Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs

1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis

Background: 1-methylnicotinamide (1-MNA), an endogenous metabolite of nicotinamide, has recently gained interest due to its anti-inflammatory and anti-thrombotic activities linked to the COX-2/PGI2 pathway. Given the previously reported anti-metastatic activity of prostacyclin (PGI2), we aimed to assess the effects of 1-MNA and its structurally related analog, 1,4-dimethylpyridine (1,4-DMP), in the prevention of cancer metastasis. Methods: All the studies on the anti-tumor and anti-metastatic activity of 1-MNA and 1,4-DMP were conducted using the model of murine mammary gland cancer (4T1) transplanted either orthotopically or intravenously into female BALB/c mouse. Additionally, the effect of the investigated molecules on cancer cell-induced angiogenesis was estimated using the matrigel plug assay utilizing 4T1 cells as a source of pro-angiogenic factors. Results: Neither 1-MNA nor 1,4-DMP, when given in a monotherapy of metastatic cancer, influenced the growth of 4T1 primary tumors transplanted orthotopically; however, both compounds tended to inhibit 4T1 metastases formation in lungs of mice that were orthotopically or intravenously inoculated with 4T1 or 4T1-luc2-tdTomato cells, respectively. Additionally, while 1-MNA enhanced tumor vasculature formation and markedly increased PGI2 generation, 1,4-DMP did not have such an effect. The anti-metastatic activity of 1-MNA and 1,4-DMP was further confirmed when both agents were applied with a cytostatic drug in a combined treatment of 4T1 murine mammary gland cancer what resulted in up to 80 % diminution of lung metastases formation. Conclusions: The results of the studies presented below indicate that 1-MNA and its structural analog 1,4-DMP prevent metastasis and might be beneficially implemented into the treatment of metastatic breast cancer to ensure a comprehensive strategy of metastasis control

Aromatic Bis[aminomethylidenebis(phosphonic)] Acids Prevent Ovariectomy-Induced Bone Loss and Suppress Osteoclastogenesis in Mice

Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates—benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid—which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy

Insights into the mechanisms involved in magnesium-dependent inhibition of primary tumor growth

International audienceWe have previously shown that a low Magnesium (Mg)-containing diet reversibly inhibits the growth of primary tumors that develop after the injection of Lewis lung carcinoma (LLC) cells in mice. Here we investigate some of the mechanisms responsible for the Mg-dependent regulation of tumor development by studying cell cycle regulation, tumor angiogenesis, and gene expression under Mg deficiency. The inhibition of LLC tumor growth in Mg-deficient mice is due to a direct effect of low Mg on LLC cell proliferation and to an impairment of the angiogenic switch. We also observed an increase of nitric oxide synthesis and oxidative DNA damage. Complementary DNA arrays reveal that Mg deficiency modulates tumor expression of genes involved in the control of cell cycle, stress response, proteolysis, and adhesion. Our results suggest that Mg has multiple and complex roles in tumor development

Structural patterns enhancing the antibacterial activity of metallacarborane-based antibiotics

Healthcare systems heavily rely on antibiotics to treat bacterial infections but widespread of the multidrug-resistant bacteria puts this strategy in danger. Novel drugs capable of overcoming current resistances are needed if our ability to treat bacterial infections is to be maintained. Boron clusters offer a valuable possibility to create a new class of antibiotics and expand antibiotic’s chemical space beyond conventional carbon-based molecules. In this work, we identified the two promising structural patterns providing cobalta bis(dicarbollide)(COSAN)-based compounds with potent and selective activity toward Staphylococcus aureus (including clinical strains): introduction of the α-amino acid amide and addition of iodine directly to the metallacarborane cage. Furthermore, we found that proper hydrophilic-lipophilic balance is crucial for the selective activity of the tested compounds toward S. aureus over mammalian cells. The patterns proposed in this paper can be useful in the development of metallacarborane-based antibiotics with potent antibacterial properties and low cytotoxicity

Additional file 1: Figure S1. of 1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis

Body weight of BALB/c Nude mice intravenously inoculated with 4T1-luc2-tdTomato cells treated with 1-MNA or 1.4-DMP in comparison to the control untreated group of animals. Table S1. The influence of 1-MNA and 1,4-DMP on the growth of selected cancer cell lines in vitro. (DOCX 1705 kb

Calcitriol Analogues Decrease Lung Metastasis but Impair Bone Metabolism in Aged Ovariectomized Mice Bearing 4T1 Mammary Gland Tumours

Calcitriol and its analogues are considered drugs supporting the anticancer treatment of breast cancer and preventing the osteoporosis that results from the development of cancer or from chemotherapy or hormone therapy. Following the orthotopic implantation of 4T1 mammary carcinoma cells into aged ovariectomized (OVX) mice, we evaluated the effects of calcitriol and its two analogues, PRI-2191 and PRI-2205, on metastatic spread and bone homeostasis. Calcitriol and its analogues temporarily inhibited the formation of metastases in the lungs. Unexpectedly, only mice treated with calcitriol analogues showed a deterioration of bone-related parameters, such as bone column density, marrow column density and the CaPO4 coefficient. These findings correlated with an increased number of active osteoclasts differentiated from bone marrow-derived macrophages in mice treated with the analogues. Interestingly, in the tumours from mice treated with PRI-2191 and PRI-2205, the expression of Tnfsf11 (RANKL) was increased. On the other hand, osteopontin (OPN) levels in plasma and tumour tissue, as well as TRAC5b levels in tumours, were diminished by calcitriol and its analogues. Despite a similar action of both analogues towards bone metabolism, their impact on vitamin D metabolism differed. In particular, PRI-2191 and calcitriol, not PRI-2205 treatment significantly diminished the levels of both 25(OH)D3 and 24,25(OH)2D3. In conclusion, though there is evident antimetastatic activity in old OVX mice, signs of increased bone metabolism and deterioration of bone mineralization during therapy with calcitriol analogues were observed

DataSheet_1_Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.docx

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.</p