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.

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.].

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol.

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC(50) values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC(50) = 199 µM) compared to the parent strain (IC(50) = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.

Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is docked within the concave surface of C3d.