Determination of Protein–Ligand Binding Constants of a Cooperatively Regulated Tetrameric Enzyme Using Electrospray Mass Spectrometry

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (<i>K</i>_D) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their <i>K</i>_Ds by nanoESI-MS. The <i>K</i>_Ds determined by ESI-MS correlate very well with IC₅₀ values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand <b>3</b> in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well

2<i>H</i>‑1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition (<i>K</i>_i values) and <i>in vitro</i> cell-growth of <i>Trypanosoma brucei rhodesiense</i> (IC₅₀ values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1<i>H</i>-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and <i>in vivo</i> half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites

2<i>H</i>‑1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition (<i>K</i>_i values) and <i>in vitro</i> cell-growth of <i>Trypanosoma brucei rhodesiense</i> (IC₅₀ values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1<i>H</i>-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and <i>in vivo</i> half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei (<i>T</i>. <i>b</i>.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors (<i>K</i>_i < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC₅₀ < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys–SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival

Identification of Potent and Selective Cathepsin S Inhibitors Containing Different Central Cyclic Scaffolds

Starting from the weakly active dual CatS/K inhibitor <b>5</b>, structure-based design supported by X-ray analysis led to the discovery of the potent and selective (>50 000-fold vs CatK) cyclopentane derivative <b>22</b> by exploiting specific ligand–receptor interactions in the S2 pocket of CatS. Changing the central cyclopentane scaffold to the analogous pyrrolidine derivative <b>57</b> decreased the enzyme as well as the cell-based activity significantly by 24- and 69-fold, respectively. The most promising scaffold identified was the readily accessible proline derivative (e.g., <b>79</b>). This compound, with an appealing ligand efficiency (LE) of 0.47, included additional structural modifications binding in the S1 and S3 pockets of CatS, leading to favorable in vitro and in vivo properties. Compound <b>79</b> reduced IL-2 production in a transgenic DO10.11 mouse model of antigen presentation in a dose-dependent manner with an ED₅₀ of 5 mg/kg

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure–activity relationship (SAR) and protease selectivity are also discussed