Strathclyde minor groove binders (S-MGBs) with activity against Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. OBJECTIVES: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. METHODS: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. RESULTS AND DISCUSSION: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTm = 1 ±â€Š0.1°C. CONCLUSIONS: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.

Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3-b]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling.

The inhibitory-kappaB kinases (IKKs) IKKα and IKKß play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKß-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.

Suppression of inflammatory arthritis by the parasitic worm product ES-62 is associated with epigenetic changes in synovial fibroblasts.

ES-62 is the major secreted protein of the parasitic filarial nematode, Acanthocheilonema viteae. The molecule exists as a large tetramer (MW, ~240kD), which possesses immunomodulatory properties by virtue of multiple phosphorylcholine (PC) moieties attached to N-type glycans. By suppressing inflammatory immune responses, ES-62 can prevent disease development in certain mouse models of allergic and autoimmune conditions, including joint pathology in collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Such protection is associated with functional suppression of "pathogenic" hyper-responsive synovial fibroblasts (SFs), which exhibit an aggressive inflammatory and bone-damaging phenotype induced by their epigenetic rewiring in response to the inflammatory microenvironment of the arthritic joint. Critically, exposure to ES-62 in vivo induces a stably-imprinted CIA-SF phenotype that exhibits functional responses more typical of healthy, Naïve-SFs. Consistent with this, ES-62 "rewiring" of SFs away from the hyper-responsive phenotype is associated with suppression of ERK activation, STAT3 activation and miR-155 upregulation, signals widely associated with SF pathogenesis. Surprisingly however, DNA methylome analysis of Naïve-, CIA- and ES-62-CIA-SF cohorts reveals that rather than simply preventing pathogenic rewiring of SFs, ES-62 induces further changes in DNA methylation under the inflammatory conditions pertaining in the inflamed joint, including targeting genes associated with ciliogenesis, to programme a novel "resolving" CIA-SF phenotype. In addition to introducing a previously unsuspected aspect of ES-62's mechanism of action, such unique behaviour signposts the potential for developing DNA methylation signatures predictive of pathogenesis and its resolution and hence, candidate mechanisms by which novel therapeutic interventions could prevent SFs from perpetuating joint inflammation and destruction in RA. Pertinent to these translational aspects of ES-62-behavior, small molecule analogues (SMAs) based on ES-62's active PC-moieties mimic the rewiring of SFs as well as the protection against joint disease in CIA afforded by the parasitic worm product.

Intranasally administered S-MGB-364 displays antitubercular activity and modulates the host immune response to Mycobacterium tuberculosis infection.

BACKGROUND: Previously, we evaluated the intracellular mycobactericidal activity of the minor groove binder, S-MGB-364 against the clinical Mycobacterium tuberculosis (Mtb) strain HN878 in macrophages. OBJECTIVES: To assess the mycobactericidal activity of S-MGB-364 in Mtb-infected mice. Further, we investigated a plausible DNA binding mechanism of action of S-MGB-364. METHODS: The anti-TB and host immune effects of intranasal S-MGB-364 or S-MGB-364 encapsulated in non-ionic surfactant vesicles (NIV) were assessed in Mtb-infected mice by cfu enumeration, ELISA, histology, and flow cytometry. DNA binding was examined using native mass spectrometry and UV-vis thermal melt determination. S-MGB interference with DNA-centric biological events was assessed using a representative panel of Mtb and human topoisomerase I, and gyrase assays. RESULTS: S-MGB-364 bound strongly to DNA as a dimer, significantly increasing the stability of the DNA:S-MGB complex compared with DNA alone. Moreover, S-MGB-364 inhibited the relaxation of Mtb topoisomerase I but not the human form. In macrophages, S-MGB-364 or S-MGB-364-NIV did not cause DNA damage as shown by the low γ-H2AX expression. Importantly, in the lungs, the intranasal administration of S-MGB-364 or S-MGB-364-NIV formulation in Mtb-infected mice was non-toxic and resulted in a ∼1 log cfu reduction in mycobacterial burden, reduced the expression of proinflammatory cytokines/chemokines, altered immune cell recruitment, and importantly reduced recruitment of neutrophils. CONCLUSIONS: Together, these data provide proof of concept for S-MGBs as novel anti-TB therapeutics in vivo.

