Publications by authors named "Michael E Johnson"

105 Publications

Baicalein Is a Phytohormone that Signals Through the Progesterone and Glucocorticoid Receptors.

Horm Cancer 2020 04 7;11(2):97-110. Epub 2020 Mar 7.

Department of Pharmaceutical Sciences, Center for Biomolecular Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, 60607, USA.

While flavonoids have been studied extensively for estrogen receptor activity, they have not been well studied for their ability to modify progesterone receptor (PR) and glucocorticoid receptor (GR) signaling. Three flavonoid compounds, tangeretin, wogonin, and baicalein, were selected for testing for PR and GR activity based on their structural similarity to known phytoprogesterone-like compounds. Each compound was docked in the binding pocket of PR and GR. Of these compounds, baicalein was predicted to be most likely to bind to both receptors. A fluorescence polarization competitive binding assay for PR and GR confirmed that baicalein binds to both the PR and GR with IC values of 15.30 μM and 19.26 μM, respectively. In Ishikawa PR-B and T47D cells, baicalein acted as a PR antagonist in a hormone response element (HRE) luciferase (Luc) assay. In OVCAR5 cells, which only express GR, baicalein was a GR agonist via an HRE/Luc assay and induced GR target genes, FKBP5 and GILZ. RU486, a PR and GR antagonist, abrogated baicalein's activity in OVCAR5 cells, confirming baicalein's activity is mediated through the GR. In vivo, baicalein administered intraperitoneally to female mice twice a week for 4 weeks at a dose of 25 mg/kg induced the GR target gene GILZ in the reproductive tract, which was blocked by RU486. In summary, baicalein has PR antagonist and GR agonist activity in vitro and demonstrates GR agonist activity in the uterus in vivo.
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http://dx.doi.org/10.1007/s12672-020-00382-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236083PMC
April 2020

Structural approaches to pathway-specific antimicrobial agents.

Transl Res 2020 06 6;220:114-121. Epub 2020 Feb 6.

Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois. Electronic address:

This perspective provides an overview of the evolution of antibiotic discovery from a largely phenotypic-based effort, through an intensive structure-based design focus, to a more holistic approach today. The current focus on antibiotic development incorporates assay and discovery conditions that replicate the host environment as much as feasible. They also incorporate several strategies, including target identification and validation within the whole cell environment, a variety of target deconvolution methods, and continued refinement of structure-based design approaches.
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http://dx.doi.org/10.1016/j.trsl.2020.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293926PMC
June 2020

Na/K-ATPase-Targeted Cytotoxicity of (+)-Digoxin and Several Semisynthetic Derivatives.

J Nat Prod 2020 03 25;83(3):638-648. Epub 2020 Feb 25.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States.

(+)-Digoxin () is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides (-) and two new analogues, (+)-8(9)-β-anhydrodigoxigenin () and (+)-17--20,22-dihydro-21α-hydroxydigoxin (), were synthesized from and evaluated for their cytotoxicity toward a small panel of human cancer cell lines. A preliminary structure-activity relationship investigation conducted indicated that the C-12 and C-14 hydroxy groups and the C-17 unsaturated lactone unit are important for to mediate its cytotoxicity toward human cancer cells, but the C-3 glycosyl residue seems to be less critical for such an effect. Molecular docking profiles showed that the cytotoxic and the noncytotoxic derivative bind differentially to Na/K-ATPase. The HO-12β, HO-14β, and HO-3'aα hydroxy groups of (+)-digoxin () may form hydrogen bonds with the side-chains of Asp121 and Asn122, Thr797, and Arg880 of Na/K-ATPase, respectively, but the altered lactone unit of results in a rotation of its steroid core, which depotentiates the binding between this compound and Na/K-ATPase. Thus, was found to inhibit Na/K-ATPase, but did not. In addition, the cytotoxic did not affect glucose uptake in human cancer cells, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na/K-ATPase but not by interacting with glucose transporters.
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http://dx.doi.org/10.1021/acs.jnatprod.9b01060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243443PMC
March 2020

KWO is a Novel Ferroelectric Nanomaterial for Application as a Room Temperature Acetone Sensor.

