Gene expression profiling unveils the temporal dynamics of CIGB-300-regulated transcriptome in AML cell lines.

BACKGROUND: Protein kinase CK2 activity is implicated in the pathogenesis of various hematological malignancies like Acute Myeloid Leukemia (AML) that remains challenging concerning treatment. This kinase has emerged as an attractive molecular target in therapeutic. Antitumoral peptide CIGB-300 blocks CK2 phospho-acceptor sites on their substrates but it also binds to CK2α catalytic subunit. Previous proteomic and phosphoproteomic experiments showed molecular and cellular processes with relevance for the peptide action in diverse AML backgrounds but earlier transcriptional level events might also support the CIGB-300 anti-leukemic effect. Here we used a Clariom S HT assay for gene expression profiling to study the molecular events supporting the anti-leukemic effect of CIGB-300 peptide on HL-60 and OCI-AML3 cell lines. RESULTS: We found 183 and 802 genes appeared significantly modulated in HL-60 cells at 30 min and 3 h of incubation with CIGB-300 for p < 0.01 and FC > = │1.5│, respectively; while 221 and 332 genes appeared modulated in OCI-AML3 cells. Importantly, functional enrichment analysis evidenced that genes and transcription factors related to apoptosis, cell cycle, leukocyte differentiation, signaling by cytokines/interleukins, and NF-kB, TNF signaling pathways were significantly represented in AML cells transcriptomic profiles. The influence of CIGB-300 on these biological processes and pathways is dependent on the cellular background, in the first place, and treatment duration. Of note, the impact of the peptide on NF-kB signaling was corroborated by the quantification of selected NF-kB target genes, as well as the measurement of p50 binding activity and soluble TNF-α induction. Quantification of CSF1/M-CSF and CDKN1A/P21 by qPCR supports peptide effects on differentiation and cell cycle. CONCLUSIONS: We explored for the first time the temporal dynamics of the gene expression profile regulated by CIGB-300 which, along with the antiproliferative mechanism, can stimulate immune responses by increasing immunomodulatory cytokines. We provided fresh molecular clues concerning the antiproliferative effect of CIGB-300 in two relevant AML backgrounds.

Broad humoral immunity generated in mice by a formulation composed of two antigens from the Delta variant of SARS-CoV-2.

Due to the rapid development of new variants of SARS-CoV-2 as well as the real threat of new coronavirus zoonosis events, the development of a preventive vaccine with a broader scope of functionality is highly desirable. Previously, we reported the functionality of a nasal formulation containing the nucleocapsid protein and the receptor-binding domain (RBD) of the spike protein of the Delta variant of SARS-CoV-2 combined with the ODN-39M adjuvant. This combination induced cross-reactive immunity in mucosal and systemic compartments at the sarbecovirus level. In the present study, we explored the magnitude of the immunity generated in BALB/c mice by the same formulation with alum added as an additional adjuvant, to enhance the humoral immunity against the two antigens. Animals were immunized with three doses of the bivalent formulation, administered by subcutaneous route. Humoral immunity was tested by ELISA, and the neutralizing capacity of the resulting antibodies (Abs) was evaluated using a surrogate test and a vesicular stomatitis virus (VSV) pseudovirus-based assay. Cell-mediated immunity was also investigated using an IFN-γ ELISpot assay. High levels of antibodies against both antigens (N and RBD) were obtained upon immunization. Anti-RBD Abs with neutralizing capacity reacted with the RBD of three SARS-CoV-2 variants tested, including Omicron. Abs recognizing the nucleocapsid proteins of SARS-CoV-1 and the SARS-CoV-2 Delta and Omicron variants were also detected. Taken together, these results suggest that this bivalent formulation could be an attractive component of a pancorona vaccine able to broaden the scope of humoral immunity against both antigens. This will be particularly important for the reinforcement of immunity in previously vaccinated and/or infected populations.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

CIGB-300 anticancer peptide regulates the protein kinase CK2-dependent phosphoproteome.

Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha' (CK2α/CK2α') and two regulatory beta subunits (CK2ß), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2ß also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.

Characterization of low-abundance species in the active pharmaceutical ingredient of CIGB-300: A clinical-grade anticancer synthetic peptide.

CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.

Pharmacologic inhibition of the CK2-mediated phosphorylation of B23/NPM in cancer cells selectively modulates genes related to protein synthesis, energetic metabolism, and ribosomal biogenesis.

B23/NPM is a multifunctional nucleolar protein frequently overexpressed, mutated, or rearranged in neoplastic tissues. B23/NPM is involved in diverse biological processes and is mainly regulated by heteroligomer association and posttranslational modification, phosphorylation being a major posttranslational event. While the role of B23/NPM in supporting and/or driving malignant transformation is widely recognized, the particular relevance of its CK2-mediated phosphorylation remains unsolved. Interestingly, the pharmacologic inhibition of such phosphorylation event by CIGB-300, a clinical-grade peptide drug, was previously associated to apoptosis induction in tumor cell lines. In this work, we sought to identify the biological processes modulated by CIGB-300 in a lung cancer cell line using subtractive suppression hybridization and subsequent functional annotation clustering. Our results indicate that CIGB-300 modulates a subset of genes involved in protein synthesis (ES = 8.4, p < 0.001), mitochondrial ATP metabolism (ES = 2.5, p < 0.001), and ribosomal biogenesis (ES = 1.5, p < 0.05). The impairment of these cellular processes by CIGB-300 was corroborated at the molecular and cellular levels by Western blot (P-S6/P-4EBP1, translation), confocal microscopy (JC-1, mitochondrial potential), qPCR (45SrRNA/p21, ribosome biogenesis), and electron microscopy (nucleolar structure, ribosome biogenesis). Altogether, our findings provide new insights on the potential relevance of the CK2-mediated phosphorylation of B23/NPM in cancer cells, revealing at the same time the potentialities of its pharmacological manipulation for cancer therapy. Finally, this work also suggests several candidate gene biomarkers to be evaluated during the clinical development of the anti-CK2 peptide CIGB-300.

"Phosphoproteomic quantification based on phosphopeptide intensity or occupancy? An evaluation based on casein kinase 2 downstream effects".

Quantitative phosphoproteomic data has mostly been reported from experiments comparing relative phosphopeptides intensities in two or more different conditions, while the ideal parameter to compare is phosphopeptides occupancies. This term is scarcely used and therefore barely implemented in phosphoproteomics studies, and this should be of concern for the scientific journals. In order to demonstrate the relevance of this issue, here we show how the method of choice affects the interpretation of the data. The phosphoproteomic profile modulated in two AML cell lines after CK2 inhibition with CIGB-300 or CX-4945 is shown. Following the downstream action of CK2 the phosphosite intensity and occupancy results were compared to validate the best approach for quantitative phosphoproteomic studies. Even when the total number of quantified phosphopeptides was higher by using the intensity calculation, in all the cases the percent of CK2 consensus sequences which were down-regulated in response to CK2 inhibition was higher using the phosphosite occupancy quantification. To note, a high number of CK2 consensus sequences was found down-regulated with at least a 10% or 15% of phosphosite occupancy variation illustrating that low thresholds of occupancy modulation might be indicative of biological effect. Additionally, several biological processes only appear significantly over-represented in the phosphoproteome quantified by occupancy. The functional enrichment analysis per ranges of occupancy variations also illustrated clear differences among AML cell lines subjected to CK2 inhibition by CX-4945. A low overlap between the phosphoproteomes quantified by intensity and occupancy was obtained illustrating that new developments in proteomics techniques are needed to improve the performance of the occupancy approach. Even in such context, results indicate that occupancy quantification performs better than phosphorylation quantification based on intensity reinforcing the importance of such quantification approach to describe phosphoproteomic data.

The Nucleocapsid Protein of SARS-CoV-2, Combined with ODN-39M, Is a Potential Component for an Intranasal Bivalent Vaccine with Broader Functionality.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

A Nasal Vaccine Candidate, Containing Three Antigenic Regions from SARS-CoV-2, to Induce a Broader Response.

