Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to Schistosoma mansoni: Structural and Mechanistic Insights into Chemotype Reactivity

Abstract

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E-to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes

“…The CO–NMe and CO–NH bond (in Gü1303 and Gü2602 , respectively) of the warhead adopted a Z -configuration in the mCatK active site. This is in line with our recent analysis of another cyanohydrazide inhibitor (with the azadipeptide nitrile scaffold) in the active site of a cysteine protease 32 which also demonstrated that the warhead with the methylated N–N axis provides atropochirality, and the E -configuration of the unbound inhibitor is transformed to a Z-configuration upon binding 32 . Therefore, such an E - to Z -conformational change in the course of its interaction with CatK is proposed for Gü1303 , containing the CO–NMe–NMe portion.…”

“…The nitrogen atom of the imidate moiety, derived from the warhead nitrile group, is stabilised by two hydrogen bonds to the backbone amide of the catalytic Cys25 and the side chain amide of Gln19 ( Figure 4(D) ) . An analogous interaction pattern was observed for the warhead of an azadipeptide nitrile inhibitor reacted with the protease SmCB1 (PDB: 6YI7) 32 . The NH of Gly66 acts as a bifurcated hydrogen bond donor for the carbonyl oxygen of the P2 phenylalanine and the noncarbonyl carbamate oxygen in the P3 position of Gü1303.…”

“…Azadipeptides such as Gü1303 are atropochiral molecules due to the restricted rotation around the methylated N–N axis, and, in the unbound state, they preferentially adopt the E -configuration of the respective CO–NMe bond 63 , 64 ( Figure 4(B) ). However, a Z -configuration at the CO–NMe bond was observed in Gü1303 bound to mCatK, suggesting an E - to Z- conformational change in Gü1303 upon binding to the enzyme, most likely due to a "configurational selection" that we recently reported for an azadipeptide nitrile inhibitor of the protease SmCB1 32 .…”

Schistosomiasis Drug Discovery in the Era of Automation and Artificial Intelligence

“…The vinyl sulfones inhibitors also showed desirable properties such as activity in phenotypic assays, selectivity for SmCB1 over human cathepsin B and metabolic stability. Besides this new SmCB1 inhibitor class, in a recent publication Jikováet al (149) showed that azanitriles chemotypes can act as potent covalent inhibitors of SmCB1. Using recombinantly expressed SmCB1, crystal structure determination, QM methods, phenotypic and target-based assays, these authors were able to identify azanitriles with nanomolar range potency.…”

Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

“…In this work, we used the fluorescence in situ hybridization (FISH) technique based on a detection of DIGlabeled RNA probes by anti-DIG antibody conjugated with horseradish peroxidase (HRP) [23] to investigate RNA distribution in adult male and female S. mansoni. The technique was validated by detecting a set of target RNA transcripts with known localization, the digestive protease cathepsin B1 of S. mansoni (SmCB1) [24][25][26][27], the surface-associated prolyl oligopeptidase (SmPOP) [28], the tegumental tetraspanin SmTsp-2 [29][30][31], and Sm29, a membrane-bound glycoprotein found at the S. mansoni tegument [31][32][33][34]. A probe targeting a bacterial neomycin gene (neo), which is absent in the S. mansoni genome, was used as a negative control.…”