Osvaldo Reyes

Antitumor Effect of a Novel Proapoptotic Peptide that Impairs the Phosphorylation by the Protein Kinase 2 (Casein Kinase 2)

Protein Kinase (casein kinase 2, CK2) is a serine-threonine kinase that is frequently dysregulated in many human tumors. Therefore we hypothesized that peptides capable of binding to the CK2 acidic domain may exhibit potential anticancer properties. By screening a random cyclic peptide phage display library, we have identified a novel peptide, P15, that abrogated CK2 phosphorylation by blocking the substrate in vitro. To verify its potential antineoplastic effect, P15 was fused to the cell-penetrating peptide derived from the HIV-Tat protein. Interestingly, P15-Tat induced apoptosis as evidenced by rapid caspase activation and cellular cytotoxicity in a variety of tumor cell lines. Furthermore, direct injection of P15-Tat into C57BL6 mice bearing day 7-established solid tumors, resulted in substantial regression of the tumor mass. Our findings describe a new proapoptotic cyclic peptide that blocks the CK2 phosphorylation and exhibits antitumor effect in vivo, indicating that the P15 peptide may potentially be used clinically to treat solid tumors or as an adjuvant for cancer therapy.

Immune response to synthetic peptides of dengue prM protein

The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P<0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines.

A Limulus anti-LPS factor-derived peptide modulates cytokine gene expression and promotes resolution of bacterial acute infection in mice.

Sepsis in experimental animals and humans has been associated with perturbed immune response. A major event contributing to the decrease in immune functions in septic disorders seems to be the inadequate balance of cytokines mediating the interactions between the innate and adaptive immune systems. We previously observed that a cyclic peptide derived from the Limulus anti-LPS factor (LALF), which partially protect mice from endotoxic shock lethality, has the ability to modulate cytokine secretion in vitro. We herein examined the effects of the LALF(31-52) peptide in an experimental model of Gram-negative peritoneal sepsis and analyzed the cytokine gene expression in the spleen and liver of peptide-treated mice. The prophylactic administration of LALF(31-52) abrogated the systemic TNF-alpha response, reduced organ damage and increased the survival of infected mice. Histological examination of spleen and liver in peptide-treated mice showed prevention of tissue damage induced by the high dose of Pseudomonas aeruginosa. This treatment modulates the cytokine gene expression in these tissues, stimulating IL-2, IL-12 and IL-13 mRNA synthesis, while IL-4 and IL-10 mRNA expression was not modified. This cytokine profile induced by the LALF-derived peptide seems to be favorable for host resistance against Gram-negative bacteria acute infection. In addition, peptide treatment was effective after the initiation of the systemic inflammatory response, promoting a significant increase in mice survival. These results further demonstrate the immunomodulatory potential of LALF(31-52) and are relevant for the design of prophylactic and therapeutic strategies for acute bacteria infection and sepsis, especially for preventing or ameliorating host immunity defects in these disorders.

Systemic administration of a peptide that impairs the protein kinase (CK2) phosphorylation reduces solid tumor growth in mice.

The antitumor efficacy of the CK2 inhibitors so far described has not been extensively evaluated in cancer animal models. We have previously demonstrated that a proapoptotic cyclic peptide termed P15 delivered into the cells by the Tat Cell Penetrating Peptide was able to abrogate the CK2‐mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice. Here we explored the antitumor effect by systemic administration of P15‐Tat in a consecutive 5‐day schedule through either intraperitoneal or intravenous route. Importantly, significant delay of tumor growth was observed at 2 mg/kg (p < 0.05), 10 mg/kg (p < 0.01) or 40 mg/kg (p < 0.001) after P15‐Tat administration both in syngeneic murine tumors and human tumors xenografted in nude mice. In line with this, the systemic administration of P15‐Tat induced apoptosis in the tumor as evidenced by in situ DNA fragmentation. Furthermore, we evidenced that 99mTc‐labeled P15‐Tat peptide was certainly accumulated on the tumors after administration by both routes. This report becomes the first describing the antitumor effect induced by systemic administration of a peptide that targets the acidic phosphorylation domain for CK2 substrates. Also, our data reinforces the perspectives of P15‐Tat for the cancer targeted therapy.

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.

A Limulus Antilipopolysaccharide Factor-Derived Peptide Exhibits a New Immunological Activity with Potential Applicability in Infectious Diseases

ABSTRACT Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of the Limulus antilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF31–52 peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-γ) and IFN-α. Analysis of the effect of LALF31–52 on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF31–52 protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-α levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.

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.

Inhibition of LPS-responses by synthetic peptides derived from LBP associates with the ability of the peptides to block LBP-LPS interaction.

The ability of LPS-binding protein (LBP) to greatly potentiate cell responses to lipopolysaccharide (LPS) may largely contribute to LPS toxicity in sepsis. The study of agents with the capacity to block the interaction between LBP and LPS might improve the understanding of the role of LBP in Gram-negative infections as well as offering new therapeutic tools for septic disorders. Here we confirm the ability of synthetic peptides comprising the human LBP amino acid region 86-108 to interfere with the LBP-LPS interaction. The analysis of selected alanine mutants of a blocking peptide corresponding to the LBP region 86-99 suggests the importance of peptide amphipathicity for the inhibitory activity. The potency of the native peptide and a selected analogue at inhibiting in vitro and in vivo LPS-induced responses was associated with their relative activity in blocking LBP-LPS interaction. It was remarkable that these peptides were at least 500-fold more active in vivo than in vitro. Also, the inhibitory activity of peptides LBP86-99 and LBPK95A seems to be independent of LBP concentrations, a behavior that may be relevant for the potential use of these peptides in septic disorders where LBP serum concentrations are considerably elevated.

A novel tick antigen shows high vaccine efficacy against the dog tick, Rhipicephalus sanguineus

Ticks are acaridae ectoparasites that, while taking a blood meal, can transmit viruses, bacteria, protozoa and filarial nematodes, which cause a variety of human and animal illnesses. The use of chemical pesticides constitutes the primary measure for control of these ectoparasites. However, the intensive use of these chemicals has drawbacks such as the contamination of food, environmental pollution and development of resistance by ectoparasites. Vaccination is considered a promising alternative for controlling infestations by ectoparasites. Although emerging tick proteins have been identified recently, and have been proposed as potential targets for generating protective molecules, only a limited number of them have been evaluated in vaccine trials. More than 80 proteins are found in eukaryotic ribosomes. The protein P0 is essential for the assembly of the 60S ribosomal subunit. We have identified an immunogenic region of the ribosomal protein P0 from Rhipicephalus sp. ticks that is not very conserved compared to host P0. The efficacy of a 20 amino acid synthetic peptide from this sequence was assayed as a vaccine antigen against Rhipicephalus sanguineus infestations in an immunization and challenge experiment on rabbits. A remarkable diminution in the viability of newly molted nymphs from larvae fed on vaccinated rabbits was observed. The number of adults and the number of eggs hatching were significantly reduced, with an overall efficacy of 90%. Our results demonstrated that immunization with an immunogenic peptide of tick protein P0 greatly reduced survival of ticks, suggesting that it has promise as an effective tick control agent.

Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.

Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial.