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. The challenges associated with biofilm infections have urged researchers to pursue a better understanding of the molecular mechanisms that are involved in the formation and dispersal of biofilms, and this has led to the identification of several steps that could be targeted in order to eradicate these challenging infections. Here we describe mechanisms that are involved in the regulation of biofilm development in Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii, and provide examples of chemical compounds that have been developed to specifically inhibit these processes. These compounds include (i) pilicides and curlicides which inhibit the initial steps of biofilm formation by E. coli; (ii) compounds that interfere with c-di-GMP signaling in P. aeruginosa and E. coli; and (iii) compounds that inhibit quorum-sensing in P. aeruginosa and A. baumannii. In cases where compound series have a defined molecular target, we focus on elucidating structure activity relationship (SAR) trends within the particular compound series.

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. This led to the identification of a small molecule that efficiently depletes P. aeruginosa for c-di-GMP, inhibits biofilm formation, and disperses established biofilm. A combination of our lead compound with standard of care antibiotics showed improved eradication of an implant-associated infection established in mice. Genetic analyses provided evidence that the anti-biofilm compound stimulates the activity of the c-di-GMP phosphodiesterase BifA in P. aeruginosa. Our work constitutes a proof of concept for c-di-GMP phosphodiesterase-activating drugs administered in combination with antibiotics as a viable treatment strategy for otherwise recalcitrant infections.

The use of nucleic acid, DNA and RNA, based strategies to disrupt gene expression as a therapeutic is quickly emerging. Indeed, synthetic oligonucleotides represent a major component of emerging gene therapeutics. However, the efficiency and specificity of intracellular uptake for non-modified oligonucleotides is rather poor. Utilizing RNA based oligonucleotides as therapeutics is even more challenging to deliver, due to extremely fast enzymatic degradation of the RNAs. Like unmodified oligonucleotides, RNAs also get rapidly degraded in vivo and demonstrate large off-target binding events when they can reach and enter the desired target cells. One approach that holds much promise is the utilization of “click chemistry” to conjugate receptor or cell specific targeting molecules directly to the effector oligonucleotides. We discuss here the applications of the breakthrough technology of CuAAC “click-chemistry” and the immense potential in utilizing “click chemistry” in the development of new age targeted oligonucleotide therapeutics.

Fast and reliable assay for amplification-free absolute quantification of DNA and RNA in biofluids.

Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 μm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major parameters that define the stability and the geometry of nucleic acid duplexes in biological samples. We also demonstrated how the fluorometry-based melting data could aid the design of a probe targeting a human BRAF gene fragment to reduce the off-target binding by a factor of seven.