Design, Synthesis, and Biological Evaluation of 2-Nitroimidazopyrazin-one/-es with Antitubercular and Antiparasitic Activity.

Tuberculosis and parasitic diseases, such as giardiasis, amebiasis, leishmaniasis, and trypanosomiasis, all urgently require improved treatment options. Recently, it has been shown that antitubercular bicyclic nitroimidazoles such as pretomanid and delamanid have potential as repurposed therapeutics for the treatment of visceral leishmaniasis. Here, we show that pretomanid also possesses potent activity against Giardia lamblia and Entamoeba histolytica, thus expanding the therapeutic potential of nitroimidazooxazines. Synthetic analogues with a novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed and synthesized, and structure-activity relationships were generated. Selected derivatives had potent antiparasitic and antitubercular activity while maintaining drug-like properties such as low cytotoxicity, good metabolic stability in liver microsomes and high apparent permeability across Caco-2 cells. The kinetic solubility of the new bicyclic derivatives varied and was found to be a key parameter for future optimization. Taken together, these results suggest that promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development.

Detection and Investigation of Eagle Effect Resistance to Vancomycin in Clostridium difficile With an ATP-Bioluminescence Assay.

Vancomycin was bactericidal against Clostridium difficile at eightfold the minimum inhibitory concentration (MIC) using a traditional minimum bactericidal concentration (MBC) assay. However, at higher concentrations up to 64 × MIC, vancomycin displayed a paradoxical "more-drug-kills-less" Eagle effect against C. difficile. To overcome challenges associated with performing the labor-intensive agar-based MBC method under anaerobic growth conditions, we investigated an alternative more convenient ATP-bioluminescence assay to assess the Eagle effect in C. difficile. The commercial BacTiter-GloTM assay is a homogenous method to determine bacterial viability based on quantification of bacterial ATP as a marker for metabolic activity. The ATP-bioluminescence assay was advantageous over the traditional MBC-type assay in detecting the Eagle effect because it reduced assay time and was simple to perform; measurement of viability could be performed in less than 10 min outside of the anaerobic chamber. Using this method, we found C. difficile survived clinically relevant, high concentrations of vancomycin (up to 2048 µg/mL). In contrast, C. difficile did not survive high concentrations of metronidazole or fidaxomicin. The Eagle effect was also detected for telavancin, but not for teicoplanin, dalbavancin, oritavancin, or ramoplanin. All four pathogenic strains of C. difficile tested consistently displayed Eagle effect resistance to vancomycin, but not metronidazole or fidaxomicin. These results suggest that Eagle effect resistance to vancomycin in C. difficile could be more prevalent than previously appreciated, with potential clinical implications. The ATP-Bioluminescence assay can thus be used as an alternative to the agar-based MBC assay to characterize the Eagle effect against a variety of antibiotics, at a wide-range of concentrations, with much greater throughput. This may facilitate improved understanding of Eagle effect resistance and promote further research to understand potential clinical relevance.

Immunomodulatory activities of pixatimod: emerging nonclinical and clinical data, and its potential utility in combination with PD-1 inhibitors.

