.

Elevated expression levels of eukaryotic initiation factor 4E (eIF4E) promote cancer development and progression. MAP kinase interacting kinases (MNKs) modulate the function of eIF4E through the phosphorylation that is necessary for oncogenic transformation. Therefore, pharmacologic MNK inhibitors may provide a nontoxic and effective anticancer strategy. MNK1b is a truncated isoform of MNK1a that is active in the absence of stimuli. Using in vitro selection, high-affinity DNA aptamers to MNK1b were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Four groups of aptamers were identified, and the affinities of one representative for rMNK1b were determined using ELONA and quantitative polymerase chain reaction. Two aptamers, named apMNK2F and apMNK3R, had a lower Kd in the nmol/l range. The secondary structure of the selected aptamers was predicted using mFold, and the QGRS Mapper indicated the presence of potential G-quadruplex structures in both aptamers. The selected aptamers were highly specific against MNK1, showing higher affinity to MNK1b than to MNK1a. Interestingly, both aptamers were able to produce significant translation inhibition and prevent tumor cell proliferation and migration and colony formation in breast cancer cells. These results indicate that MNK1 aptamers have an attractive therapeutic potential.