Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as “Doorstop” for NADPH Entry

Abstract

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

Computer-Assisted Discovery of Alkaloids with Schistosomicidal Activity

“…Four proteins of S. mansoni were downloaded from the PDB (Protein Data Bank, www. rcsb.org, accessed on 15 December 2021) [38]: Schistosoma mansoni 14 kDa fatty-acid-binding protein (Sm14) (transport protein, PDB ID 1VYF, ligand oleic acid) [39], histone deacetylase 8 (transferase, PDB ID 4BZ8, ligand J1038) [40], sulfotransferase (transferase, PDB ID 4MUB, ligand Oxamniquine) [41], and thioredoxin glutathione reductase (Flavoprotein, PDB ID 6FTC, ligand Hepes) [42].…”

First In Silico Screening of Insect Molecules for Identification of Novel Anti-Parasitic Compounds

“…Binding of 1,8-naphthyridine-2-carboxylate (A1, orange sticks in Figure 2) into this pocket prevents Tyr296 from rotating (blue sticks- NADPH bound SmTGR, magenta sticks-A1 bound SmTGR), which is needed for NADPH entry and enzyme activity. This way, the occupied pocket acts as a doorstop for the entry of NADPH [30]. Since the insect molecules from our library comprised both fragment-like and large complex natural products, and additional HEPE (1-(2-hydroxyethyl)piperazine) and Site 1 subpockets were identified adjacent to the doorstop pocket [23,30], docking analyses were performed throughout the cavity that is built by all pockets (green surface in Figure 2).…”

Schistosomiasis Drug Discovery in the Era of Automation and Artificial Intelligence

“…Most SmTGR inhibitors are reactive electrophilic compounds, such as metal derivatives or Michael acceptors, presumably targeting the nucleophilic residues (selenocysteine and low pKa redox active cysteines), which may result in low selectivity and toxicity (231,232). Attempts to obtain crystal structures of SmTGR in complex with such inhibitors have been unsuccessful, reflecting the problem of crystallizing nonhomogenous protein preparations resulting from the presence of several redox and nucleophilic centers in the SmTGR, which are the sites of action for electrophilic inhibitor (233).…”

“…The pockets are separated by the Tyr296 residue, where the aromatic ring of Tyr296 could adopt the closed (Figures 3A, D) and open (Figures 3B, C) conformations. Small molecules bound at the doorstop pocket disturb the wellknown and conserved conformational adjustments associated with NADPH binding and enzyme reduction (233). Subsequently, chimeric compounds blending the structural features of the initial fragments into single compounds were synthesized and showed improved SmTGR inhibition activity, ex vivo activity against larval and adult S. mansoni worms at low micromolar 215); (B) Fragment merging: two or more fragments sharing the same pocket are covalently merged to obtain a larger compound with higher affinity.…”

“…concentrations. In addition, the designed compounds tended to have selectivity for SmTGR, as the amino acid residues of the doorstop pocket are not conserved between members of the FAD/ NAD-linked reductase family (233). Although strictly this work was not a FBDD campaign (at least not originally designed as one), it was an interesting effort that showed the potential of the fragment-based approach to disclose new binding sites that can be explored to develop novel potent ligands.…”