Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PLpro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC50 value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds

(+)-Strebloside-Induced Cytotoxicity in Ovarian Cancer Cells Is Mediated through Cardiac Glycoside Signaling Networks

(+)-Strebloside, a cardiac glycoside isolated from the stem bark of <i>Streblus asper</i> collected in Vietnam, has shown some potential for further investigation as an antineoplastic agent. A mechanistic study using an in vitro assay and molecular docking analysis indicated that (+)-strebloside binds and inhibits Na⁺/K⁺-ATPase in a similar manner to digitoxin. Inhibition of growth of different high-grade serous ovarian cancer cells including OVCAR3, OVSAHO, Kuramochi, OVCAR4, OVCAR5, and OVCAR8 resulted from treatment with (+)-strebloside. Furthermore, this compound blocked cell cycle progression at the G2 phase and induced PARP cleavage, indicating apoptosis activation in OVCAR3 cells. (+)-Strebloside potently inhibited mutant p53 expression through the induction of ERK pathways and inhibited NF-κB activity in human ovarian cancer cells. However, in spite of its antitumor potential, the overall biological activity of (+)-strebloside must be regarded as being typical of better-known cardiac glycosides such as digoxin and ouabain. Further chemical alteration of cardiac glycosides might help to reduce negative side effects while increasing cancer cell cytotoxicity

Total Syntheses of (−)-Spirooliganones A and B and Their Diastereoisomers: Absolute Stereochemistry and Inhibitory Activity against Coxsackie Virus B3

To investigate the effects of configuration on bioactivity, spirooliganones A and B and their six diastereoisomers (<b>1</b>–<b>8</b>) were synthesized in 11 steps. The key benzopyran core was assembled by intermolecular [4 + 2] hetero-Diels–Alder cycloaddition between (−)-sabinene and <i>o</i>-quinone methide, which was generated from the corresponding <i>o</i>-hydroxybenzyl alcohol. After establishing the absolute configuration, the inhibitory activities of spirooliganones <b>1</b>–<b>8</b> against Coxsackie virus B3 were evaluated, and the primary structure–activity relationships were analyzed. Compound <b>3</b> was the most potent compound, with an IC₅₀ of 0.41 μM

The effects of 1, 9, 32 and 33 on cell cycle distribution in A549 cells.^a

a<p>These data indicate the percentage of cells in G₀/G₁, S, and G₂/M phases of the cell cycle. Each value is the mean ± SD of three determinations. (*, p<0.05 compared with control; **, p<0.01 compared with control).</p

Cytotoxic activities of deoxytylophorinine and its derivatives <i>in vitro.</i>

a<p>IC₅₀ values are the test compounds concentration (µM) that inhibited the cell growth by 50%. Data represent the mean values ± standard deviation of three dependent experiments performed in triplicate (*, p<0.05 compared with compound <b>1</b>; **, p<0.01 compared with compound <b>1</b>; ^#, p<0.05 compared with Doxorubicin; ^##, p<0.01 compared with Doxorubicin). These IC₅₀ values were all measured for 72 h treatment. IC₅₀ values for 24 h treatment were in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342.s005" target="_blank">Table S1</a>.</p

Effects of 1, 9, 12, 16, 32, 33, and 35 on phosphorylated and total proteins of Akt and ERK in A549 cells.

<p>A549 cells were untreated or treated with 500 nM of 1, 9, 12, 16, 32, 33, and 35 for 24 h. Following 24 h of recovery, cell lysates were prepared and equal amounts of protein were analyzed by SDS-PAGE. Immunoblots of cellular lysates were analyzed by antibodies of phosphorylated and total proteins of Akt and ERK, with the expression of β-actin as an internal control. A. one of the selected immunoblot analysis results of pAkt, Akt, pERK, ERK and β-actin. B. densitometric analysis results of pAkt, Akt, pERK, and ERK normalized to β-actin expression. Each histogram represents the mean values ± standard deviation of three dependent experiments. (*, p<0.05 compared with control; **, p<0.01 compared with control).</p

Synthesis of deoxytylophorinine and its derivatives.

<p>Synthesis of deoxytylophorinine and its derivatives.</p

Structures of deoxytylophorinine, its intermediate and derivatives.

a<p>Compounds <b>1</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Li1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Wang2" target="_blank">[37]</a>, <b>2</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Mulchandani2" target="_blank">[35]</a>, <b>3</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Buckley1" target="_blank">[38]</a>, <b>4</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Faber1" target="_blank">[42]</a>, <b>6</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Takashi1" target="_blank">[43]</a>, <b>7</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Takashi1" target="_blank">[43]</a>, <b>9</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Mulchandani3" target="_blank">[44]</a>, and <b>33</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030342#pone.0030342-Wang4" target="_blank">[45]</a> are known, while others are new synthesized.</p>b<p>These compounds have been claimed in a pending patent (PCT/CN2010/070832).</p

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus

Effects of 1, 9, 12, 16, 32, 33, and 35 on cyclins in A549 cells.

<p>A549 cells were untreated or treated with 500 nM of 1, 9, 12, 16, 32, 33, and 35 for 24 h. Following 24 h of recovery, cell lysates were prepared and equal amounts of protein were analyzed by SDS-PAGE. Immunoblots of cellular lysates were analyzed by antibodies of CDK2, cyclin A, cyclin B1, cyclin D1, and cyclin E, with the expression of β-actin as an internal control. A. one of the selected immunoblot analysis results of CDK2, CyclinA, CyclinB1, CyclinD1, CyclinE and β-actin. B. densitometric analysis results of CDK2, CyclinA, CyclinB1, CyclinD1, and CyclinE normalized to β-actin expression. Each histogram represents the mean values ± standard deviation of three dependent experiments. (*, p<0.05 compared with control; **, p<0.01 compared with control).</p