Publications by authors named "Judith V Hobrath"

25 Publications

  • Page 1 of 1

Ligand-dependent downregulation of MR1 cell surface expression.

Proc Natl Acad Sci U S A 2020 05 27;117(19):10465-10475. Epub 2020 Apr 27.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.
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http://dx.doi.org/10.1073/pnas.2003136117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229755PMC
May 2020

Discovery of an Allosteric Binding Site in Kinetoplastid Methionyl-tRNA Synthetase.

ACS Infect Dis 2020 05 28;6(5):1044-1057. Epub 2020 Apr 28.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.

Methionyl-tRNA synthetase (MetRS) is a chemically validated drug target in kinetoplastid parasites and . To date, all kinetoplastid MetRS inhibitors described bind in a similar way to an expanded methionine pocket and an adjacent, auxiliary pocket. In the current study, we have identified a structurally novel class of inhibitors containing a 4,6-diamino-substituted pyrazolopyrimidine core (the MetRS02 series). Crystallographic studies revealed that MetRS02 compounds bind to an allosteric pocket in MetRS not previously described, and enzymatic studies demonstrated a noncompetitive mode of inhibition. Homology modeling of the MetRS enzyme revealed key differences in the allosteric pocket between the and enzymes. These provide a likely explanation for the lower MetRS02 potencies that we observed for the enzyme compared to the enzyme. The identification of a new series of MetRS inhibitors and the discovery of a new binding site in kinetoplastid MetRS enzymes provide a novel strategy in the search for new therapeutics for kinetoplastid diseases.
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http://dx.doi.org/10.1021/acsinfecdis.9b00453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294809PMC
May 2020

Slow-Binding Inhibition of Mycobacterium tuberculosis Shikimate Kinase by Manzamine Alkaloids.

Biochemistry 2018 08 31;57(32):4923-4933. Epub 2018 Jul 31.

Department of Drug Discovery and Development, Harrison School of Pharmacy , Auburn University , 4306 Walker Building , Auburn , Alabama 36849 , United States.

Tuberculosis represents a significant public health crisis. There is an urgent need for novel molecular scaffolds against this pathogen. We screened a small library of marine-derived compounds against shikimate kinase from Mycobacterium tuberculosis ( MtSK), a promising target for antitubercular drug development. Six manzamines previously shown to be active against M. tuberculosis were characterized as MtSK inhibitors: manzamine A (1), 8-hydroxymanzamine A (2), manzamine E (3), manzamine F (4), 6-deoxymanzamine X (5), and 6-cyclohexamidomanzamine A (6). All six showed mixed noncompetitive inhibition of MtSK. The lowest K values were obtained for 6 across all MtSK-substrate complexes. Time-dependent analyses revealed two-step, slow-binding inhibition. The behavior of 1 was typical; initial formation of an enzyme-inhibitor complex (EI) obeyed an apparent K of ∼30 μM with forward ( k) and reverse ( k) rate constants for isomerization to an EI* complex of 0.18 and 0.08 min, respectively. In contrast, 6 showed a lower K for the initial encounter complex (∼1.5 μM), substantially faster isomerization to EI* ( k = 0.91 min), and slower back conversion of EI* to EI ( k = 0.04 min). Thus, the overall inhibition constants, K*, for 1 and 6 were 10 and 0.06 μM, respectively. These findings were consistent with docking predictions of a favorable binding mode and a second, less tightly bound pose for 6 at MtSK. Our results suggest that manzamines, in particular 6, constitute a new scaffold from which drug candidates with novel mechanisms of action could be designed for the treatment of tuberculosis by targeting MtSK.
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http://dx.doi.org/10.1021/acs.biochem.8b00231DOI Listing
August 2018

Oxazole and thiazole analogs of sulindac for cancer prevention.

Future Med Chem 2018 04 19;10(7):743-753. Epub 2018 Apr 19.

Division of Hematology & Oncology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

Aim: Experimental and epidemiological studies and clinical trials suggest that nonsteroidal anti-inflammatory drugs possess antitumor potential. Sulindac, a widely used nonsteroidal anti-inflammatory drug, can prevent adenomatous colorectal polyps and colon cancer, especially in patients with familial adenomatous polyposis. Sulindac sulfide amide (SSA) is an amide-linked sulindac sulfide analog that showed in vivo antitumor activity in a human colon tumor xenograft model. Results/methodology: A new analog series with heterocyclic rings such as oxazole or thiazole at the C-2 position of sulindac was prepared and screened against prostate, colon and breast cancer cell lines to probe the effect of these novel substitutions on the activity of sulindac analogs.

