Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1′ and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.


Contents
Table S1.Phenotypic effects and antischistosomal activity of gallinamides tested against S. mansoni schistosomula.Table S2.Physicochemical parameters of gallinamide A and its derivatives.Table S3.Phenotypic effects and antischistosomal activity of gallinamides tested against S. mansoni adults.Table S4.Cytotoxicity of gallinamides.Table S5.X-ray data collection and refinement statistics.Table S6.List of contacts formed between SmCB1 and gallinamide inhibitors.Table S7.Atom-atom contacts between gallinamide A and SmCB1.Table S8.Atom-atom contacts between inhibitor 1 and SmCB1.Table S9.Atom-atom contacts between inhibitor 6 and SmCB1.Table S10.Computational analysis of dual conformations of the crystallographic structure of inhibitor 6 in the SmCB1 active site.Figure S1.Analysis of the conformational flexibility of three gallinamide inhibitors in the SmCB1 active site.
a Phenotypic changes in newly transformed schistosomula (NTS) of S. mansoni induced by 1 and 10 µM compounds were recorded daily for three days.Phenotypes are reported using the following descriptors: N, normal; R, rounded; S, slow; unc, uncoordinated; Dark, dark, color altered from normal; Deg, degenerated; D, dead.b Each descriptor is assigned a value of 1, except for Deg and D, which are given the maximum score of 4. Values are then added to yield a severity score ranging from 0 (no effect) to 4 (the most severe), as described previously.

Table S6. List of contacts formed between SmCB1 and gallinamide inhibitors.
The analysis of protein-inhibitor contacts between the SmCB1 active site and the inhibitors was performed using the program CONTACT. 8The distance cutoffs were set to 4.1 Å for all contacts and 3.3 Å for hydrogen bonds.Fragmentation of the inhibitors into the positions P4 to P1' is depicted in black/red above the table; distinguishing substituents are highlighted in color (magenta, green and cyan for gallinamide A, 1 and 6, respectively).The SmCB1 residues interacting with the individual inhibitor position (P3 to P1´) are specified.The C atom of inhibitors forming a covalent bond with the enzyme is included in the analysis (P1).For each SmCB1 residue, the total number of contacts is given (∑), and the individual types of contacts are indicated, including carbon-carbon (C-C), carbon-heteroatom (Ch), and heteroatom-heteroatom (h-h) contacts.Hydrogen bonds (Hb) are indicated; residues forming Hb are in bold.For compound 6, data are shown for two alternative conformations (A and B) of the P1' position.

Table S7. Atom-atom contacts between gallinamide A and SmCB1.
The numbering of the inhibitor atoms is according to the PDB.Pairs of enzyme and inhibitor atoms forming contacts are listed.Atoms forming hydrophobic interactions and hydrogen bonds are highlighted in red and blue, respectively.See Table S6 for details.
The numbering of the inhibitor atoms is according to the PDB.Pairs of enzyme and inhibitor atoms forming contacts are listed.Atoms forming hydrophobic interactions and hydrogen bonds are highlighted in red and blue, respectively.See Table S6 for details. Cpd.1

Contact count
Enzyme residue atom Ligand atom P1'

Table S3 . Phenotypic effects of gallinamides against ex vivo S. mansoni adults. Phenotype descriptors a Time (h) 2 6 24 48 Compound (µM) 1
1,4,5notypic changes in adult S. mansoni induced by compounds at given concentrations were recorded after 2, 6, 24 and 48 h.Phenotypes are reported as the following descriptors: N, normal; S, slowed motility; Unc, uncoordinated movements; Dark, dark -color altered from normal; on sides, male worms do not adhere to dish with ventral sucker; teg dam, outer surface (tegument of the worm) is damaged; deg, degenerated.Each descriptor is assigned a value of 1, except for deg and teg dam, which are given the maximum score of 4. Values are then added to yield a severity score ranging from 0 (no effect) to 4 (the most severe), as described previously.1,4,5

Table S4 .
Cytotoxicity of gallinamides.Cytotoxicity of gallinamide A and compounds 1, 6, and 9 was tested with four human cell lines and expressed as % viability vs. untreated cells.Cells were treated with the indicated compound concentrations for 72 h (A) or with 10 µM compounds for the indicated time (B) and assayed using CellTiter Glo 2.0 (Promega) according to the manufacturer's protocol.Cytotoxicity was expressed as percent viability compared to untreated control cells; the standard deviation of the mean of quadruplicate readings is shown.Cell line abbreviations: HL-60, human promyelocytic leukemia; HeLa, human cervical carcinoma; HepG2, human hepatocellular carcinoma; NHDF, normal human dermal fibroblasts.

Table S5 . X-ray data collection and refinement statistics.
a Numbers in the parentheses refer to the highest-resolution shell.bRmerge=100∑hkl∑i|Ii(hkl) -‹I(hkl)›|/∑hkl ∑i Ii(hkl), where Ii(hkl) is an individual intensity of the i th observation of the reflection hkl and ‹I(hkl)› is the average intensity of the reflection hkl with summation over all data.cR value = ||Fo| − |Fc||/|Fo|, where Fo and Fc are the observed and calculated structure factors, respectively.dRfree is equivalent to the R value but is calculated for up to 5% of the reflections chosen at random and omitted from the refinement process. 6 AU, asymmetric unit.fADP, atomic displacement parameter, formally B-factor.gAs determined by Molprobity.7

Table S9 . Atom-atom contacts between inhibitor 6 and SmCB1.
The numbering of the inhibitor atoms is according to the PDB.Pairs of enzyme and inhibitor atoms forming contacts (CC, contact count) are listed.Atoms forming hydrophobic interactions and hydrogen bonds are highlighted in red and blue, respectively.See TableS6for details.