BACKGROUND: Pixatimod (PG545) is a novel clinical-stage immunomodulatory agent capable of inhibiting the infiltration of tumor-associated macrophages (TAMs) yet also stimulate dendritic cells (DCs), leading to activation of natural killer (NK) cells. Preclinically, pixatimod inhibits heparanase (HPSE) which may be associated with its inhibitory effect on TAMs whereas its immunostimulatory activity on DCs is through the MyD88-dependent TLR9 pathway. Pixatimod recently completed a Phase Ia monotherapy trial in advanced cancer patients. METHODS: To characterize the safety of pixatimod administered by intravenous (IV) infusion, a one month toxicology study was conducted to support a Phase Ia monotherapy clinical trial. The relative exposure (AUC) of pixatimod across relevant species was determined and the influence of route of administration on the immunomodulatory activity was also evaluated. Finally, the potential utility of pixatimod in combination with PD-1 inhibition was also investigated using the syngeneic 4T1.2 breast cancer model. RESULTS: The nonclinical safety profile revealed that the main toxicities associated with pixatimod are elevated cholesterol, triglycerides, APTT, decreased platelets and other changes symptomatic of modulating the immune system such as pyrexia, changes in WBC subsets, inflammatory changes in liver, spleen and kidney. Though adverse events such as fever, elevated cholesterol and triglycerides were reported in the Phase Ia trial, none were considered dose limiting toxicities and the compound was well tolerated up to 100 mg via IV infusion. Exposure (AUC) up to 100 mg was considered proportional with some accumulation upon repeated dosing, a phenomenon also noted in the toxicology study. The immunomodulatory activity of pixatimod was independent of the route of administration and it enhanced the effectiveness of PD-1 inhibition in a poorly immunogenic tumor model. CONCLUSIONS: Pixatimod modulates innate immune cells but also enhances T cell infiltration in combination with anti-PD-1 therapy. The safety and PK profile of the compound supports its ongoing development in a Phase Ib study for advanced cancer/pancreatic adenocarcinoma with the checkpoint inhibitor nivolumab (Opdivo®). TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02042781 . First posted: 23 January, 2014 - Retrospectively registered.

Protein-inspired antibiotics active against vancomycin- and daptomycin-resistant bacteria.

The public health threat posed by a looming 'post-antibiotic' era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature's control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the 'vancapticins', possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to 'revitalise' antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

Metronidazole-triazole conjugates: activity against Clostridium difficile and parasites.

Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window.

Improved Immunoassay Sensitivity in Serum as a Result of Polymer-Entrapped Quantum Dots: 'Papaya Particles'.

Fluorescent labels are widely employed in biomarker quantification and diagnostics, however they possess narrow Stokes shifts and can photobleach, limiting multiplexed detection applications and compromising sensitivity. In contrast, quantum dots do not photobleach and have much wider Stokes shifts, but a paucity of robust surface attachment chemistries for bioconjugation has limited their uptake in biomedical diagnostics. We report a novel class of biofunctional fluorescent labels based on trapping of ∼10(4) quantum dots within a core nanoparticle. The doped particles act as scaffolds for generation of a multilayered shell consisting of a functionalized hydrophilic polymer with covalently attached receptors for analyte capture. These constructs, which conceptually resemble a papaya fruit, are chemically stable, remain monodispersed for >6 months in buffer, and show utility in immunoassay applications. Using monoclonal antibody fragments against nonstructural protein dengue NS1, an early biomarker for dengue fever, antibody immobilization capacity was 75-fold higher compared with traditional carbodiimide protein coupling. In the model dengue immunoassay, we observed a 15-fold lower limit of detection and 4-fold higher fluorescence intensity with the "papaya particles" compared to current "best-in-class" commercial reagents. Direct deployment in human serum allowed sensitive detection of different NS1 serotypes with lower limits of detection within the clinically relevant range (1-10 ng/mL), and sufficient specificity for identification of the dengue serotype was achieved for concentrations >10 ng/mL (DV1-3) and >50 ng/mL (DV4). The combination of chemical and physical stability and high binding capacity combined with the intrinsic advantages of quantum dots may enable more simple, robust diagnostic assays in the future.

Clostridium difficile drug pipeline: challenges in discovery and development of new agents.

In the past decade Clostridium difficile has become a bacterial pathogen of global significance. Epidemic strains have spread throughout hospitals, while community acquired infections and other sources ensure a constant inoculation of spores into hospitals. In response to the increasing medical burden, a new C. difficile antibiotic, fidaxomicin, was approved in 2011 for the treatment of C. difficile-associated diarrhea. Rudimentary fecal transplants are also being trialed as effective treatments. Despite these advances, therapies that are more effective against C. difficile spores and less damaging to the resident gastrointestinal microbiome and that reduce recurrent disease are still desperately needed. However, bringing a new treatment for C. difficile infection to market involves particular challenges. This review covers the current drug discovery pipeline, including both small molecule and biologic therapies, and highlights the challenges associated with in vitro and in vivo models of C. difficile infection for drug screening and lead optimization.