Selective Anti-Leishmanial Strathclyde Minor Groove Binders Using an N-Oxide Tail-Group Modification.

The neglected tropical disease leishmaniasis, caused by Leishmania spp., is becoming more problematic due to the emergence of drug-resistant strains. Therefore, new drugs to treat leishmaniasis, with novel mechanisms of action, are urgently required. Strathclyde minor groove binders (S-MGBs) are an emerging class of anti-infective agent that have been shown to have potent activity against various bacteria, viruses, fungi and parasites. Herein, it is shown that S-MGBs have potent activity against L. donovani, and that an N-oxide derivation of the tertiary amine tail of typical S-MGBs leads to selective anti-leishmanial activity. Additionally, using S-MGB-219, the N-oxide derivation is shown to retain strong binding to DNA as a 2:1 dimer. These findings support the further study of anti-leishmanial S-MGBs as novel therapeutics.

Failure of the Anti-Inflammatory Parasitic Worm Product ES-62 to Provide Protection in Mouse Models of Type I Diabetes, Multiple Sclerosis, and Inflammatory Bowel Disease.

Parasitic helminths and their isolated secreted products show promise as novel treatments for allergic and autoimmune conditions in humans. Foremost amongst the secreted products is ES-62, a glycoprotein derived from Acanthocheilonema viteae, a filarial nematode parasite of gerbils, which is anti-inflammatory by virtue of covalently-attached phosphorylcholine (PC) moieties. ES-62 has been found to protect against disease in mouse models of rheumatoid arthritis, systemic lupus erythematosus, and airway hyper-responsiveness. Furthermore, novel PC-based synthetic small molecule analogues (SMAs) of ES-62 have recently been demonstrated to show similar anti-inflammatory properties to the parent molecule. In spite of these successes, we now show that ES-62 and its SMAs are unable to provide protection in mouse models of certain autoimmune conditions where other helminth species or their secreted products can prevent disease development, namely type I diabetes, multiple sclerosis and inflammatory bowel disease. We speculate on the reasons underlying ES-62's failures in these conditions and how the negative data generated may help us to further understand ES-62's mechanism of action.

Evaluation of minor groove binders (MGBs) as novel anti-mycobacterial agents and the effect of using non-ionic surfactant vesicles as a delivery system to improve their efficacy.

OBJECTIVES: The slow development of major advances in drug discovery for the treatment of Mycobacterium tuberculosis (Mtb) infection suggests a compelling need for evaluation of more effective drug therapies against TB. New classes of drugs are constantly being evaluated for anti-mycobacterial activity with currently a very limited number of new drugs approved for TB treatment. Minor groove binders (MGBs) have previously revealed promising antimicrobial activity against various infectious agents; however, they have not yet been screened against Mtb. METHODS: The mycobactericidal activity of 96 MGB compounds against Mtb was determined using an H37Rv-GFP microplate assay. MGB hits were screened for their intracellular mycobactericidal efficacy against the clinical Beijing Mtb strain HN878 in bone-marrow-derived macrophages using standard cfu counting. Cell viability was assessed by CellTiter-Blue assays. Selected MGBs were encapsulated into non-ionic surfactant vesicles (NIVs) for drug delivery system evaluation. RESULTS: H37Rv-GFP screening yielded a hit-list of seven compounds at an MIC99 of between 0.39 and 1.56 µM. MGB-362 and MGB-364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19 µM, respectively, whilst being non-toxic. Subsequent encapsulation into NIVs demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB-alone formulation. CONCLUSIONS: MGB anti-mycobacterial activities together with non-toxic properties indicate that MGB compounds constitute an important new class of drug/chemical entity, which holds promise in future anti-TB therapy. Furthermore, the ability of NIVs to better deliver entrapped MGB compounds to an intracellular Mtb infection suggests further preclinical evaluation is warranted.

Selective anti-malarial minor groove binders.

