Upscaling and Risk Evaluation of the Synthesis of the 3,5-Diamino-1H-Pyrazole, Disperazol.

This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound , as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA.

SAR study of 4-arylazo-3,5-diamino-1-pyrazoles: identification of small molecules that induce dispersal of biofilms.

By screening of a collection of 50 000 small-molecule compounds, we recently identified 4-arylazo-3,5-diamino-1-pyrazoles as a novel group of anti-biofilm agents. Here, we report a SAR study based on 60 analogues by examining ways in which the pharmacophore can be further optimized, for example, substitutions in the aryl ring. The SAR study revealed the very potent anti-biofilm compound 4-(2-(2-fluorophenyl)hydrazineylidene)-5-imino-4,5-dihydro-1-pyrazol-3-amine ().

Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms.

Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa.

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms.

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases.

Small Molecule Anti-biofilm Agents Developed on the Basis of Mechanistic Understanding of Biofilm Formation.

Microbial biofilms are the cause of persistent infections associated with various medical implants and distinct body sites such as the urinary tract, lungs, and wounds. Compared with their free living counterparts, bacteria in biofilms display a highly increased resistance to immune system activities and antibiotic treatment. Therefore, biofilm infections are difficult or impossible to treat with our current armory of antibiotics.

CDy14: a novel biofilm probe targeting exopolysaccharide Psl.

Detection of biofilm bacteria would be an ideal method for the physicians to diagnose chronic bacterial infections directly, but there are few imaging probes available so far. Here, we report the development of a novel biofilm detecting fluorescent probe, CDy14, through an unbiased screening of a fluorescence library and elucidated its binding partner Psl, an exopolysaccharide of the biofilm.

Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro.

The formation of biofilms in conjunction with quorum sensing (QS) regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential QS inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy.

Imaging N-Acyl Homoserine Lactone Quorum Sensing In Vivo.

In order to study N-acyl homoserine lactone (AHL)-based quorum sensing in vivo, we present a protocol using an Escherichia coli strain equipped with a luxR-based monitor system, which in the presence of exogenous AHL molecules expresses a green fluorescent protein (GFP). Lungs from mice challenged intratracheally with alginate beads containing both a Pseudomonas aeruginosa strain together with the E. coli monitor strain can be investigated at different time points postinfection.

Pseudomonas aeruginosa Biofilm Infections: Community Structure, Antimicrobial Tolerance and Immune Response.

Studies of biopsies from infectious sites, explanted tissue and medical devises have provided evidence that biofilms are the underlying cause of a variety of tissue-associated and implant-associated recalcitrant human infections. With a need for novel anti-biofilm treatment strategies, research in biofilm infection microbiology, biofilm formation mechanisms and biofilm-associated antimicrobial tolerance has become an important area in microbiology. Substantial knowledge about biofilm formation mechanisms, biofilm-associated antimicrobial tolerance and immune evasion mechanisms has been obtained through work with biofilms grown in in vitro experimental setups, and the relevance of this information in the context of chronic infections is being investigated by the use of animal models of infection.

In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a global secondary bacterial messenger that controls the formation of drug-resistant multicellular biofilms. Lowering the intracellular c-di-GMP content can disperse biofilms, and it is proposed as a biofilm eradication strategy. However, freshly dispersed biofilm cells exhibit a physiology distinct from biofilm and planktonic cells, and they might have a clinically relevant role in infections.