Nanomaterials (Basel) 2020 Jan 28;10(2). Epub 2020 Jan 28.

Materials and Nanotechnology Program, North Dakota State University, Fargo, ND 58102, USA.

A newly synthesized nanomaterial known as KWO (KWO) exhibits a stable room-temperature ferroelectric property. This unique ferroelectric property has revealed that KWO is a promising material for application in a breath sensor, which can be used for patients to monitor their daily health condition and diagnose disease at every early stage with low cost, convenience, and non-invasion. In this study, we successfully synthesized nano-structured KWO through a low cost but high yield hydrothermal method. The sensing response of KWO to acetone is examined based on a chemiresistive effect. For the first time, we systematically studied how material structures and the component, potassium (K), can affect KWO-based sensing performance. The results indicate that the low temperature ferroelectric property of KWO causes an excellent response to acetone, which is the biomarker for diabetes. The lowest detection limit can be down to 0.1 ppm and the KWO-based sensor can operate at room temperature. In addition, the K component KWO and its crystal structure also play an important role in improving its sensing performance. Our results provide advanced research in (1) exploring the study of KWO material properties by tailoring the concentration of the potassium in KWO and introducing the surfactant Pluronic L-121 in the growing process, and (2) optimizing KWO sensing performance by controlling its material properties.
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http://dx.doi.org/10.3390/nano10020225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074885PMC
January 2020

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

Bioorg Med Chem 2020 02 7;28(4):115301. Epub 2020 Jan 7.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, United States. Electronic address:

A new non-cytotoxic [(+)-17β-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na/K-ATPase. Compound 3 docks deeply in the Na/K-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na/K-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na/K-ATPase. Thus, 3 was found to inhibit Na/K-ATPase, but 1 did not. In addition, the cytotoxic and Na/K-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na/K-ATPase but not by interacting with glucose transporters.
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http://dx.doi.org/10.1016/j.bmc.2019.115301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029422PMC
February 2020

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease.

Proteins 2020 02 9;88(2):345-354. Epub 2019 Sep 9.

Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, Illinois.

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3 -helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.
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http://dx.doi.org/10.1002/prot.25809DOI Listing
February 2020

Identification of Small Molecules Exhibiting Oxacillin Synergy through a Novel Assay for Inhibition of Expression in Methicillin-Resistant Staphylococcus aureus.

Antimicrob Agents Chemother 2019 09 23;63(9). Epub 2019 Aug 23.

Center for Biomolecular Sciences and Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USA

Methicillin-resistant (MRSA) strains that are resistant to all forms of penicillin have become an increasingly common and urgent problem threatening human health. They are responsible for a wide variety of infectious diseases ranging from minor skin abscesses to life-threatening severe infections. The operon that is conserved among strains encodes a three-component signal transduction system () that is responsible for sensing and responding to cell wall stress. We developed a novel and multifaceted assay to identify compounds that potentiate the activity of oxacillin, essentially restoring efficacy of oxacillin against MRSA, and performed high-throughput screening (HTS) to identify oxacillin potentiators. HTS of 13,840 small-molecule compounds from an antimicrobial-focused Life Chemicals library, using the MRSA cell-based assay, identified three different inhibitor scaffolds. Checkerboard assays for synergy with oxacillin, reverse transcriptase PCR (RT-PCR) assays against expression, and direct confirmation of interaction with VraS by surface plasmon resonance (SPR) further verified them to be viable hit compounds. A subsequent structure-activity relationship (SAR) study of the best scaffold with diverse analogs was utilized to improve potency and provides a strong foundation for further development.
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http://dx.doi.org/10.1128/AAC.02593-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709460PMC
September 2019

Exploring small molecules with pan-genotypic inhibitory activities against hepatitis C virus NS3/4A serine protease.

Bioorg Med Chem Lett 2019 08 8;29(16):2349-2353. Epub 2019 Jun 8.

Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA; Biophysics Core at Research Resource Center, University of Illinois at Chicago, Chicago, IL 60612, USA; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, IL 60612, USA. Electronic address:

Among the many Hepatitis C virus (HCV) genotypes and subtypes, genotypes 1b and 3a are most prevalent in United States and Asia, respectively. A total of 132 commercially available analogs of a previous lead compound were initially investigated against wild-type HCV genotype 1b NS3/4A protease. Ten compounds showed inhibitory activities (IC values) below 10 µM with comparable direct binding affinities (K values) determined by surface plasmon resonance (SPR). To identify pan-genotypic inhibitors, these ten selected compounds were tested against four additional genotypes (1a, 2a, 3a, and 4) and three drug-resistant mutants (A156S, R155K, and V36M). Four new analogs have been identified with better activities against all five tested genotypes than the prior lead compound. Further, the original lead compound did not show activity against genotype 3a NS3/4A, whereas four newly identified compounds exhibited IC values below 33 µM against genotype 3a NS3/4A. Encouragingly, the best new compound F1813-0710 possessed promising activity toward genotype 3a, which is a huge improvement over the previous lead compound that had no effect on genotype 3a. This intriguing observation was further analyzed by molecular docking and molecular dynamics (MD) simulations to understand their different binding interactions, which should benefit future pan-genotypic inhibitor design and drug discovery.
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http://dx.doi.org/10.1016/j.bmcl.2019.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690791PMC
August 2019

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

Bioorg Med Chem 2019 05 26;27(10):1981-1989. Epub 2019 Mar 26.

Center for Biomolecular Sciences and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA. Electronic address:

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 (PL), 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 IC 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.
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http://dx.doi.org/10.1016/j.bmc.2019.03.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638567PMC
May 2019

Microbiome dysbiosis is associated with disease duration and increased inflammatory gene expression in systemic sclerosis skin.

Arthritis Res Ther 2019 02 6;21(1):49. Epub 2019 Feb 6.

Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.

Background: Infectious agents have long been postulated to be disease triggers for systemic sclerosis (SSc), but a definitive link has not been found. Metagenomic analyses of high-throughput data allows for the unbiased identification of potential microbiome pathogens in skin biopsies of SSc patients and allows insight into the relationship with host gene expression.

Methods: We examined skin biopsies from a diverse cohort of 23 SSc patients (including lesional forearm and non-lesional back samples) by RNA-seq. Metagenomic filtering and annotation was performed using the Integrated Metagenomic Sequencing Analysis (IMSA). Associations between microbiome composition and gene expression were analyzed using single-sample gene set enrichment analysis (ssGSEA).

Results: We find the skin of SSc patients exhibits substantial changes in microbial composition relative to controls, characterized by sharp decreases in lipophilic taxa, such as Propionibacterium, combined with increases in a wide range of gram-negative taxa, including Burkholderia, Citrobacter, and Vibrio.

Conclusions: Microbiome dysbiosis is associated with disease duration and increased inflammatory gene expression. These data provide a comprehensive portrait of the SSc skin microbiome and its association with local gene expression, which mirrors the molecular changes in lesional skin.
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http://dx.doi.org/10.1186/s13075-019-1816-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366065PMC
February 2019

Hit-to-Lead: Hit Validation and Assessment.

Methods Enzymol 2018 25;610:265-309. Epub 2018 Oct 25.

Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, United States; Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, Chicago, IL, United States. Electronic address:

High-throughput screening assays have become nearly ubiquitous in the search for small compounds or peptides that can modulate biological processes for therapeutic purposes. While many assays have become quite robust, with well-established protocols, the subsequent steps of validating the hits and choosing the best ones to take forward into leads for further chemical development are less established. In this chapter, we describe a variety of approaches, including chemical assessment, the use of various computational approaches, a variety of counter-screens, and "orthogonal" biophysical assays using nuclear magnetic resonance, surface plasmon resonance, isothermal titration calorimetry or thermal shift assays as methods for validating and assessing the quality of hits.
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http://dx.doi.org/10.1016/bs.mie.2018.09.022DOI Listing
June 2019

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions.

Bioorg Med Chem Lett 2018 09 29;28(17):2837-2841. Epub 2018 Jul 29.

Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA; Department of Bioengineering, University of Illinois at Chicago, 835 S Wolcott Ave, Chicago, IL 60612, USA; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA. Electronic address:

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.
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http://dx.doi.org/10.1016/j.bmcl.2018.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109590PMC
September 2018

The Flavonoid Apigenin Is a Progesterone Receptor Modulator with In Vivo Activity in the Uterus.