A set of 31 DNA minor groove binders (MGBs) with diverse structural features relating to both physical chemical properties and DNA binding sequence preference has been evaluated as potential drugs to treat Plasmodium falciparum infections using a chloroquine sensitive strain (3D7) and a chloroquine resistant strain (Dd2) in comparison with human embryonic kidney (HEK) cells as an indicator of mammalian cell toxicity. MGBs with an alkene link between the two N-terminal building blocks were demonstrated to be most active with IC50 values in the range 30-500nM and therapeutic ratios in the range 10->500. Many active compounds contained a C-alkylthiazole building block. Active compounds with logD7.4 values of approximately 3 or 7 were identified. Importantly the MGBs tested were essentially equally effective against both chloroquine sensitive and resistant strains. The results show that suitably designed MGBs have the potential for development into clinical candidates for antimalarial drugs effective against resistant strains of Plasmodia.

An evaluation of Minor Groove Binders as anti-lung cancer therapeutics.

A series of 47 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for anti-lung cancer activity by screening against the melanoma cancer cell line B16-F10. Five compounds have been found to possess significant activity, more so than a standard therapy, Gemcitabine. Moreover, one compound has been found to have an activity around 70-fold that of Gemcitabine and has a favourable selectivity index of greater than 125. Furthermore, initial studies have revealed this compound to be metabolically stable and thus it represents a lead for further optimisation towards a novel treatment for lung cancer.

Prophylactic and therapeutic treatment with a synthetic analogue of a parasitic worm product prevents experimental arthritis and inhibits IL-1ß production via NRF2-mediated counter-regulation of the inflammasome.

Rheumatoid arthritis (RA) remains a debilitating autoimmune condition as many patients are refractory to existing conventional and biologic therapies, and hence successful development of novel treatments remains a critical requirement. Towards this, we now describe a synthetic drug-like small molecule analogue, SMA-12b, of an immunomodulatory parasitic worm product, ES-62, which acts both prophylactically and therapeutically against collagen-induced arthritis (CIA) in mice. Mechanistic analysis revealed that SMA-12b modifies the expression of a number of inflammatory response genes, particularly those associated with the inflammasome in mouse bone marrow-derived macrophages and indeed IL-1ß was the most down-regulated gene. Consistent with this, IL-1ß was significantly reduced in the joints of mice with CIA treated with SMA-12b. SMA-12b also increased the expression of a number of genes associated with anti-oxidant responses that are controlled by the transcription factor NRF2 and critically, was unable to inhibit expression of IL-1ß by macrophages derived from the bone marrow of NRF2(-/-) mice. Collectively, these data suggest that SMA-12b could provide the basis of an entirely novel approach to fulfilling the urgent need for new treatments for RA.

Protective effect of small molecule analogues of the Acanthocheilonema viteae secreted product ES-62 on oxazolone-induced ear inflammation.

ES-62 is the major secreted protein of the rodent filarial nematode Acanthocheilonema viteae. The molecule contains covalently attached phosphorylcholine (PC) residues, which confer anti-inflammatory properties on ES-62, underpinning the idea that drugs based on this active moiety may have therapeutic potential in human diseases associated with aberrant inflammation. Here we demonstrate that two synthetic small molecule analogues (SMAs) of ES-62 termed SMA 11a and SMA 12b are protective in the oxazolone-induced acute allergic contact dermatitis mouse model of skin inflammation, as measured by a significant reduction in ear inflammation following their administration before oxazolone sensitisation and before oxazolone challenge. Furthermore, it was found that when tested, 12b was effective at reducing ear swelling even when first administered before challenge. Histological analysis of the ears showed elevated cellular infiltration and collagen deposition in oxazolone-treated mice both of which were reduced by treatment with the two SMAs. Likewise, the oxazolone-induced increase in IFNγ mRNA in the ears was reduced but no effect on other cytokines investigated was observed. Finally, no influence on the mast cell populations in the ear was observed.

Communication between the zinc and tetrahydrobiopterin binding sites in nitric oxide synthase.