Synthesis of disaccharides containing 6-deoxy-α-L-talose as potential heparan sulfate mimetics.

A 6-deoxy-α-L-talopyranoside acceptor was readily prepared from methyl α-L-rhamnopyranoside and glycosylated with thiogalactoside donors using NIS/TfOH as the promoter to give good yields of the desired a-linked disaccharide (69-90%). Glycosylation with a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor was not completely stereoselective (α:ß = 6:1), but the desired a-linked disaccharide could be isolated in good overall yield (60%) following conversion into its corresponding tribenzoate derivative. The disaccharides were designed to mimic the heparan sulfate (HS) disaccharide GlcN(2S,6S)-IdoA(2S). However, the intermediates readily derived from these disaccharides were not stable to the sulfonation/deacylation conditions required for their conversion into the target HS mimetics.

Identification of antitubercular benzothiazinone compounds by ligand-based design.

1,3-Benzothiazin-4-ones (BTZs) are a novel class of TB drug candidates with potent activity against M. tuberculosis. An in silico ligand-based model based on structure-activity data from 170 BTZ compounds was used to design a new series. Compounds were tested against a panel of mycobacterial strains and were profiled for cytotoxicity, stability, and antiproliferative effects. Several of the compounds showed improved activity against MDR-TB while retaining low toxicity with higher microsomal, metabolic, and plasma stability.

Discovery of PG545: a highly potent and simultaneous inhibitor of angiogenesis, tumor growth, and metastasis.

Increasing the aglycone lipophilicity of a series of polysulfated oligosaccharide glycoside heparan sulfate (HS) mimetics via attachment of a steroid or long chain alkyl group resulted in compounds with significantly improved in vitro and ex vivo antiangiogenic activity. The compounds potently inhibited heparanase and HS-binding angiogenic growth factors and displayed improved antitumor and antimetastatic activity in vivo compared with the earlier series. Preliminary pharmacokinetic analyses also revealed significant increases in half-life following iv dosing, ultimately supporting less frequent dosing regimens in preclinical tumor models compared with other HS mimetics. The compounds also displayed only mild anticoagulant activity, a common side effect usually associated with HS mimetics. These efforts led to the identification of 3ß-cholestanyl 2,3,4,6-tetra-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-ß-d-glucopyranoside, tridecasodium salt (PG545, 18) as a clinical candidate. Compound 18 was recently evaluated in a phase I clinical trial in cancer patients.

Structure aided design of chimeric antibiotics.

The rise of antibiotic resistance is of great clinical concern. One approach to reducing the development of resistance is to co-administer two or more antibiotics with different modes of action. However, it can be difficult to control the distribution and pharmacokinetics of two drugs to ensure both concentrations remain within the range of therapeutic efficacy whilst avoiding adverse effects. Hybrid drugs, where two drugs are linked together with a flexible linker, have been explored, but the resultant large, flexible molecules can have poor bioavailability. We have developed a chimeric approach using click chemistry where the pharmacophores of two drugs are overlapped into a single smaller, more drug-like molecule. Design and selection of compounds were assisted by in silico structural docking. We prepared a series of compounds that include candidates showing activity against the targets of both trimethoprim; dihydrofolate reductase, and ciprofloxacin; DNA gyrase and topoisomerase IV. The resultant triazole containing molecules show modest, but broad spectrum activities against drug sensitive and resistant Gram-negative and Gram-positive bacteria, with no observable cytotoxicity.

Synthesis of essramycin and comparison of its antibacterial activity.

The triazolopyrimidine natural product essramycin (1) was synthesized without the use of protecting groups via a two-step reaction scheme involving a 3-amino-1,2,4-triazole intermediate, and its structure was unequivocally determined. However, in contrast to the natural product, the synthetic essramycin (1) did not display any antibacterial activity.