Horm Cancer 2018 08 7;9(4):265-277. Epub 2018 May 7.

Department of Medicinal Chemistry and Pharmacognosy, Center for Biomolecular Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, 60607, USA.

Apigenin is a flavonoid with well-documented anti-cancer properties; however, its mechanisms of action are still unclear. We previously identified apigenin as a potential phytoprogestin, a natural product with a chemical scaffold that interacts with the progesterone receptor (PR). Our objective was to characterize the ability of apigenin to interact with PR through molecular docking studies, in vitro activity assays, and the ability of apigenin to elicit progestin-like effects in vivo. Molecular docking confirmed that apigenin could interact with PR, though with lower affinity than progesterone due to fewer van der Waals interactions. In Ishikawa cells stably expressing PR-B, apigenin significantly increased progesterone response element/luciferase (PRE/Luc) activity at 5 and 10 μM, but not in the parental Ishikawa cells that lack PR expression. In the presence of 100 nM of progesterone, 10 μM apigenin reduced PRE/Luc activity, indicative of mixed agonist activity. Apigenin also triggered degradation of PR in Ishikawa PR-B cells as measured by western blot. Apigenin reduced proliferation of Ishikawa cells, but through a PR-independent mechanism. In contrast, apigenin and progesterone both stimulated proliferation of T47D cells, an effect blocked by RU486. Apigenin activated other nuclear receptors evidenced by increased luciferase activity in MDA-MB-231 cells, which are PR negative. In vivo, apigenin blocked the genistein-stimulated increase in uterine epithelial cell height; stimulated endometrial expression of Hand2, a transcription factor stimulated by PR, and significantly reduced genistein-induced proliferation. In summary, apigenin is a phytoprogestin, with mixed agonist activity that demonstrates activity in vivo by hindering estrogen receptor-mediated uterine proliferation.
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http://dx.doi.org/10.1007/s12672-018-0333-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222006PMC
August 2018

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques.

Bioorg Med Chem Lett 2018 06 22;28(11):2074-2079. Epub 2018 Apr 22.

Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA; Novalex Therapeutics, Inc., 2242 W Harrison, Chicago, IL 60612, USA. Electronic address:

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC of benzimidazole (-)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.
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http://dx.doi.org/10.1016/j.bmcl.2018.04.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970947PMC
June 2018

Structural characterization of Porphyromonas gingivalis enoyl-ACP reductase II (FabK).

Acta Crystallogr F Struct Biol Commun 2018 02 26;74(Pt 2):105-112. Epub 2018 Jan 26.

Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 South Ashland Avenue, Suite 3100, Chicago, IL 60607-7173, USA.

Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9 Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.
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http://dx.doi.org/10.1107/S2053230X18000262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947681PMC
February 2018

Antisense Long Non-Coding RNAs Are Deregulated in Skin Tissue of Patients with Systemic Sclerosis.

J Invest Dermatol 2018 04 24;138(4):826-835. Epub 2017 Nov 24.

Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands.

Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. We studied differentially expressed coding and non-coding genes in relation to systemic sclerosis pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy-derived RNAs from 14 early systemic sclerosis patients and six healthy individuals were sequenced with ion-torrent and analyzed using DEseq2. Overall, 4,901 genes with a fold change >1.5 and a false discovery rate <5% were detected in patients versus controls. Upregulated genes clustered in immunologic, cell adhesion, and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense-antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1, and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected P-value (P < 0.0023, P = 1.1 × 10, 1.4 × 10, 1.7 × 10, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of systemic sclerosis patients suggesting a novel class of genes involved in pathogenesis of systemic sclerosis.
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http://dx.doi.org/10.1016/j.jid.2017.09.053DOI Listing
April 2018

A novel series of enoyl reductase inhibitors targeting the ESKAPE pathogens, Staphylococcus aureus and Acinetobacter baumannii.

Bioorg Med Chem 2018 01 11;26(1):65-76. Epub 2017 Nov 11.

