Application of bioisosterism in design of the semicarbazone derivatives as cruzain inhibitors: a theoretical and experimental study

<p>A series of semicarbazone, thiosemicarbazone, and aminoguanidine derivatives were synthesized and tested as antitrypanosomal agents. The theoretical NMR of the compounds was calculated using molecular modeling techniques (density functional theory (DFT) calculations) and confirmed the formation of the compounds. The ability to inhibit cruzain and <i>Trypanosoma cruzi</i> epimastigote replication was evaluated. Cruzain inhibition ranged between 70 and 75% (100 μM), and IC₅₀ values observed in epimastigote forms of <i>T. cruzi</i> ranged from 20 to 140 μM. Furthermore, the compounds did not present cytotoxicity at concentrations up to 50 and 250 μM in MTT tests. Molecular modeling studies were conducted using DFT method (B3LYP functional and the basis set 6-311G(d,p)) to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity. In docking studies, the obtained analogs showed good complementarity with cruzain active site. In addition, docking results are in accordance with the susceptibility of these analogs to nucleophilic attack of the catalytic Cys25. Taken together, this study shows that this class of compounds can be used as a prototype in the identification of new antichagasic drugs.</p

<i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor binds to elastase and impairs neutrophil function and inflammation

<div><p>Protease inhibitors have important function during homeostasis, inflammation and tissue injury. In this study, we described the role of <i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor in parasite development and as a molecule capable of regulating different models of inflammatory diseases. First, we determine that recombinant (r) <i>Sm</i>KI-1 and its Kunitz domain but not the C-terminal region possess inhibitory activity against trypsin and neutrophil elastase (NE). To better understand the molecular basis of NE inhibition by S<i>m</i>KI-1, molecular docking studies were also conducted. Docking results suggest a complete blockage of NE active site by <i>Sm</i>KI-1 Kunitz domain. Additionally, r<i>Sm</i>KI-1 markedly inhibited the capacity of NE to kill schistosomes. In order to further investigate the role of <i>Sm</i>KI-1 in the parasite, we designed specific siRNA to knockdown <i>Sm</i>KI-1 in <i>S</i>. <i>mansoni</i>. <i>SmKI-1</i> gene suppression in larval stage of <i>S</i>. <i>mansoni</i> robustly impact in parasite development <i>in vitro</i> and <i>in vivo</i>. To determine the ability of <i>Sm</i>KI-1 to interfere with neutrophil migration and function, we tested <i>Sm</i>KI-1 anti-inflammatory potential in different murine models of inflammatory diseases. Treatment with <i>Sm</i>KI-1 rescued acetaminophen (APAP)-mediated liver damage, with a significant reduction in both neutrophil recruitment and elastase activity. In the model of gout arthritis, this protein reduced neutrophil accumulation, IL-1β secretion, hypernociception, and overall pathological score. Finally, we demonstrated the ability of <i>Sm</i>KI-1 to inhibit early events that trigger neutrophil recruitment in pleural cavities of mice in response to carrageenan. In conclusion, <i>Sm</i>KI-1 is a key protein in <i>S</i>. <i>mansoni</i> survival and it has the ability to inhibit neutrophil function as a promising therapeutic molecule against inflammatory diseases.</p></div

Complementarity Between a Docking and a High-Throughput Screen in Discovering New Cruzain Inhibitors

Virtual and high-throughput screens (HTS) should have complementary strengths and weaknesses, but studies that prospectively and comprehensively compare them are rare. We undertook a parallel docking and HTS screen of 197861 compounds against cruzain, a thiol protease target for Chagas disease, looking for reversible, competitive inhibitors. On workup, 99% of the hits were eliminated as false positives, yielding 146 well-behaved, competitive ligands. These fell into five chemotypes: two were prioritized by scoring among the top 0.1% of the docking-ranked library, two were prioritized by behavior in the HTS and by clustering, and one chemotype was prioritized by both approaches. Determination of an inhibitor/cruzain crystal structure and comparison of the high-scoring docking hits to experiment illuminated the origins of docking false-negatives and false-positives. Prioritizing molecules that are both predicted by docking and are HTS-active yields well-behaved molecules, relatively unobscured by the false-positives to which both techniques are individually prone

<i>Sm</i>KI-1 treatment decreased inflammation after MSU-induced gout.

<p>Mice were treated with r<i>Sm</i>KI-1 (10 mg/kg) or PBS vehicle i.v. 15 min prior MSU injection. Then, animals were challenged with intra-articular knee injection of MSU (100μg/cavity). Mice were grouped as MSU control, PBS control, MSU+<i>Sm</i>KI-1-treatment and PBS+<i>Sm</i>KI-1-treatment. Tissue inflammation was evaluated by <b>(a)</b> relative numbers of neutrophil in periarticular tissue determined by MPO assay, <b>(b)</b> total cells and <b>(c)</b> neutrophil recruitment in the synovial cavity, <b>(d)</b> IL-1β production measured by ELISA in the periarticular knee tissue and <b>(e)</b> joint dysfunction as noted by the increase nociceptive response of mice to mechanical stimulation using an electronic paw pressure meter test 15 hrs after MSU or PBS (control vehicle) injection. <b>(f)</b> Representative photographs of hematoxylin and eosin-stained sections of knee joints of mice after 15 hrs of injection with vehicle or MSU crystals (100μg/joint). Leukocyte infiltration and hyperplasia of the synovial membrane are indicated by black arrows. (<b>g</b>) Neutrophil recruitment in the synovial cavity of mice infected with <i>S</i>. <i>mansoni</i>. Mice were grouped as PBS control, PBS infected, MSU control and MSU infected. ND = not detected. Results are the mean ± SEM of n = 6 per group. Asterisks indicate statistically significant differences of r<i>Sm</i>KI-1 compared to MSU-vehicle group *p< 0.05 or ** p< 0.005. An asterisk also indicates statistically significant differences of <i>S</i>. <i>mansoni</i> infection versus control mice that received MSU, p<0.05.</p

<i>Sm</i>KI-1 Kunitz type domain sequence and structure.

