Caracterización preclínica de Pep19-2.5 y Pep19-4LF, dos péptidos antimicrobianos en desarrollo para el tratamiento de la sepsis

Sepsis is a severe pathology caused by a systemic and disproportionate pro-inflammatory response of the immune system to an infection. Frequently, sepsis worsens and results in endotoxic (or septic) shock, which is accompanied by multi-organ failure and is usually fatal in a large number of cases. Microbial molecules triggering the most potent immune system activation are lipopolysaccharide (LPS or endotoxin) and lipoproteins (LPT) of Gram-negative and Gram-positive bacteria, respectively. Currently, there is no specific therapy against sepsis. The only treatments available are limited to fluid replacement and other life support measures. Each year, sepsis affects approximately 800,000 people in the United States alone and it has been estimated that between 28 and 50% of those affected die. Our group designed an antimicrobial peptide called Aspidasept I® (Pep19-2.5) which has high affinity in vitro for LPS and for whole cells of Staphylococcus aureus and which is capable of protecting mice against endotoxic shock. We also developed Aspidasept II® (Pep19-4LF) which has a much more potent antimicrobial activity than Pep19-2.5. Our hypothesis is that either Pep19-2.5 or Pep19-4LF, by themselves or combined with antibiotics could be effective anti-sepsis therapies in humans. To advance in the preclinical development of these compounds we characterize selected in vitro and in vivo activities of them with emphasis in those properties related to their application for the treatment of sepsis. We demonstrated that Pep19-2.5: afforded protection to mice against a lethal dose of the pro-inflammatory lipoprotein FSL-1 and efficiently cooperated with ceftriaxone to counteract sepsis-associated symptoms in a rabbit model of acute bactaeremia. However, the significant toxicity of Pep19-2.5 along with its poor half-life in blood and its sensitivity to proteases led us to focus our efforts on the preclinical development of Pep19-4LF. Compared with Pep19-2.5, we demonstrated that Pep19-4LF: i, is much more potent as antimicrobial and has a broader spectrum of activity; ii, is more capable of enhancing antibiotics and sensitizing multi-resistant strains to those compounds; iii, has a significant anti-biofilm bactericidal activity against P. aeruginosa Ps4 alone and combined with levofloxacin; iv, retains its antibacterial activity when immobilized on a polymeric matrix and prevents growth of P. aeruginosa biofilm as efficiently as colistin. Interestingly, Pep19-2.5 and Pep19-4LF synergized with each other against multi-resistant strains and showed a comparable capacity to protect mice against a lethal endotoxic shock. These results suggest that Pep19-4LF holds promise as a candidate for human sepsis therapy. In its turn, Pep19-2.5 could be developed for the topical treatment of infected wounds, since this peptide showed wound healing activity in mice as high as the reference compound

Caracterización preclínica de Pep19-2.5 y Pep19-4LF, dos péptidos antimicrobianos en desarrollo para el tratamiento de la sepsis

Sepsis is a severe pathology caused by a systemic and disproportionate pro-inflammatory response of the immune system to an infection. Frequently, sepsis worsens and results in endotoxic (or septic) shock, which is accompanied by multi-organ failure and is usually fatal in a large number of cases. Microbial molecules triggering the most potent immune system activation are lipopolysaccharide (LPS or endotoxin) and lipoproteins (LPT) of Gram-negative and Gram-positive bacteria, respectively. Currently, there is no specific therapy against sepsis. The only treatments available are limited to fluid replacement and other life support measures. Each year, sepsis affects approximately 800,000 people in the United States alone and it has been estimated that between 28 and 50% of those affected die. Our group designed an antimicrobial peptide called Aspidasept I® (Pep19-2.5) which has high affinity in vitro for LPS and for whole cells of Staphylococcus aureus and which is capable of protecting mice against endotoxic shock. We also developed Aspidasept II® (Pep19-4LF) which has a much more potent antimicrobial activity than Pep19-2.5. Our hypothesis is that either Pep19-2.5 or Pep19-4LF, by themselves or combined with antibiotics could be effective anti-sepsis therapies in humans. To advance in the preclinical development of these compounds we characterize selected in vitro and in vivo activities of them with emphasis in those properties related to their application for the treatment of sepsis. We demonstrated that Pep19-2.5: afforded protection to mice against a lethal dose of the pro-inflammatory lipoprotein FSL-1 and efficiently cooperated with ceftriaxone to counteract sepsis-associated symptoms in a rabbit model of acute bactaeremia. However, the significant toxicity of Pep19-2.5 along with its poor half-life in blood and its sensitivity to proteases led us to focus our efforts on the preclinical development of Pep19-4LF. Compared with Pep19-2.5, we demonstrated that Pep19-4LF: i, is much more potent as antimicrobial and has a broader spectrum of activity; ii, is more capable of enhancing antibiotics and sensitizing multi-resistant strains to those compounds; iii, has a significant anti-biofilm bactericidal activity against P. aeruginosa Ps4 alone and combined with levofloxacin; iv, retains its antibacterial activity when immobilized on a polymeric matrix and prevents growth of P. aeruginosa biofilm as efficiently as colistin. Interestingly, Pep19-2.5 and Pep19-4LF synergized with each other against multi-resistant strains and showed a comparable capacity to protect mice against a lethal endotoxic shock. These results suggest that Pep19-4LF holds promise as a candidate for human sepsis therapy. In its turn, Pep19-2.5 could be developed for the topical treatment of infected wounds, since this peptide showed wound healing activity in mice as high as the reference compound

Inhibition of lipopolysaccharide- and lipoprotein-induced inflammation by antitoxin peptide Pep 19-2.5

The most potent cell wall-derived inflammatory toxins (“pathogenicity factors”) of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals via toll-like receptor 4 (TLR4) and the latter via TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several in vivo models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade

Dual targeting of histone methyltransferase G9a and DNA-methyltransferase 1 for the treatment of experimental hepatocellular carcinoma

Epigenetic modifications like DNA and histone methylation functionally cooperate fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our new dual G9a histone-methyltransferase and DNA-methytransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n=268), peritumoral tissues (n=154) and HCC cell lines (n=32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cells growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1 and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia, and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. CONCLUSION: Combined targeting of G9a/DNMT1 with compounds like CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC. This article is protected by copyright. All rights reserved

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors

Fragile X mental retardation protein in intrahepatic cholangiocarcinoma: regulating the cancer cell behavior plasticity at the leading edge

Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy of the intrahepatic biliary tract with a very poor prognosis. Although some clinicopathological parameters can be prognostic factors for iCCA, the molecular prognostic markers and potential mechanisms of iCCA have not been well investigated. Here, we report that the Fragile X mental retardation protein (FMRP), a RNA binding protein functionally absent in patients with the Fragile X syndrome (FXS) and also involved in several types of cancers, is overexpressed in human iCCA and its expression is significantly increased in iCCA metastatic tissues. The silencing of FMRP in metastatic iCCA cell lines affects cell migration and invasion, suggesting a role of FMRP in iCCA progression. Moreover, we show evidence that FMRP is localized at the invasive front of human iCCA neoplastic nests and in pseudopodia and invadopodia protrusions of migrating and invading iCCA cancer cells. Here FMRP binds several mRNAs encoding key proteins involved in the formation and/or function of these protrusions. In particular, we find that FMRP binds to and regulates the expression of Cortactin, a critical regulator of invadopodia formation. Altogether, our findings suggest that FMRP could promote cell invasiveness modulating membrane plasticity and invadopodia formation at the leading edges of invading iCCA cells