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.
Intracellular localisation of fluorescent MGB in the nucleus and kinetoplast of T. b. brucei. N = nucleus; K = kinetoplast; arrow = other organelles. Bar = 10 μm.
•Minor Groove Binders (MGBs) possess activity against Trypanosma brucei brucei.•5 MGBs have been identified as leads for future investigation, each with IC50 values lower than 40 nM.•DNA remains a possible target for these MGBs in trypanosomes, similar to the case for bacteria.
Minor Groove Binders; Antiparasitic activity; African trypanosomiasis
Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP), are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT), shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT), a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.
riboswitches; triazolethiamine; click-chemistry; metal-chelating compounds; metabolic enzymes
•Small molecule analogues (SMAs) of the immunomodulator, ES-62, have been produced.•Two SMAs protect against oxazolone-induced skin inflammation in mouse ears.•Protection is associated with reduced cellular infiltration and collagen deposition.•Protection is associated with decreased IFNγ mRNA in the ears.
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.
ES-62; Immunomodulation; Oxazolone; Parasitic worm; Skin inflammation
The title compound has a disordered structure with two equally populated conformations of the amine fragment. A pair of weak C—H⋯O intermolecular interactions between the CH2 and SO2 groups gives a one-dimensional supramolecular structure running along the a-axis direction.
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 intermolecular interactions between the CH2 and SO2 groups gives a one-dimensional supramolecular structure that propagates through translation along the a-axis direction.
crystal structures; sulfone; collagen-induced arthritis; non-classical hydrogen bonding
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-1β was the most down-regulated gene. Consistent with this, IL-1β 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-1β 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.
•SMA-12b is a mimetic of the active PC-moiety of the helminth immunomodulator, ES-62.•SMA-12b effectively protects mice against collagen-induced arthritis (CIA).•SMA-12b downregulates IL-1β and inflammasome genes via activation of NRF2.•SMA-12b reduces IL-1β in the joints of mice with CIA.
Arthritis; ES-62; IL-1β; Inflammasome; NRF2; Parasitic worm
The nitric oxide synthase (NOS) dimer
is stabilized by a Zn2+ 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
Zn2+. 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 Zn2+ 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
Zn2+ binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key
residues in the Zn2+ binding motif, resulting in destabilization
of the dimer and a complete disruption of the Zn2+ site.
Addition of excess Zn2+ stabilizes the Zn2+ 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 Zn2+ site. These results illustrate a very delicate balance of forces
and structure at the dimer interface that must be maintained to properly
form the Zn2+, pterin, and substrate binding sites.
treatment of Human African trypanosomiasis remains a major
unmet health need in sub-Saharan Africa. Approaches involving new
molecular targets are important; pteridine reductase 1 (PTR1), an
enzyme that reduces dihydrobiopterin in Trypanosoma spp., has been identified as a candidate target, and it has been
shown previously that substituted pyrrolo[2,3-d]pyrimidines
are inhibitors of PTR1 from Trypanosoma brucei (J. Med. Chem.2010, 53, 221–229). In this study, 61 new pyrrolo[2,3-d]pyrimidines have been prepared, designed with input from new crystal
structures of 23 of these compounds complexed with PTR1, and evaluated
in screens for enzyme inhibitory activity against PTR1 and in vitro
antitrypanosomal activity. Eight compounds were sufficiently active
in both screens to take forward to in vivo evaluation. Thus, although
evidence for trypanocidal activity in a stage I disease model in mice
was obtained, the compounds were too toxic to mice for further development.
In spite of increasing evidence that
parasitic worms may protect
humans from developing allergic and autoimmune diseases and the continuing
identification of defined helminth-derived immunomodulatory molecules,
to date no new anti-inflammatory drugs have been developed from these
organisms. We have approached this matter in a novel manner by synthesizing
a library of drug-like small molecules based upon phosphorylcholine,
the active moiety of the anti-inflammatory Acanthocheilonema
viteae product, ES-62, which as an immunogenic protein
is unsuitable for use as a drug. Following preliminary in vitro screening
for inhibitory effects on relevant macrophage cytokine responses,
a sulfone-containing phosphorylcholine analogue (11a)
was selected for testing in an in vivo model of inflammation, collagen-induced
arthritis (CIA). Testing revealed that 11a was as effective
as ES-62 in protecting DBA/1 mice from developing CIA and mirrored
its mechanism of action in downregulating the TLR/IL-1R transducer,
MyD88. 11a is thus a novel prototype for anti-inflammatory
•Small molecule analogues of the helminth immunomodulator ES-62 have been produced.•Two analogues inhibit mast cell functions and prevent airway hypersensitivity.•The analogues are drug-like and could be considered for treatment of human allergy.
ES-62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae, exhibits anti-inflammatory properties by virtue of covalently attached phosphorylcholine moieties. Screening of a library of ES-62 phosphorylcholine-based small molecule analogues (SMAs) revealed that two compounds, termed 11a and 12b, mirrored the helminth product both in inhibiting mast cell degranulation and cytokine responses in vitro and in preventing ovalbumin-induced Th2-associated airway inflammation and eosinophil infiltration of the lungs in mice. Furthermore, the two SMAs inhibited neutrophil infiltration of the lungs when administered therapeutically. ES-62-SMAs 11a and 12b thus represent starting points for novel drug development for allergies such as asthma.
Asthma; ES-62; Parasitic helminth; Immunomodulation; Phosphorylcholine
The structural and thermodynamic basis for the strength and selectivity of the interactions of minor groove binders (MGBs) with DNA is not fully understood. In 2003, we reported the first example of a thiazole-containing MGB that bound in a phase-shifted pattern that spanned six base pairs rather than the usual four (for tricyclic distamycin-like compounds). Since then, using DNA footprinting, NMR spectroscopy, isothermal titration calorimetry, and molecular dynamics, we have established that the flanking bases around the central four being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences.
Ligand affinity; DNA minor groove; minor groove binders; DNA footprinting; NMR spectroscopy; isothermal titration calorimetry; molecular dynamics
Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.
The molecular structure of the title compound at 123 K, C12H9F3N2O, presents a rotationally disordered CF3 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.
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.