Discovery, Characterization, and Structure of a Cell Active PAD2 Inhibitor Acting through a Novel Allosteric Mechanism.

Peptidyl arginine deiminases (PADs) are important enzymes in many diseases, especially those involving inflammation and autoimmunity. Despite many years of effort, developing isoform-specific inhibitors has been a challenge. We describe herein the discovery of a potent, noncovalent PAD2 inhibitor, with selectivity over PAD3 and PAD4, from a DNA-encoded library. The biochemical and biophysical characterization of this inhibitor and two noninhibitory binders indicated a novel, Ca2+ competitive mechanism of inhibition. This was confirmed via X-ray crystallographic analysis. Finally, we demonstrate that this inhibitor selectively inhibits PAD2 in a cellular context.

The enzyme activity of sortase A is regulated by phosphorylation in Staphylococcus aureus.

In many Gram-positive bacteria, the transpeptidase enzyme sortase A (SrtA) anchors surface proteins to cell wall and plays a critical role in the bacterial pathogenesis. Here, we show that in Staphylococcus aureus, an important human pathogen, the SrtA is phosphorylated by serine/threonine protein kinase Stk1. S. aureus SrtA can also be phosphorylated by small-molecule phosphodonor acetyl phosphate (AcP) in vitro. We determined that various amino acid residues of S. aureus SrtA are subject to phosphorylation, primarily on its catalytic site residue cysteine-184 in the context of a bacterial cell lysate. Both Stk1 and AcP-mediated phosphorylation inhibited the enzyme activity of SrtA in vitro. Consequently, deletion of gene (i.e. stp1) encoding serine/threonine phosphatase Stp1, the corresponding phosphatase of Stk1, caused an increase in the phosphorylation level of SrtA. The stp1 deletion mutant mimicked the phenotypic traits of srtA deletion mutant (i.e. attenuated growth where either haemoglobin or haem as a sole iron source and reduced liver infections in a mouse model of systemic infection). Importantly, the phenotypic defects of the stp1 deletion mutant can be alleviated by overexpressing srtA. Taken together, our finding suggests that phosphorylation plays an important role in modulating the activity of SrtA in S. aureus.

Discovery of Benzocycloalkane Derivatives Efficiently Blocking Bacterial Virulence for the Treatment of Methicillin-Resistant S. aureus (MRSA) Infections by Targeting Diapophytoene Desaturase (CrtN).

Antivirulence strategies are now attracting interest for the inherent mechanism of action advantages. In our previous work, diapophytoene desaturase (CrtN) was identified to be an attractive and drugable target for fighting pigmented S. aureus infections. In this research, we developed a series of effective benzocycloalkane-derived CrtN inhibitors with submicromolar IC50. Analogue 8 blocked the pigment biosynthesis of three MRSA strains with a nanomolar IC50 value. Corresponding to its mode of action, 8 did not function as a bactericidal agent. 8 could sensitize S. aureus to immune clearance. In vivo, 8 was proven to be efficacious in an S. aureus Newman sepsis model and abscess formation model. For two typical MRSAs, USA400 MW2 and Mu50, 8 significantly decreased the staphylococcal loads in the liver and kidneys. Moreover, 8 showed minimal antifungal activity compared to that of NTF. In summary, 8 has the potential to be developed as a therapeutic drug, especially against intractable MRSA issues.

Discovery of Potent Benzofuran-Derived Diapophytoene Desaturase (CrtN) Inhibitors with Enhanced Oral Bioavailability for the Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) Infections.

Blocking the staphyloxanthin biosynthesis process has emerged as a new promising antivirulence strategy. Previously, we first revealed that CrtN is a druggable target against infections caused by pigmented Staphylococcus aureus (S. aureus) and that naftifine was an effective CrtN inhibitor. Here, we identify a new type of benzofuran-derived CrtN inhibitor with submicromolar IC50 values that is based on the naftifine scaffold. The most potent analog, 5m, inhibits the pigment production of S. aureus Newman and three MRSA strains, with IC50 values of 0.38-5.45 nM, without any impact on the survival of four strains (up to 200 µM). Notably, compound 5m (1 µM) could significantly sensitize four strains to immune clearance and could effectively attenuate the virulence of three strains in vivo. Moreover, 5m was determined to be a weak antifungal reagent (MIC > 16 µg/mL). Combined with good oral bioavailability (F = 42.2%) and excellent safety profiles, these data demonstrate that 5m may be a good candidate for the treatment of MRSA infections.

Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence.

The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.

The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.

Rhamnolipid acts as a virulence factor during Pseudomonas aeruginosa infection. Here, we show that deletion of the catabolite repression control (crc) gene in P. aeruginosa leads to a rhamnolipid-negative phenotype. This effect is mediated by the down-regulation of rhl quorum sensing (QS). We discover that a disruption of the gene encoding the Lon protease entirely offsets the effect of crc deletion on the production of both rhamnolipid and rhl QS signal C4-HSL. Crc is unable to bind lon mRNA in vitro in the absence of the RNA chaperon Hfq, while Crc contributes to Hfq-mediated repression of the lon gene expression at a posttranscriptional level. Deletion of crc, which results in up-regulation of lon, significantly reduces the in vivo stability and abundance of the RhlI protein that synthesizes C4-HSL, causing the attenuation of rhl QS. Lon is also capable of degrading the RhlI protein in vitro. In addition, constitutive expression of rhlI suppresses the defects of the crc deletion mutant in rhamnolipid, C4-HSL and virulence on lettuce leaves. This study therefore uncovers a novel posttranscriptional regulatory cascade, Crc-Hfq/Lon/RhlI, for the regulation of rhamnolipid production and rhl QS in P. aeruginosa.

Metabolic sensor governing bacterial virulence in Staphylococcus aureus.

An effective metabolism is essential to all living organisms, including the important human pathogen Staphylococcus aureus. To establish successful infection, S. aureus must scavenge nutrients and coordinate its metabolism for proliferation. Meanwhile, it also must produce an array of virulence factors to interfere with host defenses. However, the ways in which S. aureus ties its metabolic state to its virulence regulation remain largely unknown. Here we show that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, binds to and activates the catabolite control protein E (CcpE) of S. aureus. Using structural and site-directed mutagenesis studies, we demonstrate that two arginine residues (Arg145 and Arg256) within the putative inducer-binding cavity of CcpE are important for its allosteric activation by citrate. Microarray analysis reveals that CcpE tunes the expression of 126 genes that comprise about 4.7% of the S. aureus genome. Intriguingly, although CcpE is a major positive regulator of the TCA-cycle activity, its regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Moreover, inactivation of CcpE results in increased staphyloxanthin production, improved ability to acquire iron, increased resistance to whole-blood-mediated killing, and enhanced bacterial virulence in a mouse model of systemic infection. This study reveals CcpE as an important metabolic sensor that allows S. aureus to sense and adjust its metabolic state and subsequently to coordinate the expression of virulence factors and bacterial virulence.