Discovery of an Allosteric Binding Site in Kinetoplastid Methionyl-tRNA Synthetase.

Methionyl-tRNA synthetase (MetRS) is a chemically validated drug target in kinetoplastid parasites Trypanosoma brucei and Leishmania donovani. To date, all kinetoplastid MetRS inhibitors described bind in a similar way to an expanded methionine pocket and an adjacent, auxiliary pocket. In the current study, we have identified a structurally novel class of inhibitors containing a 4,6-diamino-substituted pyrazolopyrimidine core (the MetRS02 series). Crystallographic studies revealed that MetRS02 compounds bind to an allosteric pocket in L. major MetRS not previously described, and enzymatic studies demonstrated a noncompetitive mode of inhibition. Homology modeling of the Trypanosoma cruzi MetRS enzyme revealed key differences in the allosteric pocket between the T. cruzi and Leishmania enzymes. These provide a likely explanation for the lower MetRS02 potencies that we observed for the T. cruzi enzyme compared to the Leishmania enzyme. The identification of a new series of MetRS inhibitors and the discovery of a new binding site in kinetoplastid MetRS enzymes provide a novel strategy in the search for new therapeutics for kinetoplastid diseases.

Rapid and scale-independent microfluidic manufacture of liposomes entrapping protein incorporating in-line purification and at-line size monitoring.

Within this paper we present work that has the ability to de-risk the translation of liposomes from bench to the clinic. We have used microfluidics for the rapid and scale-independent manufacture of liposomes and have incorporated in-line purification and at-line monitoring of particle size. Using this process, we have manufactured a range of neutral and anionic liposomes incorporating protein. Factors investigated include the microfluidics operating parameters (flow rate ratio (FRR) and total flow rate (TFR)) and the liposome formulation. From these studies, we demonstrate that FRR is a key factor influencing liposome size, protein loading and release profiles. The liposome formulations produced by microfluidics offer high protein loading (20-35%) compared to production by sonication or extrusion (<5%). This high loading achieved by microfluidics results from the manufacturing process and is independent of lipid selection and concentration across the range tested. Using in-line purification and at-line size monitoring, we outline the normal operating range for effective production of size controlled (60-100 nm), homogenous (PDI <0.2) high load liposomes. This easy microfluidic process provides a translational manufacturing pathway for liposomes in a wide-range of applications.