Conclusion: In general, replacement of the amide function of SSA analogs had a negative impact on the cell lines tested. A small number of hits incorporating rigid oxazole or thiazole groups in the sulindac scaffold in place of the amide linkage show comparable activity to our lead agent SSA.
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http://dx.doi.org/10.4155/fmc-2017-0182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367724PMC
April 2018

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone.

Biochim Biophys Acta Proteins Proteom 2018 May - Jun;1866(5-6):731-739. Epub 2018 Apr 12.

Department of Drug Discovery and Development, Harrison School of Pharmacy, 3306 Walker Building, Auburn University, Auburn, AL 36849, USA. Electronic address:

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M s. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.
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http://dx.doi.org/10.1016/j.bbapap.2018.04.007DOI Listing
July 2018

Amine Containing Analogs of Sulindac for Cancer Prevention.

Open Med Chem J 2018 31;12:1-12. Epub 2018 Jan 31.

Division of Hematology and Oncology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

Background: Sulindac belongs to the chemically diverse family of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) that effectively prevent adenomatous colorectal polyps and colon cancer, especially in patients with familial adenomatous polyposis. Sulindac sulfide amide (SSA), an amide analog of sulindac sulfide, shows insignificant COX-related activity and toxicity while enhancing anticancer activity and demonstrating xenograft activity.

Objective: Develop structure-activity relationships in the sulindac amine series and identify analogs with promising anticancer activities.

Method: A series of sulindac amine analogs were designed and synthesized and then further modified in a "libraries from libraries" approach to produce amide, sulfonamide and N,N-disubstituted sulindac amine sub-libraries. All analogs were screened against three cancer cell lines (prostate, colon and breast).

Results: Several active compounds were identified cancer cell line screening with the most potent compound () in the nanomolar range.

Conclusion: Compound and analogs showing the most potent inhibitory activity may be considered for further design and optimization efforts as anticancer hit scaffolds.
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http://dx.doi.org/10.2174/1874104501812010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817852PMC
January 2018

A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase.

Bioorg Med Chem Lett 2018 02 5;28(4):802-808. Epub 2017 Dec 5.

Department of Drug Discovery and Development, Harrison School of Pharmacy, 4306 Walker Building, Auburn University, Auburn, AL 36849, USA. Electronic address:

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.
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http://dx.doi.org/10.1016/j.bmcl.2017.12.002DOI Listing
February 2018

Synthesis and preliminary assessment of the anticancer and Wnt/β-catenin inhibitory activity of small amide libraries of fenamates and profens.

Med Chem Res 2017;26(11):3038-3045. Epub 2017 Aug 5.

Division of Hematology and Oncology, The University of Alabama at Birmingham, Birmingham, Alabama 35294 USA.

As part of an ongoing program to study the anticancer activity of non-steroidal anti-inflammatory drugs (NSAIDs) through generating diversity libraries of multiple NSAID scaffolds, we synthesized a series of NSAID amide derivatives and screened these sets against three cancer cell lines (prostate, colon and breast) and Wnt/β-catenin signaling. The evaluated amide analog libraries show significant anticancer activity/cell proliferation inhibition, and specific members of the sets show inhibition of Wnt/β-catenin signaling.
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http://dx.doi.org/10.1007/s00044-017-2001-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656725PMC
August 2017

Diverse amide analogs of sulindac for cancer treatment and prevention.

Bioorg Med Chem Lett 2017 10 13;27(20):4614-4621. Epub 2017 Sep 13.

Division of Hematology and Oncology, The University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address:

Sulindac is a non-steroidal anti-inflammatory drug (NSAID) that has shown significant anticancer activity. Sulindac sulfide amide (1) possessing greatly reduced COX-related inhibition relative to sulindac displayed in vivo antitumor activity that was comparable to sulindac in a human colon tumor xenograft model. Inspired by these observations, a panel of diverse sulindac amide derivatives have been synthesized and their activity probed against three cancer cell lines (prostate, colon and breast). A neutral analog, compound 79 was identified with comparable potency relative to lead 1 and activity against a panel of lymphoblastic leukemia cell lines. Several new series also show good activity relative to the parent (1), including five analogs that also possess nanomolar inhibitory potencies against acute lymphoblastic leukemia cells. Several new analogs identified may serve as anticancer lead candidates for further development.
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http://dx.doi.org/10.1016/j.bmcl.2017.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022754PMC
October 2017

Characterization of a new pathway that activates lumisterol in vivo to biologically active hydroxylumisterols.