Novalex Therapeutics, 2242 W. Harrison, Chicago, IL 60612, United States. Electronic address:

S. aureus and A. baumannii are among the ESKAPE pathogens that are increasingly difficult to treat due to the rise in the number of drug resistant strains. Novel therapeutics targeting these pathogens are much needed. The bacterial enoyl reductase (FabI) is as potentially significant drug target for developing pathogen-specific antibiotics due to the presence of alternate FabI isoforms in many other bacterial species. We report the identification and development of a novel N-carboxy pyrrolidine scaffold targeting FabI in S. aureus and A. baumannii, two pathogens for which FabI essentiality has been established. This scaffold is unrelated to other known antibiotic families, and FabI is not targeted by any currently approved antibiotic. Our data shows that this scaffold displays promising enzyme inhibitory activity against FabI from both S. aureus and A. baumannii, as well as encouraging antibacterial activity in S. aureus. Compounds also display excellent synergy when combined with colistin and tested against A. baumannii. In this combination the MIC of colistin is reduced by 10-fold. Our first generation compound displays promising enzyme inhibition, targets FabI in S. aureus with a favorable selectivity index (ratio of cytotoxicity to MIC), and has excellent synergy with colistin against A. baumannii, including a multidrug resistant strain.
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http://dx.doi.org/10.1016/j.bmc.2017.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733704PMC
January 2018

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

J Nat Prod 2017 03 24;80(3):659-669. Epub 2017 Feb 24.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago , Chicago, Illinois 60612, United States.

(+)-Strebloside, a cardiac glycoside isolated from the stem bark of Streblus asper 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.
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http://dx.doi.org/10.1021/acs.jnatprod.6b01150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768141PMC
March 2017

Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus.

Antiviral Res 2017 Mar 26;139:49-58. Epub 2016 Dec 26.

Novalex Therapeutics, Inc., 2242 W Harrison Suite 201, Chicago, IL 60612, USA. Electronic address:

Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain-Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activity (IC) and binding affinity (K) of ∼5-10 μM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a K value of 9.5 μM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a "pre-open conformation", a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.
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http://dx.doi.org/10.1016/j.antiviral.2016.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627664PMC
March 2017

Benzimidazole-Based FabI Inhibitors: A Promising Novel Scaffold for Anti-staphylococcal Drug Development.

ACS Infect Dis 2017 01 27;3(1):54-61. Epub 2016 Oct 27.

Novalex Therapeutics , 2242 W. Harrison, Chicago, Illinois 60612, United States.

The enoyl-ACP reductase (FabI) enzyme is a well validated target for anti-staphylococcal drug discovery and development. With the goal of finding alternate therapeutics for drug-resistant strains of Staphylococcus aureus, such as methicillin-resistant S. aureus (MRSA), our previously published series of benzimidazole-based inhibitors of the FabI enzyme from Francisella tularensis (FtFabI) have been evaluated against FabI from S. aureus (SaFabI). We report here the preliminary structure-activity relationship of this series and the prioritization of compounds toward lead optimization. Mutational studies have identified key residues that contribute toward stabilizing the inhibitors in the active site of FabI. Mutations that do not significantly impact enzyme function but destabilize inhibitor binding are more likely to occur in nature as organisms evolve to evade the action of antibiotics leading to resistance. Identifying these residues provides guidance for minimizing susceptibility to resistance. Additionally, we have identified compounds that elicit antibacterial activity through off-target effects and observe that close analogs can display differing modes of action (on-target vs off-target) and need to be individually evaluated early on to prioritize compounds for lead optimization. Overall, our data suggest that the benzimidazole scaffold is a promising scaffold for anti-staphylococcal drug development.
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http://dx.doi.org/10.1021/acsinfecdis.6b00123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659837PMC
January 2017

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase).

Bioorg Med Chem 2016 10 29;24(19):4536-4543. Epub 2016 Jul 29.

Center for Biomolecular Sciences and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S, Ashland, IL 60607, USA. Electronic address:

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045712PMC
http://dx.doi.org/10.1016/j.bmc.2016.07.055DOI Listing
October 2016

Stress granules and RNA processing bodies are novel autoantibody targets in systemic sclerosis.

Arthritis Res Ther 2016 Jan 22;18:27. Epub 2016 Jan 22.

Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.