The nitric oxide synthase (NOS) dimer is stabilized by a Zn(2+) ion coordinated to four symmetry-related Cys residues exactly along the dimer 2-fold axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is ~15 Å from the Zn(2+). We have determined the crystal structures of the bovine endothelial NOS dimer oxygenase domain bound to three different pterin analogues, which reveal an intimate structural communication between the H4B and Zn(2+) sites. The binding of one of these compounds, 6-acetyl-2-amino-7,7-dimethyl-7,8-dihydro-4(3H)-pteridinone (1), to the pterin site and Zn(2+) binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key residues in the Zn(2+) binding motif, resulting in destabilization of the dimer and a complete disruption of the Zn(2+) site. Addition of excess Zn(2+) stabilizes the Zn(2+) site at the expense of weakened binding of 1. The unique structural features of 1 that disrupt the dimer interface are extra methyl groups that extend into the dimer interface and force a slight opening of the dimer, thus resulting in disruption of the Zn(2+) site. These results illustrate a very delicate balance of forces and structure at the dimer interface that must be maintained to properly form the Zn(2+), pterin, and substrate binding sites.

Recognition of the DNA minor groove by thiazotropsin analogues.

Solution-phase self-association characteristics and DNA molecular-recognition properties are reported for three close analogues of minor-groove-binding ligands from the thiazotropsin class of lexitropsin molecules; they incorporate isopropyl thiazole as a lipophilic building block. Thiazotropsin B (AcImPy(iPr) ThDp) shows similar self-assembly characteristics to thiazotropsin A (FoPyPy(iPr) ThDp), although it is engineered, by incorporation of imidazole in place of N-methyl pyrrole, to swap its DNA recognition target from 5'-ACTAGT-3' to 5'-ACGCGT-3'. Replacement of the formamide head group in thiazotropsin A by nicotinamide in AIK-18/51 results in a measureable difference in solution-phase self-assembly character and substantially enhanced DNA association characteristics. The structures and associated thermodynamic parameters of self-assembled ligand aggregates and their complexes with their respective DNA targets are considered in the context of cluster targeting of DNA by minor-groove complexes.

The allure of targets for novel drugs.

The challenges of bringing new medicines to patients have been extensively discussed and debated, including consideration of the contribution that academic laboratories can make. At the University of Strathclyde, drug discovery has been a continuing focal activity since the 1960s, and in the past 30 years, the author has led or contributed to many projects of different character and for diverse diseases. A feature common to these projects is the extension of concepts of molecular and biological targets in drug discovery research. In mechanistic terms, these have included compounds that are activators and not inhibitors, and in particular multitargeted compounds. With respect to relevance to disease, schizophrenia, pulmonary disfunction, autoimmune, and infectious disease are most relevant. These projects are discussed in the context of classical medicinal chemistry and more recent concepts in and approaches to drug discovery.

MptpB Inhibitor Improves the Action of Antibiotics against Mycobacterium tuberculosis and Nontuberculous Mycobacterium avium Infections.

Treatment of Mycobacterium tuberculosis and Mycobacterium avium infections requires multiple drugs for long time periods. Mycobacterium protein-tyrosine-phosphatase B (MptpB) is a key M. tuberculosis virulence factor that subverts host antimicrobial activity to promote intracellular survival. Inhibition of MptpB reduces the infection burden in vivo and offers new opportunities to improve current treatments. Here, we demonstrate that M. avium produces an MptpB orthologue and that the MptpB inhibitor C13 reduces the M. avium infection burden in macrophages. Combining C13 with the antibiotics rifampicin or bedaquiline showed an additive effect, reducing intracellular infection of both M. tuberculosis and M. avium by 50%, compared to monotreatment with antibiotics alone. This additive effect was not observed with pretomanid. Combining C13 with the minor groove-binding compounds S-MGB-362 and S-MGB-363 also reduced the M. tuberculosis intracellular burden. Similar additive effects of C13 and antibiotics were confirmed in vivo using Galleria mellonella infections. We demonstrate that the reduced mycobacterial burden in macrophages observed with C13 treatments is due to the increased trafficking to lysosomes.

The diversity-oriented synthesis of pteridines - achievements and potential for development.

The importance of pteridines in the key cofactors, tetrahydrofolate and tetrahydrobiopterin, has encouraged the development of the chemistry and chemical biology of pteridines. In order to investigate the latter, versatile synthetic methods are required to prepare designed relatives of the natural cofactors for use as potential drugs or biological probes. Recently, diversity-oriented synthesis has become a particularly powerful and versatile approach to the synthesis of pteridines and related heterocyclic compounds. This article describes and discusses available methods using radical, electrophilic, nucleophilic, and organometal mediated substitution reactions.

