Discovery of Small-Molecule VapC1 Nuclease Inhibitors by Virtual Screening and Scaffold Hopping from an Atomic Structure Revealing Protein-Protein Interactions with a Native VapB1 Inhibitor.

Nontypeable Haemophilus influenzae (NTHi) are clinically important Gram-negative bacteria that are responsible for various human mucosal diseases, including otitis media (OM). Recurrent OM caused by NTHi is common, and infections that recur less than 2 weeks following antimicrobial therapy are largely attributable to the recurrence of the same strain of bacteria. Toxin-antitoxin (TA) modules encoded by bacteria enable rapid responses to environmental stresses and are thought to facilitate growth arrest, persistence, and tolerance to antibiotics. The vapBC-1 locus of NTHi encodes a type II TA system, comprising the ribonuclease toxin VapC1 and its cognate antitoxin VapB1. The activity of VapC1 has been linked to the survival of NTHi during antibiotic treatment both in vivo and ex vivo. Therefore, inhibitors of VapC1 might serve as adjuvants to antibiotics, preventing NTHi from entering growth arrest and surviving; however, none have been reported to date. A truncated VapB1 peptide from a crystal structure of the VapBC-1 complex was used to generate pharmacophore queries to facilitate a scaffold hopping approach for the identification of small-molecule VapC1 inhibitors. The National Center for Advancing Translational Sciences small-molecule library was virtually screened using the shape-based method rapid overlay of chemical structures (ROCS), and the top-ranking hits were docked into the VapB1 binding pocket of VapC1. Two hundred virtual screening hits with the best docking scores were selected and tested in a biochemical VapC1 activity assay, which confirmed eight compounds as VapC1 inhibitors. An additional 60 compounds were selected with structural similarities to the confirmed VapC1 inhibitors, of which 20 inhibited VapC1 activity. Intracellular target engagement of five inhibitors was indicated by the destabilization of VapC1 within bacterial cells from a cellular thermal shift assay; however, no impact on bacterial growth was observed. Thus, this virtual screening and scaffold hopping approach enabled the discovery of VapC1 ribonuclease inhibitors that might serve as starting points for preclinical development.

Structural Basis for Toxin Inhibition in the VapXD Toxin-Antitoxin System.

Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogenicity; however, the mode of action of VapD and the mechanism of inhibition by the VapX antitoxin remain unknown. Here, we report the structure of the intact H. influenzae VapXD complex, revealing an unusual 2:1 TA molecular stoichiometry where a Cas2-like homodimer of VapD binds a single VapX antitoxin. VapX consists of an oligonucleotide/oligosaccharide-binding domain that docks into an asymmetrical cavity on the toxin dimer. Structures of isolated VapD further reveal how a symmetrical toxin homodimer adapts to interacting with an asymmetrical antitoxin and suggest how a primordial TA system evolved to become part of CRISPR-Cas immunity systems.

Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection.

Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivoIMPORTANCE Herein the crystal structure of the VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in E. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues ex vivo Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in cis wild-type toxin under the same conditions.

Better living through chemistry: Addressing emerging antibiotic resistance.

The increasing emergence of multidrug-resistant bacteria is recognized as a major threat to human health worldwide. While the use of small molecule antibiotics has enabled many modern medical advances, it has also facilitated the development of resistant organisms. This minireview provides an overview of current small molecule drugs approved by the US Food and Drug Administration (FDA) for use in humans, the unintended consequences of antibiotic use, and the mechanisms that underlie the development of drug resistance. Promising new approaches and strategies to counter antibiotic-resistant bacteria with small molecules are highlighted. However, continued public investment in this area is critical to maintain an edge in our evolutionary "arms race" against antibiotic-resistant microorganisms. Impact statement The alarming increase in antibiotic-resistant microorganisms is a rapidly emerging threat to human health throughout the world. Historically, small molecule drugs have played a major role in controlling bacterial infections and they continue to offer tremendous potential in countering resistant organisms. This minireview provides a broad overview of the relevant issues, including the diversity of FDA-approved small molecule drugs and mechanisms of drug resistance, unintended consequences of antibiotic use, the current state of development for small molecule antibacterials and financial challenges that impact progress towards novel therapies. The content will be informative to diverse stakeholders, including clinicians, basic scientists, translational scientists and policy makers, and may be used as a bridge between these key players to advance the development of much-needed therapeutics.