Sci Rep 2017 09 12;7(1):11434. Epub 2017 Sep 12.

School of Molecular Sciences, The University of Western Australia, Perth, WA, Australia.

Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.
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http://dx.doi.org/10.1038/s41598-017-10202-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595834PMC
September 2017

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as "biased" agonists on VDR and inverse agonists on RORα and RORγ.

J Steroid Biochem Mol Biol 2017 10 28;173:42-56. Epub 2016 Sep 28.

Cell Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)D3, 20,22(OH)D3 and 17,20,23(OH)D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)D3, 20,23(OH)D3, 17,20,23(OH)D3, 1,20(OH)D3, 1,20,23(OH)D3, 1,20,22(OH)D3, 20,24(OH)D3, 1,20,24(OH)D3, 20,25(OH)D3, 1,20,25(OH)D3, 20,26(OH)D3 and 1,20,26(OH)D3. 20(OH)D3 and 20,23(OH)D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as "biased" agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)D3 have been tested in a cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as "biased" agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)D3 and VDR.
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http://dx.doi.org/10.1016/j.jsbmb.2016.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373926PMC
October 2017

Design and Synthesis of Nonpeptide Inhibitors of Hepatocyte Growth Factor Activation.

ACS Med Chem Lett 2016 Feb 22;7(2):177-81. Epub 2015 Dec 22.

Departments of Chemistry, Oncology and High Throughput Screening, Drug Discovery Division, and Toxicology and Pathology Services, Drug Development Division, Southern Research , 2000 Ninth Avenue S., Birmingham, Alabama 35205, United States.

In this letter we report first nonpeptide inhibitors of hepatocyte growth factor (HGF) activation. These compounds inhibit the three proteases (matriptase, hepsin, and HGF activator) required for HGF maturation. We show that 6, 8a, 8b, and 8d block activation of fibroblast-derived pro-HGF, thus preventing fibroblast-induced scattering of DU145 prostate cancer cells. Compound 6 (SRI 31215) is very soluble (91 μM) and has excellent microsome stability (human t 1/2 = 162 min; mouse t 1/2 = 296 min). In mouse 6 has an in vivo t 1/2 = 5.8 h following IV administration. The high solubility of 6 and IV t 1/2 make this compound a suitable prototype "triplex inhibitor" for the study of the inhibition of HGF activation in vivo.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753539PMC
February 2016

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

PLoS One 2014 19;9(11):e113568. Epub 2014 Nov 19.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at (535)GAC>(535)AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113568PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237437PMC
December 2015

Design, synthesis, and structure-activity relationship studies of a series of [4-(4-carboxamidobutyl)]-1-arylpiperazines: insights into structural features contributing to dopamine D3 versus D2 receptor subtype selectivity.

J Med Chem 2014 Aug 15;57(16):7042-60. Epub 2014 Aug 15.

Organic Chemistry Department, Southern Research Institute , Birmingham, Alabama 35205, United States.

Antagonist and partial agonist modulators of the dopamine D3 receptor (D3R) have emerged as promising therapeutics for the treatment of substance abuse and neuropsychiatric disorders. However, development of druglike lead compounds with selectivity for the D3 receptor has been challenging because of the high sequence homology between the D3R and the dopamine D2 receptor (D2R). In this effort, we synthesized a series of acylaminobutylpiperazines incorporating aza-aromatic units and evaluated their binding and functional activities at the D3 and D2 receptors. Docking studies and results from evaluations against a set of chimeric and mutant receptors suggest that interactions at the extracellular end of TM7 contribute to the D3R versus D2R selectivity of these ligands. Molecular insights from this study could potentially enable rational design of potent and selective D3R ligands.
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http://dx.doi.org/10.1021/jm500801rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148173PMC
August 2014

Identification of shikimate kinase inhibitors among anti-Mycobacterium tuberculosis compounds by LC-MS.