<p>(<b>a</b>) Multiple Sequence Alignment between the SmKI-1 protein from <i>Schistosoma mansoni</i>, and its homologous proteins performed using Clustal Omega, refined using BoxShade and then manually. The residues that are similar are shaded in gray, identical in dark-black and in yellow absolutely conserved cysteine residues. (<b>b</b>) Disordered Probability Prediction showing the structured Kunitz domain and unstructured C-terminal region. Analysis performed using COILS algorithms available at the Expasy website. (<b>c</b>) Schematic representation and linear view of the domains of the full-length SmKI-1 protein showing the Kunitz domain with the three disulfide bounds arrangements. (<b>d</b>) 3D protein structure of the <i>Sm</i>KI-1 Kunitz domain modeled using MODELLER v9.17.</p

Recombinant <i>Sm</i>KI-1 and its Kunitz domain inhibit serine proteases and protect <i>S</i>. <i>mansoni</i> against neutrophil elastase.

<p>Recombinant <i>Sm</i>KI-1, its Kunitz or C-terminal domains (100 nM) were tested as inhibitor of serino proteases: (<b>a</b>) Bovine Trypsin activity (100nM), (<b>b</b>) Human Neutrophil Elastase activity (300nM) and (<b>c</b>) Neutrophil-secreted elastase activity. In all experiments, bovine serum albumin (BSA, 300nM) was used as a negative control. Enzyme inhibition was detected over two-hour incubation with r<i>Sm</i>KI-1 or its Kunitz domain. Bars indicate each enzyme activity mean ± standard deviation. (<b>d</b>) Protective effect of rSmKI-1 (0.15 mg/mL) in cultured schistosomula treated with purified elastase (0.05 mg/mL). Bars represent live parasites ± standard deviation. Data are representative of at least three independent experiments. For (<b>a</b>) and (<b>b</b>), an asterisk indicate statistically significant differences of r<i>Sm</i>KI-1 or Kunitz domain compared to control group p< 0.05. For (<b>c</b>) and (<b>d</b>), ** asterisks indicate statistically significant differences of r<i>Sm</i>KI-1, compared to control group or elastase group p< 0.005. <b>(e)</b> Binding mode of <i>Sm</i>KI-1 Kunitz domain (purple) to neutrophil elastase (gray) predicted by docking with CLUSPRO 2.0. Residues from elastase catalytic triad (His⁷⁰, Asp¹¹⁷ and Ser²⁰²) are highlighted in orange sticks. (<b>f</b>) Detailed analysis of the docking predicted interface reveals residues involved in hydrogen bonds, a salt bridge and a π-stacking interaction (all interactions shown as green dashes). <i>Sm</i>KI-1 residues are represented and labeled in purple, NE residues in gray.</p

<i>Sm</i>KI-1 reduces hepatic APAP-induced injury.

<p><b>(a)</b> MPO and <b>(b)</b> Elastase activities were measured in r<i>Sm</i>KI-1 treated mice during liver APAP-induced hepatotoxicity. Treated-mice received <i>Sm</i>KI-1 (10 mg/kg) or PBS vehicle i.v. 15 min prior APAP administration (600 mg/kg). <b>(c)</b> Number of neutrophils per field of view (FOV) in the liver of r<i>Sm</i>KI-1 treated animals. <b>(d)</b> Liver confocal intravital microscopy showing neutrophil (anti-GR1 PE in red) migration into necrotic sites (Sytox green staining) following 24 hours of APAP challenge. Scale bar = 100 μm. <b>(e)</b> Left panels represent histology of hematoxylin and eosin-stained liver sections, scale bar = 300μm. In right panels, liver damage is highlighted. <b>(f)</b> serum ALT levels confirmed severe liver damage in APAP group and reduction of liver necrosis in mice treated with APAP+<i>Sm</i>KI-1. Results are the mean ± SEM of n = 6 per group. An asterisk indicate statistically significant differences of r<i>Sm</i>KI-1 compared to APAP group (p< 0.05) or ** p< 0.005.</p

<i>Sm</i>KI-1 treatment reduces neutrophil migration into pleural cavity in response to carrageenan injection.

<p>After carrageenan injection (2mg/mL) into pleural cavity, animals received an intravenous dose of <i>Sm</i>KI-1 (10 mg/kg) or PBS (vehicle). Four hours later, we recovered cells by washing pleural cavity with PBS. Counting of (<b>a</b>) total cells and (<b>b</b>) neutrophils were performed by cytospin preparations. Specific cell populations in pleural fluid were also evaluated by flow cytometry, being the percentage of (<b>c</b>) neutrophils (Ly6G⁺CD11b⁺), (<b>d</b>) macrophages (F4/80⁺CD11b⁺), and (<b>e</b>) T lymphocytes (CD3⁺ cells) calculated from the total cell numbers. Results are expressed as the number of cells per cavity or percentage of cell subpopulations (mean ± SD) for each treated group (5–6 mice each). An Asterisk indicates statistically significant differences of carrageenan+<i>Sm</i>KI-1 compared to carrageenan vehicle group (p< 0.05).</p