S-MGBs bearing amidine tail groups are potent, selective antiplasmodial agents

Abstract

There were an estimated 249 million cases of malaria globally in 2022, causing approximately 608 000 deaths. Most of these are attributed to infection by P. falciparum. 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, including P. falciparum. A panel of 25 S-MGBs was synthesised, including those bearing an amidine tail group, and their antiplasmodial activity against 3D7 and Dd2 strains was determined in vitro using an asexual P. falciparum imaging assay. Determination of activity against HEK293 cells allowed for selective cytotoxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. A comparison of 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant) potency showed no evidence of cross-resistance across the S-MGB set. S-MGB-356, S-MGB-368 and S-MGB-359, amidine tail containing S-MGBs, were identified as the most promising hit compounds based on their selectivity indices (HEK293/3D7) of >612.6, >335.8 and >264.8, respectively. S-MGB-356, S-MGB-368 and S-MGB-359 were confirmed to bind to DNA as dimers, with gDNA thermal shifts (ΔT _m) of 12 °C, 3 °C and 16 °C, respectively. Together, these data demonstrate that amidine tail bearing S-MGBs are promising hit compounds against P. falciparum, and can be further optimised into lead compounds.

Fig. 1. Distamycin and the variety of S-MGBs examined in this study. For R¹, R = m-OMe, p-CF₃, p-N(Me)₂, o-OH, p-OMe, p-F, m-F, m-CF₃. For R², n = 1 or 2. Ar¹ X = C or N.

Scheme 1. Synthesis of 5 novel S-MGBs, S-MGB-365, S-MGB-368, S-MGB-359, S-MGB-388 and S-MGB-361 (4a–e, respectively).

Fig. 2. Panel A, a comparison of the cytotoxicity amidine tail and non-amidine tail S-MGBs. Panel B, selectivity index (HEK293/3D7) compared to potency (3D7) of S-MGBs to identify three most promising compounds, S-MGB-365 (4a), S-MGB-368 (4b) and S-MGB-359 (4c).

Fig. 3. P. falciparum (3D7 and Dd2) and HEK293 dose–response curves of S-MGB-365 (Panel A), S-MGB368 (Panel B) and S-MGB-359 (Panel C). Panel D shows the key. Curves labelled as 3D7_A and 3D7_B are for the P. falciparum 3D7 strain, two independent experiments in duplicate. Curves labelled as Dd2_A and Dd2_B are for the P. falciparum Dd2 strain, two independent experiments in duplicate. Curves labelled as cytox represent the HEK293 assay (duplicate data).

Fig. 4. nESI-MS characterisation of S-MGBs binding to double stranded DNA oligo 5′-CGCATATATGCG-3′. 9 μM DNA, (100 μM KCl, 1% DMSO) sprayed from ammonium acetate (150 mM, pH 7) in the absence (a) and presence (b) of 100 μM S-MGBs. For each panel A) S-MGB-365 (4a), B) S-MGB-368 (4b) and C) S-MGB-359 (4c), subpanels show the following: a) single stranded DNA (denoted [SS]) were present in charge states 4- and 3-, and double stranded DNA (denoted [DS]) were present in charge states 5-⁻ and 4-. b) [SS] was present in charge state 3- and 4-. Each [DS] molecule bound 2xS-MGB molecules (denoted [DS+2 M]) and was present in charge states 5-and 4-. For S-MGB-359 (4c), panel C), a small amount of bound [DS] molecules was bound to 1xS-MGB molecule (denoted [DS+1 M]), present in charge state 5-. Expected and measured masses of each species are provided in Tables S2–S4.

Fig. 5. Thermal melt analysis of S-MGB-365 (4a), 368 (4b), 359 (4c) bound to gDNA including exemplar melt curve from one experimental repeat, fitted with a Boltzmann distribution (A) and computed melting temperatures for N = 3 experiments, with all values within ±1 °C (B).