Background: Autoantibody profiles represent important patient stratification markers in systemic sclerosis (SSc). Here, we performed serum-immunoprecipitations with patient antibodies followed by mass spectrometry (LC-MS/MS) to obtain an unbiased view of all possible autoantibody targets and their associated molecular complexes recognized by SSc.

Methods: HeLa whole cell lysates were immunoprecipitated (IP) using sera of patients with SSc clinically positive for autoantibodies against RNA polymerase III (RNAP3), topoisomerase 1 (TOP1), and centromere proteins (CENP). IP eluates were then analyzed by LC-MS/MS to identify novel proteins and complexes targeted in SSc. Target proteins were examined using a functional interaction network to identify major macromolecular complexes, with direct targets validated by IP-Western blots and immunofluorescence.

Results: A wide range of peptides were detected across patients in each clinical autoantibody group. Each group contained peptides representing a broad spectrum of proteins in large macromolecular complexes, with significant overlap between groups. Network analyses revealed significant enrichment for proteins in RNA processing bodies (PB) and cytosolic stress granules (SG) across all SSc subtypes, which were confirmed by both Western blot and immunofluorescence.

Conclusions: While strong reactivity was observed against major SSc autoantigens, such as RNAP3 and TOP1, there was overlap between groups with widespread reactivity seen against multiple proteins. Identification of PB and SG as major targets of the humoral immune response represents a novel SSc autoantigen and suggests a model in which a combination of chronic and acute cellular stresses result in aberrant cell death, leading to autoantibody generation directed against macromolecular nucleic acid-protein complexes.
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http://dx.doi.org/10.1186/s13075-016-0914-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724133PMC
January 2016

Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

Bioorg Med Chem 2016 Feb 18;24(4):596-605. Epub 2015 Dec 18.

Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland Ave., IL 60607, USA. Electronic address:

The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme.
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http://dx.doi.org/10.1016/j.bmc.2015.12.029DOI Listing
February 2016

Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl-ACP reductase (FabI) benzimidazole inhibitors.

J Comput Chem 2016 Apr 15;37(9):836-47. Epub 2015 Dec 15.

Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, 60607.

Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme.
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http://dx.doi.org/10.1002/jcc.24274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769659PMC
April 2016

Over-expression, purification, and confirmation of Bacillus anthracis transcriptional regulator NprR.

Protein Expr Purif 2016 09 3;125:83-9. Epub 2015 Sep 3.

Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA. Electronic address:

Quorum sensing (QS) has been recognized as an important biological phenomenon in which bacterial cells communicate and coordinate their gene expression and cellular processes with respect to population density. Bacillus anthracis is the etiological agent of fatal pulmonary anthrax infections, and the NprR/NprX QS system may be involved in its pathogenesis. NprR, renamed as aqsR for anthrax quorum sensing Regulator, is a transcriptional regulator that may control the expression of genes required for proliferation and survival. Currently, there is no protocol reported to over-express and purify B. anthracis AqsR. In this study, we describe cloning, purification, and confirmation of functional full-length B. anthracis AqsR protein. The AqsR gene was cloned into the pQE-30 vector with an HRV 3C protease recognition site between AqsR and the N-terminal His6-tag in order to yield near native AqsR after the His-tag cleavage, leaving only two additional amino acid residues at the N-terminus.
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http://dx.doi.org/10.1016/j.pep.2015.08.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853309PMC
September 2016

Gene expression profiling offers insights into the role of innate immune signaling in SSc.

Semin Immunopathol 2015 Sep 30;37(5):501-9. Epub 2015 Jul 30.

Department of Genetics, Geisel School of Medicine at Dartmouth, 7400 Remsen, Hanover, NH, 03755, USA.