Tuberculosis (Edinb) 2014 Mar 31;94(2):152-8. Epub 2013 Dec 31.

Department of Pharmacal Sciences, Harrison School of Pharmacy, 4306 Walker Building, Auburn University, Auburn, AL 36849, USA. Electronic address:

Increasing drug resistance has challenged the control and treatment of tuberculosis, sparking recent interest in finding new antitubercular agents with different chemical scaffolds and mechanisms of action. Mycobacterium tuberculosis shikimate kinase (MtSK), an enzyme present in the shikimate pathway in bacteria, is essential for the survival of the tubercle bacillus, representing an ideal target for therapeutic intervention given its absence in mammals. In this study, a small library of 404 synthetic antimycobacterial compounds identified and supplied through the NIH Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) high throughput screening program against whole cell M. tuberculosis H37Rv was further screened using a mass spectrometry-based functional assay in order to identify a potential enzymatic target. Fourteen compounds containing an oxadiazole-amide or a 2-aminobenzothiazole core scaffold showed MtSK inhibitory activity at 50 μM, with the lowest giving an IC50 of 1.94 μM. Induced fit docking studies suggested that the scaffolds shared by these compounds fit well in the shikimate binding pocket of MtSK. In summary, we report new early discovery stage lead scaffolds targeting the essential protein MtSK that can be further pursued in a rational drug design program for the discovery of more selective antitubercular drugs.
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http://dx.doi.org/10.1016/j.tube.2013.12.004DOI Listing
March 2014

Combining computational methods for hit to lead optimization in Mycobacterium tuberculosis drug discovery.

Pharm Res 2014 Feb 17;31(2):414-35. Epub 2013 Oct 17.

Collaborative Drug Discovery, 1633 Bayshore Highway, Suite 342, Burlingame, California, 94010, USA,

Purpose: Tuberculosis treatments need to be shorter and overcome drug resistance. Our previous large scale phenotypic high-throughput screening against Mycobacterium tuberculosis (Mtb) has identified 737 active compounds and thousands that are inactive. We have used this data for building computational models as an approach to minimize the number of compounds tested.

Methods: A cheminformatics clustering approach followed by Bayesian machine learning models (based on publicly available Mtb screening data) was used to illustrate that application of these models for screening set selections can enrich the hit rate.

Results: In order to explore chemical diversity around active cluster scaffolds of the dose-response hits obtained from our previous Mtb screens a set of 1924 commercially available molecules have been selected and evaluated for antitubercular activity and cytotoxicity using Vero, THP-1 and HepG2 cell lines with 4.3%, 4.2% and 2.7% hit rates, respectively. We demonstrate that models incorporating antitubercular and cytotoxicity data in Vero cells can significantly enrich the selection of non-toxic actives compared to random selection. Across all cell lines, the Molecular Libraries Small Molecule Repository (MLSMR) and cytotoxicity model identified ~10% of the hits in the top 1% screened (>10 fold enrichment). We also showed that seven out of nine Mtb active compounds from different academic published studies and eight out of eleven Mtb active compounds from a pharmaceutical screen (GSK) would have been identified by these Bayesian models.

Conclusion: Combining clustering and Bayesian models represents a useful strategy for compound prioritization and hit-to lead optimization of antitubercular agents.
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http://dx.doi.org/10.1007/s11095-013-1172-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946937PMC
February 2014

Identification of novel Mt-Guab2 inhibitor series active against M. tuberculosis.

PLoS One 2012 29;7(3):e33886. Epub 2012 Mar 29.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD(+)). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL(-1). Among the identified ligands, two inhibitors have nanomolar K(i)s against the Mt-GuaB2 enzyme.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033886PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315515PMC
November 2012

Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2012 Mar 4;92(2):139-47. Epub 2012 Jan 4.

Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, AL 35294, USA.

5'-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 2.4.2.28) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in Mycobacterium tuberculosis, was expressed in and purified from Escherichia coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-l-homocysteine were poor substrates and no activity was detected with 5'-methylthioinosine, the other natural purines, or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the K(m) value for MTA was 9 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicated that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure-activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
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http://dx.doi.org/10.1016/j.tube.2011.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288397PMC
March 2012

High throughput screening of a library based on kinase inhibitor scaffolds against Mycobacterium tuberculosis H37Rv.

