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.

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.

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.

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.].

A new synthesis of alkene-containing minor-groove binders and essential hydrogen bonding in binding to DNA and in antibacterial activity.

A practical synthesis of alkene-containing minor-groove binders for DNA, related to distamycin, with potential for wide structural diversity is described, based upon the Wittig chemistry of N-alkylpyrrole aldehydes. The compounds prepared have been evaluated for binding to DNA by physical methods (melting temperature and NMR) and for their antibacterial activity. Significantly, it was found that alkenes linking the aryl head group of the minor-groove binder promote strong binding to DNA and high antibacterial activity against Gram-positive bacteria. Conversely, a minor-groove binder containing an alkene located towards the alkylamino tail group has a low affinity for DNA and does not show antibacterial activity. These observations suggest an important role for specific hydrogen bonds in the binding of compounds of this type to DNA, and in their antibacterial activity.

A detailed binding free energy study of 2:1 ligand-DNA complex formation by experiment and simulation.

In 2004, we used NMR to solve the structure of the minor groove binder thiazotropsin A bound in a 2:1 complex to the DNA duplex, d(CGACTAGTCG)2. In this current work, we have combined theory and experiment to confirm the binding thermodynamics of this system. Molecular dynamics simulations that use polarizable or non-polarizable force fields with single and separate trajectory approaches have been used to explore complexation at the molecular level. We have shown that the binding process invokes large conformational changes in both the receptor and ligand, which is reflected by large adaptation energies. This is compensated for by the net binding free energy, which is enthalpy driven and entropically opposed. Such a conformational change upon binding directly impacts on how the process must be simulated in order to yield accurate results. Our MM-PBSA binding calculations from snapshots obtained from MD simulations of the polarizable force field using separate trajectories yield an absolute binding free energy (-15.4 kcal mol(-1)) very close to that determined by isothermal titration calorimetry (-10.2 kcal mol(-1)). Analysis of the major energy components reveals that favorable non-bonded van der Waals and electrostatic interactions contribute predominantly to the enthalpy term, whilst the unfavorable entropy appears to be driven by stabilization of the complex and the associated loss of conformational freedom. Our results have led to a deeper understanding of the nature of side-by-side minor groove ligand binding, which has significant implications for structure-based ligand development.

2,2,2-Trifluoro-N-(isoquinolin-5-ylmeth-yl)acetamide.

The mol-ecular structure of the title compound at 123 K, C(12)H(9)F(3)N(2)O, presents a rotationally disordered CF(3) group. Hydrogen bonds between the amide NH group and the N atom of the isoquinoline form a chain in the b-axis direction. The packed structure forms alternate layers of isoquinoline and amide groups parallel to the ab plane.

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.

Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups.

The synthesis and properties of 80 short minor groove binders related to distamycin and the thiazotropsins are described. The design of the compounds was principally predicated upon increased affinity arising from hydrophobic interactions between minor groove binders and DNA. The introduction of hydrophobic aromatic head groups, including quinolyl and benzoyl derivatives, and of alkenes as linkers led to several strongly active antibacterial compounds with MIC for Staphylococcus aureus, both methicillin-sensitive and -resistant strains, in the range of 0.1-5 microg mL-1, which is comparable to many established antibacterial agents. Antifungal activity was also found in the range of 20-50 microg mL-1 MIC against Aspergillus niger and Candida albicans, again comparable with established antifungal drugs. A quinoline derivative was found to protect mice against S. aureus infection for a period of up to six days after a single intraperitoneal dose of 40 mg kg-1.

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-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.

DNA sequence recognition by an isopropyl substituted thiazole polyamide.

We have used DNA footprinting and fluorescence melting experiments to study the sequence-specific binding of a novel minor groove binding ligand (thiazotropsin A), containing an isopropyl substituted thiazole polyamide, to DNA. In one fragment, which contains every tetranucleotide sequence, sub-micromolar concentrations of the ligand generate a single footprint at the sequence ACTAGT. This sequence preference is confirmed in melting experiments with fluorescently labelled oligonucleotides. Experiments with DNA fragments that contain variants of this sequence suggest that the ligand also binds, with slightly lower affinity, to sequences of the type XCYRGZ, where X is any base except C, and Z is any base except G.

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.