Mechanistic exploration of the palladium-catalyzed process for the synthesis of benzoxazoles and benzothiazoles.

A convenient one-pot palladium-catalyzed cascade process for the preparation of both benzoxazoles and benzothiazoles has been developed. While these reactions proceed to give similar compounds the mechanisms governing the processes are different as are the experimental conditions employed.

Protection against lung pathology during obesity-accelerated ageing in mice by the parasitic worm product ES-62.

Mice develop pathology in the lungs as they age and this may be accelerated by a high calorie diet (HCD). ES-62 is a protein secreted by the parasitic worm Acanthocheilonema viteae that is immunomodulatory by virtue of covalently attached phosphorylcholine (PC) moieties. In this study, we show that weekly treatment of C57BL/6J mice with ES-62 protected against pathology in the lungs in male but not female mice fed a HCD from 10 weeks of age as shown by reductions in cellular infiltration and airway remodelling, particularly up to 160 days of age. ES-62 also reduced gene expression of the cytokines IL-4 and IL-17 and in addition the TLR/IL-1R adaptor MyD88, in the lungs of male mice although HCD-induced increases in these inflammatory markers were not detected until between 340 and 500 days of age. A combination of two drug-like ES-62 PC-based small molecule analogues (SMAs), produced broadly similar protective effects in the lungs of male mice with respect to both lung pathology and inflammatory markers, in addition to a decrease in HCD-induced IL-5 expression. Overall, our data show that ES-62 and its SMAs offer protection against HCD-accelerated pathological changes in the lungs during ageing. Given the targeting of Th2 cytokines and IL-17, we discuss this protection in the context of ES-62's previously described amelioration of airway hyper-responsiveness in mouse models of asthma.

Multitargeted anti-infective drugs: resilience to resistance in the antimicrobial resistance era.

The standard drug discovery paradigm of single molecule - single biological target - single biological effect is perhaps particularly unsuitable for anti-infective drug discovery. This is due to the rapid evolution of resistance likely to be observed with single target drugs. Multitargeted anti-infective drugs are likely to be superior due to their lower susceptibility to target-related resistance mechanisms. Strathclyde minor groove binders are a class of compounds which have been developed by adopting the multitargeted anti-infective drugs paradigm, and their effectiveness against a wide range of pathogenic organisms is discussed. The renaming of this class to Strathclyde nucleic acid binders is also presented due to their likely targets including both DNA and RNA.

Ratiometric imaging of minor groove binders in mammalian cells using Raman microscopy.

Quantitative drug imaging in live cells is a major challenge in drug discovery and development. Many drug screening techniques are performed in solution, and therefore do not consider the impact of the complex cellular environment in their result. As such, important features of drug-cell interactions may be overlooked. In this study, Raman microscopy is used as a powerful technique for semi-quantitative imaging of Strathclyde-minor groove binders (S-MGBs) in mammalian cells under biocompatible imaging conditions. Raman imaging determined the influence of the tail group of two novel minor groove binders (S-MGB-528 and S-MGB-529) in mammalian cell models. These novel S-MGBs contained alkyne moieties which enabled analysis in the cell-silent region of the Raman spectrum. The intracellular uptake concentration, distribution and mechanism were evaluated as a function of the pK a of the tail group, morpholine and amidine, for S-MGB-528 and S-MGB-529, respectively. Although S-MGB-529 had a higher binding affinity to the minor groove of DNA in solution-phase measurements, the Raman imaging data indicated that S-MGB-528 showed a greater degree of intracellular accumulation. Furthermore, using high resolution stimulated Raman scattering (SRS) microscopy, the initial localisation of S-MGB-528 was shown to be in the nucleus before accumulation in the lysosome, which was demonstrated using a multimodal imaging approach. This study highlights the potential of Raman spectroscopy for semi-quantitative drug imaging studies and highlights the importance of imaging techniques to investigate drug-cell interactions, to better inform the drug design process.