Validated Preclinical Mouse Model for Therapeutic Testing against Multidrug-Resistant Pseudomonas aeruginosa Strains.

The rise in infections caused by antibiotic-resistant bacteria is outpacing the development of new antibiotics. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are a group of clinically important bacteria that have developed resistance to multiple antibiotics and are commonly referred to as multidrug resistant (MDR). The medical and research communities have recognized that, without new antimicrobials, infections by MDR bacteria will soon become a leading cause of morbidity and death. Therefore, there is an ever-growing need to expedite the development of novel antimicrobials to combat these infections. Toward this end, we set out to refine an existing mouse model of pulmonary Pseudomonas aeruginosa infection to generate a robust preclinical tool that can be used to rapidly and accurately predict novel antimicrobial efficacy. This refinement was achieved by characterizing the virulence of a panel of genetically diverse MDR P. aeruginosa strains in this model, by both 50% lethal dose (LD50) analysis and natural history studies. Further, we defined two antibiotic regimens (aztreonam and amikacin) that can be used as comparators during the future evaluation of novel antimicrobials, and we confirmed that the model can effectively differentiate between successful and unsuccessful treatments, as predicted by in vitro inhibitory data. This validated model represents an important tool in our arsenal to develop new therapies to combat MDR P. aeruginosa strains, with the ability to provide rapid preclinical evaluation of novel antimicrobials and support data from clinical studies during the investigational drug development process. IMPORTANCE The prevalence of antibiotic resistance among bacterial pathogens is a growing problem that necessitates the development of new antibiotics. Preclinical animal models are important tools to facilitate and speed the development of novel antimicrobials. Successful outcomes in animal models not only justify progression of new drugs into human clinical trials but also can support FDA decisions if clinical trial sizes are small due to a small population of infections with specific drug-resistant strains. However, in both cases the preclinical animal model needs to be well characterized and provide robust and reproducible data. Toward this goal, we have refined an existing mouse model to better predict the efficacy of novel antibiotics. This improved model provides an important tool to better predict the clinical success of new antibiotics.

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State.

Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.

Development and evaluation of murine lung-specific disease models for Pseudomonas aeruginosa applicable to therapeutic testing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing a wide range of disease manifestations, including severe bacterial pneumonia. Recently, clinics have reported a rise in nosocomial infections with multidrug resistant (MDR) species, including MDR strains of P. aeruginosa. In order to quickly evaluate the efficacy of new therapeutics for MDR infections, highly reproducible and validated animal models need to be developed for pre-clinical testing. Here, we describe the characterization of two murine models to study MDR P. aeruginosa respiratory disease. We evaluated and compared these models using a non-invasive intratracheal instillation method and established the 50% lethal dose, course of infection, biometric parameters of disease and degree of pneumonia development for each model. Further, we tested meropenem as a proof-of-concept therapeutic and report efficacy data that suggests that the leukopenic model could serve a robust pre-clinical model to test novel therapeutics.

Development of (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide, ML336: Novel 2-amidinophenylbenzamides as potent inhibitors of venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 = 0.8 µM), limited cytotoxic liability (CC50 > 50 µM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 µM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC50 = 0.02-0.04 µM, CC50 > 50 µM) while limiting in vitro viral replication (EC90 = 0.17 µM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg(-1) day(-1) and viral replication appeared to be inhibited through interference of viral nonstructural proteins.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain.

Parotid secretory protein (PSP) (C20orf70) is a salivary protein of unknown function. The protein belongs to the palate, lung, and nasal epithelium clone (PLUNC) family of mucosal secretory proteins that are predicted to be structurally similar to lipid-binding and host-defense proteins including bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein. However, the PLUNC proteins exhibit significant sequence variation and different biological functions have been proposed for different family members. This study tested the functional implications of the proposed similarity of PSP to the acute phase protein lipopolysaccharide-binding protein (LBP). PSP was identified in human saliva and was soluble in 70% ethanol, as shown for other PLUNC proteins. PSP binds lipopolysaccharide and can be eluted by non-ionic detergent, but not by urea or high salt. A synthetic PSP peptide, GL13NH2, which corresponds to a lipopolysaccharide-inhibiting peptide from LBP, inhibited the binding of lipopolysaccharide to both PSP and lipopolysaccharide-binding protein. Peptides from other regions of PSP and the control peptide polymyxin B showed no effect on the binding of PSP to lipopolysaccharide. GL13NH2 also inhibited lipopolysaccharide-stimulated secretion of tumor necrosis factor from macrophages. The other PSP peptides had no effect in this assay. PSP peptides had no or only minor effect on macrophage cell viability. These results indicate that PSP is a lipopolysaccharide-binding protein that is functionally related to LBP, as suggested by their predicted structural similarities.

Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein.

PSP (parotid secretory protein)/SPLUNC2 (short palate, lung and nasal epithelium clone 2) is expressed in human salivary glands and saliva. The protein exists as an N-glycosylated and non-glycosylated form and both appear to induce agglutination of bacteria, a major antibacterial function for salivary proteins. Both forms of PSP/SPLUNC2 bind LPS (lipopolysaccharide), suggesting that the protein may also play an anti-inflammatory role. Based on the predicted structure of PSP/SPLUNC2 and the location of known antibacterial and anti-inflammatory peptides in BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), we designed GL13NH2 and GL13K, synthetic peptides that capture these proposed functions of PSP/SPLUNC2. GL13NH3 agglutinates bacteria, leading to increased clearance by macrophages and reduced spread of infection in a plant model. GL13K kills bacteria with a minimal inhibitory concentration of 5-10 µg/ml, kills bacteria in biofilm and retains activity in 150 mM NaCl and 50% saliva. Both peptides block endotoxin action, but only GL13K appears to bind endotoxin. The peptides do not cause haemolysis, haemagglutination in serum, inhibit mammalian cell proliferation or induce an inflammatory response in macrophages. These results suggest that the GL13NH2 and the modified peptide GL13K capture the biological activity of PSP/SPLUNC2 and can serve as lead compounds for the development of novel antimicrobial and anti-inflammatory peptides.

Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal/permeability increasing-like protein family.

Parotid secretory protein (PSP) (SPLUNC2), a potential host-defense protein related to bactericidal/permeability-increasing protein (BPI), was used as a template to design antibacterial peptides. Based on the structure of BPI, new PSP peptides were designed and tested for antibacterial activity. The peptides did not exhibit significant bactericidal activity or inhibit growth but the peptide GL-13 induced bacterial matting, suggesting passive agglutination of bacteria. GL-13 was shown to agglutinate the Gram negative bacteria Pseudomonas aeruginosa and Aggregatibacter (Actinobacillus) actinomycetemcomitans, Gram positive Streptococcus gordonii and uncoated sheep erythrocytes. Bacterial agglutination was time and dose-dependent and involved hydrophobic interactions. Variant forms of GL-13 revealed that agglutination also depended on the number of amine groups on the peptide. GL-13 inhibited the adhesion of bacteria to plastic surfaces and the peptide prevented the spread of P. aeruginosa infection in a lettuce leaf model, suggesting that GL-13 is active in vivo. Moreover, GL-13-induced agglutination enhanced the phagocytosis of P. aeruginosa by RAW 264.7 macrophage cells. These results suggest that GL-13 represents a class of antimicrobial peptides, which do not directly kill bacteria but instead reduce bacterial adhesion and promote agglutination, leading to increased clearance by host phagocytic cells. Such peptides may cause less bacterial resistance than traditional antibiotic peptides.