Systemic sclerosis (SSc) is characterized by inflammation, vascular dysfunction, and ultimately fibrosis. Progress in understanding disease pathogenesis and developing effective disease treatments has been hampered by an incomplete understanding of SSc heterogeneity. To clarify this, we have used genomic approaches to identify distinct patient subsets based on gene expression patterns in SSc skin and other end-target organs. Here, we review what is known about the gene expression-based subsets in SSc, currently defined as the inflammatory, fibroproliferative, limited, and normal-like subsets. The inflammatory subset of patients is characterized by infiltrating immune cells that include T cells, macrophages, and possibly dendritic cells, although little is known about the mediators these cells secrete and the pathways that govern cell activation. Prior studies have suggested a role for pathogens as a trigger of immune responses in SSc, and recent data have identified viral and mycobiome components as potential environmental triggers. We present a model based on analyses of gene expression data and a review of the literature, which suggests that the gene expression subsets observed in patients possibly represent distinct, interconnected molecular states of disease, to which an innate immune response is central that results in the generation of clinical disease.
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http://dx.doi.org/10.1007/s00281-015-0512-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722533PMC
September 2015

Comparison of radii sets, entropy, QM methods, and sampling on MM-PBSA, MM-GBSA, and QM/MM-GBSA ligand binding energies of F. tularensis enoyl-ACP reductase (FabI).

J Comput Chem 2015 Sep 27;36(25):1859-73. Epub 2015 Jul 27.

Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, 60607.

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA, and QM/MM-GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GB(neck2) , PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r(2)  = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called "multiple independent sampling"), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB(HCT) and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r(2)  = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive "multiple independent sampling method" may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors' relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program.
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http://dx.doi.org/10.1002/jcc.24011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688044PMC
September 2015

Indole trimers with antibacterial activity against Gram-positive organisms produced using combinatorial biocatalysis.

AMB Express 2015 Dec 26;5(1):125. Epub 2015 Jun 26.

CB&I Federal Services, 17 Princess Rd, Lawrenceville, NJ, 08648, USA,

The I100V isoform of toluene-4-monooxygenase was used to catalyze the oxidative polymerization of anthranil and various indoles under mildly acidic conditions, favoring the production of trimers. Compounds produced in sufficient yield were purified and tested for their ability to inhibit the growth of B. anthracis, E. faecalis, L. monocytogenes, S. aureus, and in some cases, F. tularensis. 15 of the compounds displayed promising antibacterial activity (MIC < 5 µg/ml) against one or more of the strains tested, with the best MIC values being <0.8 µg/ml. All of these compounds had good selectivity, showing minimal cytotoxicity towards HepG2 cells. The structure was solved for six of the compounds that could be crystallized, revealing that minimally two classes of indole based trimers were produced. One compound class produced was a group of substituted derivatives of the natural product 2,2-bis(3-indolyl) indoxyl. The other group of compounds identified was classified as tryptanthrin-like compounds, all having multi-ring pendant groups attached at position 11 of tryptanthrin. One compound of particular interest, SAB-J85, had a structure that suggests that any compound, with a ring structure that can be activated by an oxygenase, might serve as a substrate for combinatorial biocatalysis.
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http://dx.doi.org/10.1186/s13568-015-0125-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480272PMC
December 2015

Inhibitor recognition specificity of MERS-CoV papain-like protease may differ from that of SARS-CoV.

ACS Chem Biol 2015 Jun 16;10(6):1456-65. Epub 2015 Mar 16.

†Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 900 S. Ashland, Chicago, Illinois 60607, United States.

The Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) blocking loop 2 (BL2) structure differs significantly from that of SARS-CoV PLpro, where it has been proven to play a crucial role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead inhibitors were tested against MERS-CoV PLpro, none of which were effective against MERS-CoV PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV PLpro, making critical binding interactions difficult to form for similar types of inhibitors. High-throughput screening (HTS) of 25 000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics and competition surface plasmon resonance (SPR) analyses suggested that this compound acts as a competitive inhibitor with an IC50 of 6 μM against MERS-CoV PLpro, indicating that it binds to the active site, whereas it acts as an allosteric inhibitor against SARS-CoV PLpro with an IC50 of 11 μM. These results raised the possibility that inhibitor recognition specificity of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition, inhibitory activity of this compound was selective for SARS-CoV and MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).
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http://dx.doi.org/10.1021/cb500917mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845099PMC
June 2015

Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity.

Bioorg Med Chem 2015 Apr 16;23(7):1492-9. Epub 2015 Feb 16.

Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, Chicago, IL 60607, United States. Electronic address:

N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15μg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.
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http://dx.doi.org/10.1016/j.bmc.2015.02.016DOI Listing
April 2015