Tuberculosis (Edinb) 2012 Jan 25;92(1):72-83. Epub 2011 Jun 25.

Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA.

Kinase targets are being pursued in a variety of diseases beyond cancer, including immune and metabolic as well as viral, parasitic, fungal and bacterial. In particular, there is a relatively recent interest in kinase and ATP-binding targets in Mycobacterium tuberculosis in order to identify inhibitors and potential drugs for essential proteins that are not targeted by current drug regimens. Herein, we report the high throughput screening results for a targeted library of approximately 26,000 compounds that was designed based on current kinase inhibitor scaffolds and known kinase binding sites. The phenotypic data presented herein may form the basis for selecting scaffolds/compounds for further enzymatic screens against specific kinase or other ATP-binding targets in Mycobacterium tuberculosis based on the apparent activity against the whole bacteria in vitro.
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http://dx.doi.org/10.1016/j.tube.2011.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183257PMC
January 2012

Identification of novel small molecule activators of nuclear factor-κB with neuroprotective action via high-throughput screening.

J Neurosci Res 2011 Jan;89(1):58-72

Laboratory of Neuropharmacology, Department of Biochemistry and Molecular Biology, Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35205, USA.

Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.
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http://dx.doi.org/10.1002/jnr.22526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280078PMC
January 2011

A high-throughput screen for chemical inhibitors of exocytic transport in yeast.

Chembiochem 2010 Jun;11(9):1291-301

Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA.

Most of the components of the membrane and protein traffic machinery were discovered by perturbing their functions, either with bioactive compounds or by mutations. However, the mechanisms responsible for exocytic transport vesicle formation at the Golgi and endosomes are still largely unknown. Both the exocytic traffic routes and the signaling pathways that regulate these routes are highly complex and robust, so that defects can be overcome by alternate pathways or mechanisms. A classical yeast genetic screen designed to account for the robustness of the exocytic pathway identified a novel conserved gene, AVL9, which functions in late exocytic transport. We now describe a chemical-genetic version of the mutant screen, in which we performed a high-throughput phenotypic screen of a large compound library and identified novel small-molecule secretory inhibitors. To maximize the number and diversity of our hits, the screen was performed in a pdr5Delta snq2Delta mutant background, which lacks two transporters responsible for pleiotropic drug resistance. However, we found that deletion of both transporters reduced the fitness of our screen strain, whereas the pdr5Delta mutation had a relatively small effect on growth and was also the more important transporter mutation for conferring sensitivity to our hits. In this and similar chemical-genetic yeast screens, using just a single pump mutation might be sufficient for increasing hit diversity while minimizing the physiological effects of transporter mutations.
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http://dx.doi.org/10.1002/cbic.200900681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3090732PMC
June 2010

Antituberculosis activity of the molecular libraries screening center network library.

Tuberculosis (Edinb) 2009 Sep 26;89(5):354-63. Epub 2009 Sep 26.

Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA.

There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against Mycobacterium tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein.
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http://dx.doi.org/10.1016/j.tube.2009.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792876PMC
September 2009

High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv.

Tuberculosis (Edinb) 2009 Sep 15;89(5):334-53. Epub 2009 Sep 15.

Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA.

There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein.
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http://dx.doi.org/10.1016/j.tube.2009.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255569PMC
September 2009

Assay development and high-throughput antiviral drug screening against Bluetongue virus.

Antiviral Res 2009 Sep 24;83(3):267-73. Epub 2009 Jun 24.

Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, AL 35205, United States.

Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z'-values > or = 0.70, Coefficient of Variations > or = 5.68 and signal-to-background ratio > or = 7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194,950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC(50) < or =100 microM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease.
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http://dx.doi.org/10.1016/j.antiviral.2009.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727572PMC
September 2009

A novel sulindac derivative that does not inhibit cyclooxygenases but potently inhibits colon tumor cell growth and induces apoptosis with antitumor activity.

Cancer Prev Res (Phila) 2009 Jun 26;2(6):572-80. Epub 2009 May 26.

Drug Discovery and Development Divisions, Southern Research Institute, Birmingham, Alabama 35205, USA.

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC(50) values of 2 to 5 micromol/L compared with 73 to 85 micromol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC(50) for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.
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http://dx.doi.org/10.1158/1940-6207.CAPR-09-0001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227417PMC
June 2009
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