A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.

On the gene expression landscape of cancer.

Kauffman picture of normal and tumor states as attractors in an abstract state space is used in order to interpret gene expression data for 15 cancer localizations obtained from The Cancer Genome Atlas. A principal component analysis of this data unveils the following qualitative aspects about tumors: 1) The state of a tissue in gene expression space can be described by a few variables. In particular, there is a single variable describing the progression from a normal tissue to a tumor. 2) Each cancer localization is characterized by a gene expression profile, in which genes have specific weights in the definition of the cancer state. There are no less than 2500 differentially-expressed genes, which lead to power-like tails in the expression distribution functions. 3) Tumors in different localizations share hundreds or even thousands of differentially expressed genes. There are 6 genes common to the 15 studied tumor localizations. 4) The tumor region is a kind of attractor. Tumors in advanced stages converge to this region independently of patient age or genetic characteristics. 5) There is a landscape of cancer in gene expression space with an approximate border separating normal tissues from tumors.

Cross-Reactive Profile Against Two Conserved Coronavirus Antigens in Sera from SARS-CoV-2 Hybrid and Vaccinated Immune Donors.

Since the beginning of the pandemic, the pre-existing immunity against SARS-CoV-2 has been postulated as one possible cause of asymptomatic infections. Later, various works reported that pre-existing immune response against the two structural conserved antigens: S2 subunit and the nucleocapsid protein, were associated to some level of asymptomatic profile in infected individuals. To explore the Ab background against these two antigens, in the context of vaccine-elicited and hybrid (natural infection plus vaccination induced) immunity of SARS-CoV-2, in this work, we tested sera from inactivated vaccine-immunized donors and from vaccinated and subsequent natural infected donors upon the Omicron variant wave in Guangdong province, China. Serum samples were collected from 27 COVID-19 convalescent, 25 SARS-CoV-2 vaccinated, and 10 negative donors. The IgG cross-reactivity response against these two antigens from another relevant human coronavirus (HCoV) was also evaluated. The findings indicate that IgG response against S2 and N protein was particularly higher in sera with hybrid immunity. The cross-reactive Abs were more significant against SARS-CoV-1, while a wide cross-reactivity was detected for N antigen for one human Alpha coronavirus HCoV-229E even in the negative control samples. The presence of cross-reactive Abs against the two conserved antigens N and S2, particularly in the context of hybrid immunity, could pave the way for future boosted vaccines carrying these conserved regions.

Sensitivity of tumor cells towards CIGB-300 anticancer peptide relies on its nucleolar localization.

CIGB-300 is a novel anticancer peptide that impairs the casein kinase 2-mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB-300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB-300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB-300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB-300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB-300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull-down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB-300-treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB-300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide-based drug should entail its more efficient intracellular delivery at such subcellular localization.

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo.

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.

CIGB-300 Peptide Targets the CK2 Phospho-Acceptor Domain on Human Papillomavirus E7 and Disrupts the Retinoblastoma (RB) Complex in Cervical Cancer Cells.

CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate's phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7-pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells.

CIGB-300-Regulated Proteome Reveals Common and Tailored Response Patterns of AML Cells to CK2 Inhibition.

