Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

The throughput of a biomimetic lipid-mediated assay used to investigate the effects of inhibitors on the kinetics of β-hematin formation has been optimized through the use of 24-well microplates. The rate constant for β-hematin formation mediated by monopalmitoyl-rac-glycerol was reduced from 0.17 ± 0.04 min−1 previously measured in Falcon tubes to 0.019 ± 0.002 min−1 in the optimized assay. While this necessitated longer incubation times, transferring aliquots from multiple 24-well plates to a single 96-well plate for final absorbance measurements actually improved the overall turnaround time per inhibitor. This assay has been applied to investigate the effects of four clinically-relevant antimalarial drugs (chloroquine, amodiaquine, quinidine and quinine) as well as several short-chain 4-aminoquinoline derivatives and non-quinoline (benzamide) compounds on the kinetics of β-hematin formation. The adsorption strength of these inhibitors to crystalline β-hematin (Kads) was quantified using a theoretical kinetic model that is based on the Avrami equation and the Langmuir isotherm. Statistically-significant linear correlations between lipid-mediated β-hematin inhibitory activity and Kads values for quinoline (r2 = 0.76, P-value = 0.0046) and non-quinoline compounds (r2 = 0.99, P-stat = 0.0006), as well as between parasite inhibitory activity (D10) and Kads values for quinoline antimalarial drugs and short-chain chloroquine derivatives (r2 = 0.64, P-value = 0.0098) provides a strong indication that drug action involves adsorption to the surface of β-hematin crystals. Independent support in this regard is provided by experiments that spectrophotometrically monitor the direct adsorption of antimalarial drugs to pre-formed β-hematin.

In silico adsorption of eight antimalarials that inhibit β‐hematin (synthetic hemozoin) formation identified a primary binding site on the (001) face, which accommodates inhibitors via formation of predominantly π‐π interactions. A good correlation (r2=0.64, P=0.017) between adsorption energies and the logarithm of β‐hematin inhibitory activity was found for this face. Of 53 monocyclic, bicyclic and tricyclic scaffolds, the latter yielded the most favorable adsorption energies. Five new amino‐phenoxazine compounds were pursued as β‐hematin inhibitors based on adsorption behaviour. The 2‐substituted phenoxazines show good to moderate β‐hematin inhibitory activity (<100 μM) and Plasmodium falciparum blood stage activity against the 3D7 strain. N1,N1‐diethyl‐N4‐(10H‐phenoxazin‐2‐yl)pentane‐1,4‐diamine (P2a) is the most promising hit with IC50 values of 4.7±0.6 and 0.64±0.05 μM, respectively. Adsorption energies are predictive of β‐hematin inhibitory activity, and thus the in silico approach is a beneficial tool for structure‐based development of new non‐quinoline inhibitors.

In this study we provide deeper insight into both the structural and physicochemical properties of the privileged triarylimidazole required for inhibition of β-hematin formation.