DISCOVERY AND CHARACTERIZATION OF PROMISING CRUZAIN INHIBITORS THROUGH PATHOGEN BOX SCREENING

Currently, there are only two approved drugs for the treatment of Chagas disease - Benznidazole and
Nifurtimox. Both have limited efficacy in the chronic phase of the disease and are associated with
significant adverse effects. Despite the exhaustive search for new drug candidates, the development
of safer and more effective drugs is still necessary. The aim of this study is to determine the IC50 values
and, when possible, the mechanism of enzymatic inhibition of 10 hits obtained from screening of the
Pathogen Box library in a previous work of our research group. Proteolytic activity was measured by
monitoring the cleavage of a fluorogenic substrate. Enzymatic activities were calculated by comparing
the initial reaction rate of the dimethyl sulfoxide (DMSO) control with the velocities in the compound's
presence. IC50 curves were determined in two independent experiments, each involving at least seven
compound concentrations in triplicates. To determine the mechanism of inhibition, cruzain activity was
measured at seven different substrate concentrations (0.1-10 μM) and five inhibitor concentrations.
Docking was performed in AutoDock Vina 1.2.3. The crystallographic structure of cruzain was acquired
from RSCB PDB (ID: 3KKU) and ten poses were generated and subjected to visual inspection,
considering the ligand-binding site complementarity, the presence of hydrogen bonds and polar
interactions. Inhibition mechanisms against cruzain were investigated for MMV688179 and
MMV687246. For MMV687246, a mixed inhibition pattern was observed with an α = 0.7 ± 0.2 and a Ki
of 57 ± 6 μM. Analysis of the Km app at varying concentrations of MMV687246 suggests absence of
substrate competition. On the other hand, for MMV688179, clear competition with the substrate was
observed, as determined by combined analysis of the Michaelis-Menten and Lineweaver-Burk plots.
The Ki was estimated to be 206 nM from the linear regression of Km app. Due to the competitive
mechanism and the high potency of MMV688179, molecular docking was employed to propose a
binding mode for this molecule. The best-scored pose was selected through visual inspection as the
most plausible binding mode of MMV688179. In this binding pose, one of the protonated guanidines is
buried in the S2 subsite, forming an ion-ion interaction with Glu208. The other protonated guanidine
forms hydrogen bonds with the main chain of residues Gly20 and Cys22, both at the S1 subsite. The
docking pose spans over a large part of the catalytic cleft, displaying good spatial complementarity to
the protein site, and also suggests that the furan ring acts as a linker. The results suggest that
MMV687246 is a mixed inhibitor of cruzain and MMV688179 is a competitive inhibitor. Further studies
are necessary to elucidate the structure-activity relationship of MMV688179, however, the molecules
identified in this study can be considered candidates for optimization