Identification of chlamydial T3SS inhibitors through virtual screening against T3SS ATPase

Abstract

Chlamydia trachomatis is a widespread sexually transmitted pathogen that resides within a special vacuole inside host cells. Although acute infection can be treated with antibiotics, chlamydia can enter persistent state, leading to chronic infection that is difficult to cure. Thus, novel anti-chlamydial compounds active against persistent chlamydia are required. Chlamydiae rely upon type III secretion system (T3SS) to inject effector proteins into host cell cytoplasm, and T3SS inhibitors are viewed as promising compounds for treatment of chlamydial infections. C. trachomatis ATPase SctN is an important T3SS component and has not been targeted before. We thus used virtual screening against homology modeled SctN structure to search for SctN inhibitors. Selected compounds were tested for their ability to inhibit chlamydial survival and development within eukaryotic cells, and for the ability to suppress normal T3SS functioning. We identified two compounds that were able to block normal protein translocation through T3SS and inhibit chlamydial survival within eukaryotic cells in 50-100 μm concentrations. These two novel T3SS inhibitors also possessed relatively low toxicity toward eukaryotic cells. A small series of derivatives was further synthesized for the most active of two inhibitors to probe SAR properties.

Structural Insight Into Conformational Changes Induced by ATP Binding in a Type III Secretion-Associated ATPase From Shigella flexneri

“…Such information could facilitate the construction of a general model for designing small inhibitors targeting T3SS ATPases in various Gram-negative bacteria. In recent years, many small inhibitors targeting T3SS ATPases have been identified and developed against pathogens (Swietnicki et al, 2011 ; Gong et al, 2015 ; Grishin et al, 2018 ). Small-molecule compounds that bind to T3SS ATPases based on the nucleotide-bound Spa47 structure determined in the present work could obstruct the ATP-binding site and effectively inhibit catalytic activity.…”

On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome

“…Using the structure of EscN (SctN for EPEC; PDB ID: 2OBL), FliI (flagellar SctN; PDB ID: 2DPY) and a bovine F-type ATPase (PDB ID: 2JDI) as ATPase templates, Grishin et al (2018) screened a library of compounds that could potentially inhibit the Chlamydia trachomatis ATPase homologue, CdsN, which has no known structure but approximately 50% sequence identity with the SctN's of known structure. 103 The ATPase is the only conserved enzymatic activity of the T3SS apparatus itself, and is thus an attractive drug target. 104 Grishin and colleagues identified 16 top-scoring candidates in their virtual screening process, with 2 compounds exhibiting significant suppression of the C. trachomatis development cycle in mammalian cells at low μM concentrations without displaying significant toxicity in mammalian cells.…”

“…The authors probed potential structure-activity relationships, but no improvements were found. 103 Case et al (2018) identified 3 non-competitive inhibitors of Spa47 (SctN of Shigella flexneri) through virtual screening techniques and were predicted to bind at the interface between protomers based on modelling the oligomeric structure of Spa47 after a heterohexameric F-type ATPase. 105 Two inhibitors functioned in vivo, exhibiting almost no secretion without compromising S. flexneri or HeLa viability.…”

“…18 Previously, inhibitors against the ATPase of several T3SS's have been designed, based off of high throughput or virtual screening using homology modelling and notably in the absence of the central stalk component, which defined the observed asymmetry. [103][104][105]127 As the active site(s) sits between adjacent protomers, the precise architecture provided by the novel atomic model of EscN/O may assist with optimization of previously designed inhibitors through SAR techniques.…”

“…Grishin et al (2018) used the homology model of the Chlamydia trachomatis ATPase to screen a virtual library of compounds that may target this family of enzymes. 103 With atomic resolution data now possible via cryoEM for the assembled T3SS ATPase, 18 identification of precise interfaces of these new inhibitors and their structure-guided optimization is now on the horizon. Other recent examples include, Dey et al (2017) who recombinantly expressed and purified the needle tip protein from Shigella flexneri (IpaD); they employed a high-throughput surface plasmon resonance screening strategy to monitor interactions of IpaD with 288 different compounds in a defined library.…”