New drugs for visceral leishmaniasis that are safe, low cost and adapted to the field are urgently required. Despite concerted efforts over the last several years, the number of new chemical entities with novel mechanisms of action that are suitable for clinical development remains low. Here, we describe the development of DNDI-6174, an inhibitor of Leishmania cytochrome b that originated from a phenotypically-identified pyrrolopyrimidine series. This compound fulfills all Target Candidate Profile criteria required for progression into preclinical development. In addition to good metabolic stability and pharmacokinetic properties, DNDI-6174 demonstrates potent in vitro activity against a variety of Leishmania species and can reduce parasite burden in animal models of infection, with potential for sterile cure. No significant flags were identified in preliminary safety studies, including an exploratory 14-day toxicology study in the rat. DNDI-6174 represents the first cytochrome b inhibitor to enter preclinical development for visceral leishmaniasis.

Molecular modelling

Ligand and enzyme preparation: The 3D model of DNDI-6174 was created using LigPrep (Schrödinger Release 2021-2, LigPrep, Schrödinger, LLC, New York, NY, 2021), generating the most probable ionization state and tautomers at neutral pH (7.4 ± 0.2). Our previously reported homology model of L. donovani cytochrome b (8) was processed using the protein preparation module in the Schrödinger suite to provide starting points for subsequent molecular modelling studies. New structures were generated incorporating each of the five mutations found to drive drug resistance. For every mutant, the hydrogen atoms’ positions were optimized using the H-bond assignment/sample water orientation tool, and the resulting structures were subjected to a restrained minimisation procedure with the OPLS3e force field. This was achieved using the minimisation protocol of the protein preparation module of the Schrödinger’s suite of software.

Molecular docking: Molecular docking was performed using the six cytochrome b structures (wild-type and mutated) with ubiquinone bound in the Qi site alongside one conserved water. Molecular docking studies were carried out using Glide (Schrödinger Release 2021-2, Glide, Schrödinger, LLC, New York, NY, 2021) in standard precision (SP). The docking energy grids for wild-type and mutant cytochrome b were prepared using the default value of the protein atom scaling factor (1.0 Å) within a cubic box centered on the ubiquinone (UQ2) bound in the wild-type model. The conserved water molecule interacting with Phe34 was also retained. After grid generation, DNDI-6174 was docked into cytochrome b. The number of poses entered to post-docking minimization was set to 10. In order to assess the validity of the docking protocol, UQ2 was used as a reference ligand for a redocking procedure with all 6 enzymes (WT and 5 mutants).

Molecular dynamics: DNDI-6174: cytochrome b complexes generated during molecular docking studies underwent 100 ns molecular dynamic (MD) simulations using Desmond
(Schrödinger Release 2021-2: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2021. Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2021). The C-terminal carboxylic acid of all model proteins was capped with Nmethylamine, and the N-terminal position acetylated. Bilayers of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) membranes were added to the model systems to mimic the integral membrane environment of cytochrome b. The obtained systems were placed in orthorhombic boxes filled with simple point-charge (SPC) water molecules. To ensure an electrically neutral system for the simulation, counter ions were added to the system in the form of a 0.15 M salt solution. The ions were placed 10 Å away from the ligands as a buffer zone. Prior to running simulations, the systems were allowed to relax. The NPγT ensemble class was chosen, so that fixing the surface tension ensured that the simulation box does not deform significantly in the plane of the membrane while the pressure is applied normal to the membrane surface. The analysis of all the MD trajectories was performed within the Schrödinger’s simulation interactions diagram panel.