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Mucosal-associated invariant T cells (MAIT cells) represent a potential therapeutic target as they can tune or enhance immune responses. They recognise and become activated by antigens, presented by the monomorphic MHC-I related molecule, MR1. To assess the significance of MAIT cells in human diseases, a better understanding of the MAIT cell-MR1-antigen interaction is imperative. Easy access to MR1 ligands and MAIT cells activators can help achieve this. In this review, we summarise current literature that has identified the natural ligands and drug-like molecules that activate MAIT cells and provide insight into their key molecular interactions with MR1 and MAIT T cell receptors (TCRs). We focus on the progress made in synthesizing and isolating 5-amino-6-d-ribitylaminouracil (5-A-RU), a key precursor in the synthesis of the known natural ligands, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil(5-OP-RU) and 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU), and also on the stabilisation and optimisation of the latter compounds.

Significance MR1 is a monomorphic major histocompatibility complex (MHC) class I-like molecule that presents ligands to Mucosal Associated Invariant T cells. MR1 antigen presentation at the cell surface is tightly regulated by ligand availability. Although previously described MR1 ligands facilitate translocation of ER-resident MR1 to the cell surface, we describe nonmicrobial ligands, DB28 and its ester analogue NV18.1, which retain MR1 in the ER in an immature ligand-receptive form and competitively inhibit stimulatory ligands. We provide the molecular and functional basis underpinning the interactions of this class of ligands with MR1., 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.

Methionyl-tRNA synthetase (MetRS) is a chemically validated drug target in kinetoplastid parasites Trypanosoma brucei and Leishmania donovani. 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 L. major MetRS not previously described, and enzymatic studies demonstrated a noncompetitive mode of inhibition. Homology modeling of the Trypanosoma cruzi MetRS enzyme revealed key differences in the allosteric pocket between the T. cruzi and Leishmania enzymes. These provide a likely explanation for the lower MetRS02 potencies that we observed for the T. cruzi enzyme compared to the Leishmania 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.

Activation of invariant natural killer T lymphocytes (iNKT cells) by α-galactosylceramide (α-GC) elicits a range of pro-inflammatory or anti-inflammatory immune responses. We report the synthesis and characterization of a series of α-GC analogues with acyl chains of varying length and a terminal benzophenone. These bound efficiently to the glycolipid antigen presenting protein CD1d, and upon photoactivation formed stable CD1d-glycolipid covalent conjugates. Conjugates of benzophenone α-GCs with soluble or cell-bound CD1d proteins retained potent iNKT cell activating properties, with biologic effects that were modulated by acyl chain length and the resulting affinities of conjugates for iNKT cell antigen receptors. Analysis by mass spectrometry identified a unique covalent attachment site for the glycolipid ligands in the hydrophobic ligand binding pocket of CD1d. The creation of covalent conjugates of CD1d with α-GC provides a new tool for probing the biology of glycolipid antigen presentation, as well as opportunities for developing effective immunotherapeutics.

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 KI 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 KI of ∼30 μM with forward ( k5) and reverse ( k6) rate constants for isomerization to an EI* complex of 0.18 and 0.08 min-1, respectively. In contrast, 6 showed a lower KI for the initial encounter complex (∼1.5 μM), substantially faster isomerization to EI* ( k5 = 0.91 min-1), and slower back conversion of EI* to EI ( k6 = 0.04 min-1). Thus, the overall inhibition constants, KI*, 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.

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−1 s−1. 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.

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., Graphical abstract

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, 1HNMR, 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.

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 activityin vitroand demonstratingin vivoxenograft 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 identifiedviain vitrocancer cell line screening with the most potent compound (26) in the nanomolar range.Conclusion:Compound26and analogs showing the most potent inhibitory activity may be considered for further design and optimization efforts as anticancer hit scaffolds.

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.

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.

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.

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.

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.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.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.Combining clustering and Bayesian models represents a useful strategy for compound prioritization and hit-to lead optimization of antitubercular agents.

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)2D3 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)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3. 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)2D3, 20,23(OH)2D3, 17,20,23(OH)3D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,22(OH)3D3, 20,24(OH)2D3, 1,20,24(OH)3D3, 20,25(OH)2D3, 1,20,25(OH)3D3, 20,26(OH)2D3 and 1,20,26(OH)3D3. 20(OH)D3 and 20,23(OH)2D3 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)2D3. 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)2D3 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)2D3 and VDR.

Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca(2+) homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca(2+) from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR.

A highly reproducible and robust cell-based high-throughput screening (HTS) assay was adapted for screening of small molecules for antiviral activity against influenza virus strain A/Vietnam/1203/2004 (H5N1). The NIH Molecular Libraries Small Molecule Repository (MLSMR) Molecular Libraries Screening Centers Network (MLSCN) 100,000-compound library was screened at 50 µM. The “hit” rate (>25% inhibition of the viral cytopathic effect) from the single-dose screen was 0.32%. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen yielded 5 active compounds (SI value >3). One compound showed an SI 50 value of greater than 3, 3 compounds had SI values ranging from greater than 14 to 34, and the most active compound displayed an SI value of 94. The active compounds represent 2 different classes of molecules, benzoquinazolinones and thiazoloimidazoles, which have not been previously identified as having antiviral/anti-influenza activity. These molecules were also effective against influenza A/ California/04/2009 virus (H1N1) and other H1N1 and H5N1 virus strains in vitro but not H3N2 strains. Real-time qRT-PCR results reveal that these chemotypes significantly reduced M1 RNA levels as compared to the no-drug influenza-infected Madin Darby canine kidney cells. (Journal of Biomolecular Screening XXXX:xx-xx)

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.

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.

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.

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.

A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics.

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.

Summary 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.

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.

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.

The PTEN tumor suppressor gene is one of the most commonly mutated genes in human cancer. Because inactivation of PTEN is a somatic event, PTEN mutations represent an important genetic difference between cancer cells and normal cells and therefore a potential anticancer drug target. However, it remains a substantial challenge to identify compounds that target loss-of-function events such as mutations of tumor suppressors. In an effort to identify small molecules that preferentially kill cells with mutations of PTEN, the authors developed and implemented a high-throughput, paired cell-based screen composed of parental HCT116 cells and their PTEN gene-targeted derivatives. From 138 758 compounds tested, two hits were identified, and one, N'-[(1-benzyl-1H-indol-3-yl)methylene]benzenesulfonohydrazide (CID1340132), was further studied using a variety of cell-based models, including HCT116, MCF10A, and HEC1A cells with targeted deletion of either their PTEN or PIK3CA genes. Preferential killing of PTEN and PIK3CA mutant cells was accompanied by DNA damage, inhibition of DNA synthesis, and apoptosis. Taken together, these data validate a cell-based screening approach for identifying lead compounds that target cells with specific tumor suppressor gene mutations and describe a novel compound with preferential killing activity toward PTEN and PIK3CA mutant cells.

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.

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 IC50 values of 2 to 5 μmol/L compared with 73 to 85 μmol/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 IC50 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.

In addition to the rhodopsin crystal structure, high-resolution crystal structures of ligand-mediated G-protein-coupled receptors (GPCRs) have recently become available, and these have become attractive templates for developing homology models of several GPCRs of therapeutic interest. These crystal structures and the homology models derived from them have provided significant insights into ligand–receptor interactions. Moreover, several studies have demonstrated that the structural models are indeed suitable for virtual screening of compound databases to identify new ligands for various GPCRs. Recent examples of such virtual screening against GPCRs are discussed in this review.