Screening of selected wood-damaging fungi for the HIV-1 reverse transcriptase inhibitors

Iz 57 vrst lesnih gliv sta bili pripravljeni dve seriji izvlečkov. V prvi so bili izvlečki, pripravljeni z metanolom, v drugi pa z diklormetanom. Preizkušenih je bilo 63 vzorcev, saj so bile nekatere vrste zastopane z večjim številom izolatov. Izvlečkom smo in vitro preizkusili inhibitorno aktivnost na HIV-1 reverzno transkriptazo s pomočjo neradioaktivne metode. 13 metanolnih izvlečkov je inhibiralo encim več kot 40-odstotno, med njimi sta dva inhibirala encim več kot 80-odstotno. Najbolj učinkovita sta bila izvlečka gliv Laetiporus sulphuerus in Poria monticola, sledita jim izvlečka vrst Poria vaillanti in Chondrostereum purpureum. V nadaljevanju raziskave smo ugotavljali inhibitorno aktivnost različitih izolatov glive Laetiporus sulphureus. Najbolj aktivne izvlečke smo frakcionirali s pomočjo preparativne tekočinske kromatografije. Domnevamo, da bi bila lahko v najbolj aktivni frakciji prisotna spojina ali spojine, ki so kisle narave in imajo v strukturi amino skupino.Extracts obtained by using methanol and dichloromethane from 57 species of wood damaging fungi were investigated for their ability to inhibit HIV-1 reverse transcriptase activity in vitro using non-radioactive assay. Sixty tree samples were tested all together; some species were represented by more than one isolate. Thirteen methanol extracts exhibited more than 40% inhibition and two among them inhibited the enzyme more than 80%. All extracts obtained with dichloromethane were inferior to methanolic extracts in their inhibitory activity. The most active fungal species discovered in the first screening were Laetiporus sulphureus and Poria monticola, followed by Poria vaillanti and Chondrostereum purpureum. In the second screening, Laetiporus sulphureus was selected for detailed examination and different isolates were tested. Preliminary findings confirmed a presence of acidic compound with amino group in the most active fraction

Selective cytotoxicity of amidinopiperidine based compounds towards Burkitt's lymphoma cells involves proteasome inhibition.

Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-based compounds manifest cytotoxic activity, we have selected serine protease inhibitors designed and synthesized in-house with large hydrophobic naphthalene moiety for screening. The cytotoxic potential of screened molecules was correlated to modifications of R(1) residues. The most cytotoxic were compounds with greater basicity; amidinopiperidines, piperidines and benzamidines. Amidinopiperidine-based compounds exert cytotoxicity in low µM range, with IC(50) 18 µM and 22 µM for inhibitors 15 and 16 respectively. These compounds exhibited selective cytotoxicity towards the Burkitt's lymphoma cells Ramos and Daudi, and proved nontoxic to PMBC, Jurkat and U937. They induce caspase-dependent apoptotic cell death, as demonstrated by the use of a pan-caspase inihibitor, zVADfmk, which was able to rescue Ramos cells from compound(s)-induced apoptosis. We confirm a disruption of the pro-survival pathway in Burkitt's lymphoma through NFκB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IκB) molecules with no subsequent release of active NFκB implicated the involvement of proteasome. Indeed, we show that the amidinopiperidine-based compounds inhibit all three proteolytical activities of the human 20S proteasome, with the most prominent effect being on the trypsin-like activity. Consistently, treatment of Ramos cells with these compounds led to an increase in ubiquitinated proteins. The amidinopiperidine-based serine protease inhibitors presented are, as selective inducers of apoptosis in Burkitt's lymphoma cells, promising leads for the development of novel chemotherapeutics

zVADfmk inhibits amidinopiperidine-based compounds induced apoptosis.

<p>(A) The determination of Annexin V and 7-AAD positive Ramos cells treated with compounds <b>15</b> or <b>16</b> for 24 h. The data present the percentage of gated cells. (B) Caspase 3/7 activity was determined in cell lysates of Ramos cells treated for 4 h, 8 h, 16 h and 24 h with either 50 µM inhibitor or 10 µM TPCK, used as a positive control. Cleavage of Ac-DEVD-AFC in whole cell lysates was determined spectrofluorometrically. The results are presented as changes in fluorescence as a function of time. (C) Western blot analysis of the caspase-3 processing. Cells were treated for indicated time periods in the presence of zVADfmk (50 µM) and/or compound 16 (50 µM). (D) Analysis of Annexin V/7-AAD positive cells after <b>16</b> h treatment with compound <b>16</b> in the absence or presence of zVADfmk. NT, non-treated cells.</p

K_m values of substrates and K_i values of compounds 15 and 16 for chymotrypsin-, trypsin- and caspase-like activities of human 20S proteasome.

<p>K_m values of substrates and K_i values of compounds 15 and 16 for chymotrypsin-, trypsin- and caspase-like activities of human 20S proteasome.</p

The cytotoxicity screening of serine protease inhibitors.

<p>(A) Ramos cells (1×10⁵ cells/ml) were incubated in the presence of inhibitors at 100 µM (<b>1</b>–<b>13</b>, <b>15</b>, <b>16</b>) or 50 µM (<b>14</b>) for 24 h. (B) PBMC (1×10⁶ cells/ml), Jurkat, Daudi, Ramos and U937 (10⁵ cells/ml) were incubated for 24 h with 50 µM of compounds. Data present the residual metabolic activity as a percentage relative to control cells incubated in growth media supplemented with DMSO vehicle (mean ± SD) from three independent experiments, each conducted in triplicate.</p

Molecular modelling.

<p>The binding mode of compound <b>16</b> in the trypsin-like (β2) subunit of the 20S proteasome. The compound is presented as a stick structure (carbon atoms are colored in pink, oxygen in red, nitrogen in blue and sulfur in yellow).</p

Inhibition of the proteasome.

<p>Western blot analysis of ubiquitinated proteins accumulation in Ramos cell protein extracts after 6 h of treatment with compound <b>16</b>. Actin was used as a loading control.</p

K_i values of tested compounds for thrombin, factor Xa, trypsin and α-chymotrypsin.

<p>ND, not determined.</p