Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group

This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease—staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme

Composites based on gellan gum, alginate and nisin-enriched lipid nanoparticles for the treatment of infected wounds

Due to growing antimicrobial resistance to antibiotics, novel methods of treatment of infected wounds are being searched for. The aim of this research was to develop a composite wound dressing based on natural polysaccharides, i.e., gellan gum (GG) and a mixture of GG and alginate (GG/Alg), containing lipid nanoparticles loaded with antibacterial peptide—nisin (NSN). NSN-loaded stearic acid-based nanoparticles (NP_NSN) were spherical with an average particle size of around 300 nm and were cytocompatible with L929 fibroblasts for up to 500 µg/mL. GG and GG/Alg sponges containing either free NSN (GG + NSN and GG/Alg + NSN) or NP_NSN (GG + NP_NSN and GG/Alg + NP_NSN) were highly porous with a high swelling capacity (swelling ratio above 2000%). Encapsulation of NSN within lipid nanoparticles significantly slowed down NSN release from GG-based samples for up to 24 h (as compared to GG + NSN). The most effective antimicrobial activity against Gram-positive Streptococcus pyogenes was observed for GG + NP_NSN, while in GG/Alg it was decreased by interactions between NSN and Alg, leading to NSN retention within the hydrogel matrix. All materials, except GG/Alg + NP_NSN, were cytocompatible with L929 fibroblasts and did not cause an observable delay in wound healing. We believe that the developed materials are promising for wound healing application and the treatment of bacterial infections in wounds

Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

<div><p>Background</p><p>Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study.</p><p>Methods</p><p>We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed.</p><p>Findings</p><p>Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC.</p><p>Conclusion</p><p>Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.</p></div

Comparison of cytotoxic and anti-clonogenic activities of highly potent natural compounds from Natural Products Set II.

<p>Comparison of cytotoxic and anti-clonogenic activities of highly potent natural compounds from Natural Products Set II.</p

Selected natural compounds induced diverse effects on the expression of <i>MITF</i> and <i>c-MYC</i>.

<p>(A) Cell viability was measured for highly cytotoxic compounds to choose a concentration not reducing cell viability below 40% of control after 24 h of treatment. (B) qRT-PCR was used to assess the fold change in the expression of <i>MITF</i> and <i>c-MYC</i> after treatment with selected compounds at indicated concentrations and time of exposure. (*P<0.05; **P<0.01; ***P<0.001).</p

The summary of natural compound activities at 5 µM.

<p>After initial screening, compounds were grouped based on their activities. A compound was defined as anti-clonogenic when it reduced the percentage of clones formed in soft agar to less than 20% of control treated with vehicle (0.05% DMSO). A compound was named cytostatic when it reduced the viable cell number to less than 50% of control using viable cell number counting, and cytotoxic using the flow cytometry after PI-staining. Numbers corresponding to compounds that accumulated melanoma cells in subG₁ are underlined. Compounds that caused cell cycle arrest are marked in green for G₀/G₁ phase, blue for S phase and red for G₂/M phase. Several compounds (46) were not active in any assay. Few compounds were cytotoxic but not markedly influenced the numbers of colonies formed in agar. Few other compounds exerted their effects only on clonogenic cells or reduced clonogenicity below 20% of control, caused cytostatic/cytostatic effect without inducing substantial cell death. Several compounds were cytostatic and cytotoxic and in addition they efficiently eradicated cells with clonogenic potential. They were in the focus of the further study. See Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090783#pone.0090783.s001" target="_blank">File SI</a> for the names of the compounds corresponding to numbers shown in the Figure.</p

Viability of melanoma cells was substantially reduced by several natural compounds used at 5 µM.

<p>Viability was measured after 45% of vehicle (0.05% DMSO)-treated control. For comparison, the leukemia cell line K562 was used. Each square represents the response of melanoma or leukemia cells to one out of 120 compounds. Colors indicate the level of cell response. For clarity the numbers designated in the present study to the tested compounds (Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090783#pone.0090783.s001" target="_blank">File SI</a>) are put in the first raw and the first column. See Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090783#pone.0090783.s001" target="_blank">File SI</a> for quantitative data.</p

Anti-clonogenic compounds did not induce apoptosis in melanoma cells.

<p>Induction of apoptosis was determined by flow cytometry after Annexin V/propidium iodide staining. Typical contour plots of melanoma cells treated with compounds at indicated concentrations are shown. Numbers in the rectangles indicate the mean percentages of all Annexin V-positive cells, both PI negative (early apoptosis) and PI positive (late apoptosis/necrosis) (n = 2). Several compounds e.g., nanaomycin A or pentoxifylline, used at the concentrations that were effective against clonogenic cells, did not induce apoptosis. The percentages of Annexin V-positive cells in vehicle-treated cells did not exceed 6% (not shown).</p