Inhibiting Helicobacter pylori HtrA protease by addressing a computationally predicted allosteric ligand binding site

Abstract

Helicobacter pylori is associated with inflammatory diseases and can cause gastric cancer and mucosa-associated lymphoma. One of the bacterium's key proteins is high temperature requirement A (HpHtrA) protein, an extracellular serine protease that cleaves E-cadherin of gastric epithelial cells, which leads to loss of cell-cell adhesion. Inhibition of HpHtrA may constitute an intervention strategy against H. pylori infection. Guided by the computational prediction of hypothetical ligand binding sites on the surface of HpHtrA, we performed residue mutation experiments that confirmed the functional relevance of an allosteric region. We virtually screened for potential ligands addressing this surface cleft located between the catalytic and PDZ1 domains. Our receptor-based computational method represents protein surface pockets in terms of graph frameworks and retrieves small molecules that satisfy the constraints given by the pocket framework. A new chemical entity was identified that blocked E-cadherin cleavage in vitro by direct binding to HpHtrA, and efficiently blocked pathogen transmigration across the gastric epithelial barrier. A preliminary crystal structure of HpHtrA confirms the validity of a comparative "homology" model of the enzyme, which we used for the computational study. The results of this study demonstrate that addressing orphan protein surface cavities of target macromolecules can lead to new bioactive ligands.More than 50% of the human population is infected by the gram-negative bacterium Helicobacter pylori (H. pylori). 1 It is associated with several inflammatory diseases such as ulceration and gastritis. 2,3 H. pylori colonizes the host's gastric epithelium where it is able to destroy mucosal integrity and therefore can pass the epithelial barrier. 4 In severe cases, an infection can lead to gastric cancer and mucosa-associated lymphoid tissue lymphoma, which is why H. pylori has been rated as a class 1 carcinogen by the World Health gisbert.schneider@pharma.ethz.ch. ( 1) where F is the set of all ligand graph potentials, v(f) corresponds to the value of property f in vertex v, and comp(f) is the complementary graph potential to f. The fit between two vertices in feature f is weighted according to predetermined correlation values c f , which we obtained from observed property distributions in a set of known ligand-protein complexes (PDBbind core set). 23 Briefly, graph representations of all binding sites and their bound ligands were calculated, and all vertices of the ligand graphs were assigned to their closest vertex of the respective pocket graph. The correlation values between the resulting paired property distributions were taken as weighting factors c f . To be consistent with the vertex-fit calculations of the other modules of PoLiMorph that employ z-scoring procedures, the vertex-fit distributions of all assigned vertices within the PDBbind core set were determined. Means and standard deviations of these distributions were used for rescoring of the matched graphs (for technic...

Proteases and protease inhibitors in infectious diseases

“…9A). 148,152 In a similar fashion, Huston and co-workers discovered C. trachomatis HtrA (CtHtrA) to be crucial for the survival of the organism and likely to be important for the establishment of infections in humans as it enables the pathogen to withstand stress and maintains its cellular morphology. 24,34,153 Subsequently they identified two inhibitors JO146 (IC 50 = 12.5 ± 2.94 M) and JCP83 (IC 50 = 47.2 ± 7.4 M) ( Fig.…”

“…Additionally, overall potency was found to be enhanced by noncovalent interactions between the benzothiazole ring system and Phe209 of the enzyme, as well as similar interactions between the terminal ethyl and IIe253 and Met257 of the enzyme's subpocket 11 (Fig. A) …”

Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis

“…HtrA, and it efficiently inhibited cleavage of E-cadherin (Perna et al, 2014). A peptide-based inhibitor was also designed from an E-cadherin cleavage site; however, its mode of action has not yet been determined (Schmidt, Perna, et al, 2016).…”

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

“…However, most allosteric modulators were discovered by high-throughput screening experiment, which is time-consuming and serendipitous to some extent due to the lack of the comprehensive understanding about the allosteric sites and mechanisms [22]. With the development of computational allosteric prediction methods including the structure-based virtual screening and protein-modulator interactions assessment, an integrated computational and experimental approach provide a robust tool to aid in the allosteric inhibitors discovery [23][24][25]. In this study, we aimed to explore the discovery of novel allosteric inhibitors targeting the αD pocket of CK2α using structure-based virtual screening method.…”