The gene repertoire of the main cysteine protease of Trypanosoma cruzi, cruzipain, reveals four sub-types with distinct active sites

Abstract

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.

Coumarin-based derivatives targetingTrypanosoma cruzicruzain andTrypanosoma bruceicathepsin L-like proteases

“…It is possible to verify that its 8-methoxy-substituted coumarin nucleus is found at the S2 subpocket entry since it has a preference for van der Waals interactions with hydrophobic substituents, 74–76 while its thiosemicarbazone moiety is oriented toward the shallow S1 subpocket, which is typically associated with inhibitors and better accommodates non-bulky groups. 77,78 It may explain the better activity of FN-27 towards CRZ in our experimental assays. Additionally, it was observed that despite the nucleophilic Cys 25 residue being found near the methylamine double bond of FN-27 (a potential electrophilic moiety at a distance of 4.02 Å), it probably did not undergo covalent bonding, although an anion–π interaction could be possible for it.…”

Screening the Pathogen Box to Discover and Characterize New Cruzain and TbrCatL Inhibitors

“…Cruzain, a cysteine protease of Trypanosoma cruzi , is a validated [ 11 , 27 , 28 , 29 ] and well-explored pharmacological target (see [ 30 ] for a recent review). Cruzain is the truncated, recombinant-expressed cruzipain 1, part of a multigenic family comprising of four subtypes of cruzipains [ 31 ]. Tbr CatL, a homologous protease from Trypanosoma brucei, is also a validated target for discovering trypanocidal compounds [ 32 , 33 ].…”

Computational approaches towards the discovery and optimisation of cruzain inhibitors

“…Analysis of cruzain crystal structures reveals that the S3 to S1’ sub-pockets usually interact with inhibitors, being the most relevant for drug development. 2 Among these pockets, only S2 is well defined, whilst the S1’, S1, and S3 pockets are shallow. Studies of cruzain’s substrate specificity indicate that S3 accommodates better positively charged residues and bulky aromatic groups; S2 accommodates hydrophobic aas, favoring the aromatic ones, and also accepting basic aas; S1 accommodates better positively charged residues and small aromatic groups; S1’ lacks an amino acid (aa) preference.…”

“…Despite most of the drug studies focusing on cruzain, there are other cruzipain sub-types expressed by T. cruzi . Santos et al 2 identified two cruzipain families organised in two clusters in the parasite genome and subdivided the sequences into four sub-types. Family I embraces the czp1 sub-type, the target of drug design projects, represented by cruzain.…”

“…On the other hand, Family II sequences are more expressed in the trypomastigotes, which, together with amastigotes, are more relevant in the drug design context. 2 These findings raise a red flag to groups working with cruzain as a drug target. Researchers have known the existence of over one cruzipain for a long time.…”