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

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

<p>ND, not determined.</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

General structure of compounds.

<p>All compound possess <i>N'</i>-acyl-2-naphthohydrazide or <i>N'</i>-acyl-naphthalene-2-sulfonohydrazide moiety.</p

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

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

Accumulation of phospho-IκB and phospho-p105 with no subsequent increase in NFκB transcriptional activity.

<p>(A) Western blot analysis of Ramos cell lysates after treatment with compound <b>15</b> for the indicated time periods. Phospho-p105, p105, phospho-IκB and IκB were determined; with p50 or actin being used as loading controls. The relative quantifications of protein expression compared to control are graphically presented below each western blot. (B) Western blot analysis of cytoplasm and nuclear protein fractions after treating cells for indicated lengths of time with inhibitor <b>15</b> or TNFα (100 ng/mL, 6 h, the latter used as a positive control. (C) The determination of NFκB transcriptional activity. SEAP activity was measured in Ramos-Blue™ cells after pre-treating them for 1 h with compounds <b>15</b> or <b>16</b> (50 µM) and subsequently adding TNFα (100 ng/mL). After 8 h, the supernatant was collected and SEAP activity was determined. Results present 3 independent experiments. * P<0.05; NT, non-treated cells.</p

Cobalt-Catalyzed Cross-Coupling of Grignards with Allylic and Vinylic Bromides: Use of Sarcosine as a Natural Ligand

Sarcosine was discovered to be an excellent ligand for cobalt-catalyzed carbon–carbon cross-coupling of Grignard reagents with allylic and vinylic bromides. The Co­(II)/sarcosine catalytic system is shown to perform efficiently when phenyl and benzyl Grignards are coupled with alkenyl bromides. Notably, previously unachievable Co-catalyzed coupling of allylic bromides with Grignards to linearly coupled α-products was also realized with Co­(II)/sarcosine catalyst. This method was used for efficient preparation of the key intermediate in an alternative synthesis of the antihyperglycemic drug sitagliptin

Inhibitor Design Strategy Based on an Enzyme Structural Flexibility: A Case of Bacterial MurD Ligase

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound <b>3</b> from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound <b>3</b> were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process