A highly lipophilic sulfated tetrasaccharide glycoside related to muparfostat (PI-88) exhibits virucidal activity against herpes simplex virus.

Although sulfated polysaccharides potently inhibit the infectivity of herpes simplex virus (HSV) and human immunodeficiency virus in cultured cells, these compounds fail to show protective effects in humans, most likely due to their poor virucidal activity. Herein we report on sulfated oligosaccharide glycosides related to muparfostat (formerly known as PI-88) and their assessment for anti-HSV activity. Chemical modifications based on the introduction of specific hydrophobic groups at the reducing end of a sulfated oligosaccharide chain enhanced the compound's capability to inhibit the infection of cells by HSV-1 and HSV-2 and abrogated the cell-to-cell transmission of HSV-2. Furthermore, modification with a highly lipophilic cholestanyl group provided a compound with virucidal activity against HSV. This glycoside targeted the viral particle and, to a lesser degree, the cell, and exhibited an antiviral mode of action typical for sulfated polysaccharides and virucides, i.e., interference with the virus attachment to cells and irreversible inactivation of virus infectivity, respectively. The virucidal activity was decreased in the presence of human cervical secretions suggesting that higher doses of this glycoside might be needed for in vivo application. Altogether, the sulfated oligosaccharide-cholestanyl glycoside exhibits potent anti-HSV activity and is, therefore, a good candidate for development as a virucide.

Synthesis and biological evaluation of polysulfated oligosaccharide glycosides as inhibitors of angiogenesis and tumor growth.

A series of polysulfated penta- and tetrasaccharide glycosides containing alpha(1-->3)/alpha(1-->2)-linked mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2, and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor activity in vivo in a mouse melanoma (solid tumor) model resistant to the phase III HS mimetic 1 (muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoagulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate.

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy-beta-D-glucopyranose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from D-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.

Design, synthesis, FGF-1 binding, and molecular modeling studies of conformationally flexible heparin mimetic disaccharides.

Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a D-galactose donor with a methyl beta-D-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solution in equilibrium between the (4)C1 and (1)C4 conformers, as confirmed by 1H NMR spectroscopy, thus mimicking the conformationally flexible L-iduronic acid found in heparin. Docking calculations showed that the predicted locations of disaccharide sulfo groups in the binding site of FGF-1 are consistent with the positions observed for co-crystallized heparin-derived oligosaccharides. Predicted binding affinities are in accord with experimental Kd values obtained from binding assays and are similar to the predicted values for a model heparin disaccharide.

PI-88 and novel heparan sulfate mimetics inhibit angiogenesis.

The heparan sulfate (HS) mimetic PI-88 is a promising inhibitor of tumor growth and metastasis expected to commence phase III clinical evaluation in 2007 as an adjuvant therapy for postresection hepatocellular carcinoma. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents.

Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88).

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as its anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

The synthesis of phosphorylated disaccharide components of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448.

Methods for the stereoselective synthesis of alpha-(1-->2)- and alpha-(1-->3)-linked 6(II)-O-phosphomannobiosides were developed. Two strategies were successfully employed: a D-mannosyl acceptor was coupled with a phosphorylated D-mannosyl trichloroacetimidate donor, or alternatively with a differentially 6-O-protected D-mannosyl trichloroacetimidate donor which, after glycosylation, was selectively deprotected and phosphorylated. Two target phosphomannobiosides intended for use in SAR studies of the antiangiogenic drug candidate PI-88, 2-O-(6-O-phospho-alpha-D-mannopyranosyl)-D-mannopyranose and methyl 3-O-(6-O-phospho-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, were synthesized. The former is a minor component of the side-chain repeating unit of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448, whilst the latter represents a nonreducing end fragment of the phosphomannan.

Synthesis and activity of analogues of the isoleucyl tRNA synthetase inhibitor SB-203207.

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.