Protein kinase CK2 is a highly pleiotropic and ubiquitously expressed Ser/Thr kinase with instrumental roles in normal and pathological states, including neoplastic phenotype in solid tumor and hematological malignancies. In line with previous reports, CK2 has been suggested as an attractive prognostic marker and molecular target in acute myeloid leukemia (AML), a blood malignant disorder that remains as an unmet medical need. Accordingly, this work investigates the complex landscape of molecular and cellular perturbations supporting the antileukemic effect exerted by CK2 inhibition in AML cells. To identify and functionally characterize the proteomic profile differentially modulated by the CK2 peptide-based inhibitor CIGB-300, we carried out LC-MS/MS and bioinformatic analysis in human cell lines representing two differentiation stages and major AML subtypes. Using this approach, 109 and 129 proteins were identified as significantly modulated in HL-60 and OCI-AML3 cells, respectively. In both proteomic profiles, proteins related to apoptotic cell death, cell cycle progression, and transcriptional/translational processes appeared represented, in agreement with previous results showing the impact of CIGB-300 in AML cell proliferation and viability. Of note, a group of proteins involved in intracellular redox homeostasis was specifically identified in HL-60 cell-regulated proteome, and flow cytometric analysis also confirmed a differential effect of CIGB-300 over reactive oxygen species (ROS) production in AML cells. Thus, oxidative stress might play a relevant role on CIGB-300-induced apoptosis in HL-60 but not in OCI-AML3 cells. Importantly, these findings provide first-hand insights concerning the CIGB-300 antileukemic effect and draw attention to the existence of both common and tailored response patterns triggered by CK2 inhibition in different AML backgrounds, a phenomenon of particular relevance with regard to the pharmacologic blockade of CK2 and personalized medicine.

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection.

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

CIGB-300 Anticancer Peptide Differentially Interacts with CK2 Subunits and Regulates Specific Signaling Mediators in a Highly Sensitive Large Cell Lung Carcinoma Cell Model.

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.

Positive effects of Phycocyanobilin on gene expression in glutamate-induced excitotoxicity in SH-SY5Y cells and animal models of multiple sclerosis and cerebral ischemia.

Background: Oxidative stress has a predominant role in the pathogenesis of neurodegenerative diseases and therefore the modulation of genes and the identification of biological pathways associated with antioxidant therapies, have an impact on its treatment. Objective: The objective of this study was the comparison of 2 methods for the analysis of real-time PCR (qPCR) data, through the use of the evaluation of genes that mediate the effect of Phycocyanobilin (PCB) and its validation in animal models. Methods: We evaluated the effect of PCB:" in vitro" on gene modulation through qPCR analyzed by parametric ANOVA and multivariate principal component analysis (PCA) in a model of glutamate-induced excitotoxicity in the SH-SY5Y cell line and" in vivo"; in animal models of multiple sclerosis (MS) and cerebral ischemia (CI). Results: The results showed that PCA is a robust and powerful method that allows the assessment of gene expression profiles. We detected the significant down-regulation of the CYBB (NOX2), and HMOX1 by the action of PCB in SH-5YSH cell line insulted with Glutamate. The decrease in pro-inflammatory cytokines and markers related to apoptosis and innate immune response, mediated the effect of PCB in the animal models of MS and CI, respectively. Conclusion: We concluded that the mechanisms by which PCB protected cells included the reduction of oxidative stress damage, which could contribute to its clinical efficacy for the treatment of neurodegenerative diseases.

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research.

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.

Evaluation of Cell-Penetrating Peptides as Mucosal Immune Enhancers for Nasal Vaccination.

Cell-penetrating peptides (CPPs) have been evaluated as enhancers in drug delivery, their addition in medical formulations favors drug absorption allowing obtaining the pharmacological effect with lower doses. In vaccine formulations their inclusion has been also explored with interesting results. Currently mucosal vaccination constitutes a promising alternative with the main advantage of inducing both systemic and mucosal immune responses, which are crucial for control tumors and infections at mucosal tissues. In the present work the nasal immune-enhancing effect of four CPPs was evaluated in Balb/c mice. Animals were intranasally immunized with CPP and the recombinant hepatitis B surface protein (HBsAg) as model antigen. The antibody response in sera and mucosal tissue was measured by ELISA. The IFN-γ secretion response at spleen was also evaluated by ELISPOT and ELISA. Among the CPPs studied one novel peptide stand out by its ability to potentiate the humoral and cellular immune response against the co-administered antigen. Considering that the use of mucosal routes is a promising strategy in vaccination, which are gaining special relevance nowadays in the development of novel candidates against SARS-CoV-2 and other potential emerging respiratory virus, the searching and development of safe mucosal adjuvants constitute a current need.