Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas’ disease drug discovery

Chagas' disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments

Model comparison from LIGO–Virgo data on GW170817’s binary components and consequences for the merger remnant

GW170817 is the very first observation of gravitational waves originating from the coalescence of two compact objects in the mass range of neutron stars, accompanied by electromagnetic counterparts, and offers an opportunity to directly probe the internal structure of neutron stars. We perform Bayesian model selection on a wide range of theoretical predictions for the neutron star equation of state. For the binary neutron star hypothesis, we find that we cannot rule out the majority of theoretical models considered. In addition, the gravitational-wave data alone does not rule out the possibility that one or both objects were low-mass black holes. We discuss the possible outcomes in the case of a binary neutron star merger, finding that all scenarios from prompt collapse to long-lived or even stable remnants are possible. For long-lived remnants, we place an upper limit of 1.9 kHz on the rotation rate. If a black hole was formed any time after merger and the coalescing stars were slowly rotating, then the maximum baryonic mass of non-rotating neutron stars is at most 3.05 M-circle dot, and three equations of state considered here can be ruled out. We obtain a tighter limit of 2.67 M-circle dot for the case that the merger results in a hypermassive neutron star