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.

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.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

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.

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.

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.

Truncated S-MGBs: towards a parasite-specific and low aggregation chemotype.

This paper describes the design and synthesis of Strathclyde minor groove binders (S-MGBs) that have been truncated by the removal of a pyrrole ring in order to mimic the structure of the natural product, disgocidine. S-MGBs have been found to be active against many different organisms, however, selective antiparasitic activity is required. A panel of seven truncated S-MGBs was prepared and the activities examined against a number of clinically relevant organisms including several bacteria and parasites. The effect of the truncation strategy on S-MGB aggregation in aqueous environment was also investigated using 1H inspection and DOSY experiments. A lead compound, a truncated S-MGB, which possesses significant activity only against trypanosomes and Leishmania has been identified for further study and was also found to be less affected by aggregation compared to its full-length analogue.

Correction: Truncated S-MGBs: towards a parasite-specific and low aggregation chemotype.

[This corrects the article DOI: 10.1039/D1MD00110H.].

Cyrene™ is a green alternative to DMSO as a solvent for antibacterial drug discovery against ESKAPE pathogens.

Dimethyl sulfoxide (DMSO) is currently employed across the biomedical field, from cryopreservation to in vitro assays, despite the fact that it has been shown to have an assortment of biologically relevant effects. The amphiphilic nature of DMSO along with its relatively low toxicity at dilute concentrations make it a challenging solvent to replace. A possible alternative is Cyrene™ (dihydrolevoglucosenone), an aprotic dipolar solvent that is derived from waste biomass. In addition to being a green solvent, Cyrene™ has comparable solvation properties and is reported to have low toxicity. Herein the abilities of the two solvents to solubilize drug compounds and to act as non-participatory vehicles in drug discovery for antibacterials are compared. It was demonstrate that the results of standardised antimicrobial susceptibility testing do not differ between drugs prepared from either Cyrene™ or DMSO stock. Moreover, in contrast to DMSO, Cyrene™ does not offer protection from ROS mediated killing of bacteria and may therefore be an improvement over DMSO as a vehicle in antimicrobial drug discovery.

Novel Minor Groove Binders Cure Animal African Trypanosomiasis in an in Vivo Mouse Model.

Animal African trypanosomiasis (AAT) is a significant socioeconomic burden for sub-Saharan Africa because of its huge impact on livestock health. Existing therapies including those based on minor groove binders (MGBs), such as the diamidines, which have been used for decades, have now lost efficacy in some places because of the emergence of resistant parasites. Consequently, the need for new chemotherapies is urgent. Here, we describe a structurally distinct class of MGBs, Strathclyde MGBs (S-MGBs), which display excellent in vitro activities against the principal causative organisms of AAT: Trypanosoma congolense, and Trypanosoma vivax. We also show the cure of T. congolense-infected mice by a number of these compounds. In particular, we identify S-MGB-234, compound 7, as curative by using two applications of 50 mg/kg intraperitoneally. Crucially, we demonstrate that S-MGBs do not show cross-resistance with the current diamidine drugs and are not internalized via the transporters used by diamidines. This study demonstrates that S-MGBs have significant potential as novel therapeutic agents for AAT.

Selective in vitro anti-cancer activity of non-alkylating minor groove binders.

Traditional cytotoxic agents which act through a DNA-alkylating mechanism are relatively non-specific, resulting in a small therapeutic window and thus limiting their effectiveness. In this study, we evaluate a panel of 24 non-alkylating Strathclyde Minor Groove Binders (S-MGBs), including 14 novel compounds, for in vitro anti-cancer activity against a human colon carcinoma cell line, a cisplatin-sensitive ovarian cancer cell line and a cisplatin-resistant ovarian cancer cell line. A human non-cancerous retinal epithelial cell line was used to measure selectivity of any response. We have identified several S-MGBs with activities comparable to cis-platin and carboplatin, but with better in vitro selectivity indices, particularly S-MGB-4, S-MGB-74 and S-MGB-317. Moreover, a comparison of the cis-platin resistant and cis-platin sensitive ovarian cancer cell lines reveals that our S-MGBs do not show cross resistance with cisplatin or carboplatin and that they likely have a different mechanism of action. Finally, we present an initial investigation into the mechanism of action of one compound from this class, S-MGB-4, demonstrating that neither DNA double strand breaks nor the DNA damage stress sensor protein p53 are induced. This indicates that our S-MGBs are unlikely to act through an alkylating or DNA damage response mechanism.

Synthetic analogues of the parasitic worm product ES-62 reduce disease development in in vivo models of lung fibrosis.

Parasitic worms are receiving much attention as a potential new therapeutic approach to treating autoimmune and allergic conditions but concerns remain regarding their safety. As an alternative strategy, we have focused on the use of defined parasitic worm products and recently taken this one step further by designing drug-like small molecule analogues of one such product, ES-62, which is anti-inflammatory by virtue of covalently attached phosphorylcholine moieties. Previously, we have shown that ES-62 mimics are efficacious in protecting against disease in mouse models of rheumatoid arthritis, systemic lupus erythematosus and skin and lung allergy. Given the potential role of chronic inflammation in fibrosis, in the present study we have focused our attention on lung fibrosis, a debilitating condition for which there is no cure and which in spite of treatment slowly gets worse over time. Two mouse models of fibrosis - bleomycin-induced and LPS-induced - in which roles for inflammation have been implicated were adopted. Four ES-62 analogues were tested - 11a and 12b, previously shown to be active in mouse models of allergic and autoimmune disease and 16b and AIK-29/62 both of which are structurally related to 11a. All four compounds were found to significantly reduce disease development in both fibrosis models, as shown by histopathological analysis of lung tissue, indicating their potential as treatments for this condition.

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.

An evaluation of Minor Groove Binders as anti-fungal and anti-mycobacterial therapeutics.

This study details the synthesis and biological evaluation of a collection of 19 structurally related Minor Groove Binders (MGBs), derived from the natural product distamycin, which were designed to probe antifungal and antimycobacterial activity. From this initial set, we report several MGBs that are worth more detailed investigation and optimisation. MGB-4, MGB-317 and MGB-325 have promising MIC80s of 2, 4 and 0.25 µg/mL, respectively, against the fungus C. neoformans.MGB-353 and MGB-354 have MIC99s of 3.1 µM against the mycobacterium M. tuberculosis. The selectivity and activity of these compounds is related to their physicochemical properties and the cell wall/membrane characteristics of the infective agents.

Four pyrrole derivatives used as building blocks in the synthesis of minor-groove binders.

The title nitro-pyrrole-based compounds, C7H8N2O4, (I) (ethyl 4-nitro-1H-pyrrole-2-carboxyl-ate), its derivative C12H14N2O4, (II) [ethyl 4-nitro-1-(4-pent-yn-yl)-1H-pyrrole-2-carboxyl-ate], C15H26N4O3, (III) {N-[3-(di-methyamino)prop-yl]-1-isopentyl-4-nitro-1H-pyrrole-2-carboxamide}, and C20H27N9O5, (IV) {1-(3-azido-prop-yl)-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-N-[2-(morpholin-4-yl)eth-yl]-1H-pyrrole-2-carboxamide}, are inter-mediates used in the synthesis of modified DNA minor-groove binders. In all four compounds, the nitro groups lie in the plane of the pyrrole ring. In compounds (I) and (II), the ester groups also lie in the plane of the pyrrole ring. In compound (III), both of the other substituents lie out of the plane of the pyrrole ring. In the case of compound (IV), the coplanarity extends to the second pyrrole ring and through both amide groups. In the crystals of all four compounds, layer-like structures are formed, via a combination of N-H⋯O and C-H⋯O hydrogen bonds for (I), (III) and (IV), but by only C-H⋯O hydrogen bonds for (II).

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.

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-Trypanosoma brucei brucei therapeutics.

A series of 32 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for activity against Trypanosoma brucei brucei. Four compounds have been found to possess significant activity, in the nanomolar range, and represent hits for further optimisation towards novel treatments for Human and Animal African Trypanosomiases. Moreover, SAR indicates that the head group linking moiety is a significant modulator of biological activity.

Crystal structure of N,N-dimethyl-2-[(4-methyl-benz-yl)sulfon-yl]ethanamine.

In the crystal, the title compound, C12H19NO2S, has a disordered structure with two equally populated conformations of the amine fragment. A pair of weak C-H⋯O inter-molecular inter-actions between the CH2 and SO2 groups gives a one-dimensional supra-molecular structure that propagates through translation along the a-axis direction.