Design, synthesis and biological evaluation of novel inhibitors of key enzymes in c-di-GMP metabolism as potential antibiofilm drugs

Bacterial biofilm is responsible for numerous chronic infections, which are characterized by persisting inflammation and tissue damage. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses, antibiotic therapy and disinfectant chemicals. 3',5'-Cyclic diguanylic acid (c-di-GMP) is a widely conserved second-messenger signal that regulates a wide range of functions including motility, synthesis of virulence factors, adhesion and biofilm formation. In particular, high levels of intracellular cyclic di-GMP promote biofilm formation and sessility, whereas low levels induce biofilm dispersion and motility. Since the pathways involved in c-di-GMP signaling are not present in mammalians, they represent attractive targets for the development of anti-virulence drugs. Synthesis of c-di-GMP occurs via diguanylate cyclase (DGC) enzymes, encoding GGDEF domains, while degradation of c-di-GMP occurs via phosphodiesterase (PDE) enzymes, encoding either an EAL or a HD-GYP domain. The identification of compounds reducing c-di-GMP levels in bacteria would allow the development of new antibiofilm drugs. In my PhD thesis, an array of c-di-GMP-based molecules has been synthesized and tested on both DGC and PDE enzymes, in particular RocR (PDE), WspR and YfiN (DGCs) from P. aeruginosa, and PleD (DGC) from C. crescentus. Three series of compounds have been rationally designed as linear c-di-GMP analogues using a molecular simplification strategy and a click chemistry method. In particular, a unique scaffold was identified, where two guanine bases and their derivatives were directly linked by a triazole moiety. The most potent inhibitor of the series resulted to inhibit PleD (89%, at 100 μM inhibitor) and RocR (59%, at 100 of μM inhibitor) with IC50 values of 17.5 +- 1.1 μM and 66.3 +- 1.3 μM, respectively. The novel inhibitors possess a number of properties that are attractive from a drug development point of view, compared with other c-di-GMP analogues. They are low-molecular-weight compounds, stable and easily synthesizable. More importantly, this study allowed us to identify the minimal structural determinants required for specifically targeting the I-site of DGCs. Although none of these compounds were able to inhibit biofilm formation in vivo, they could become potential tools for mechanicistic biochemical studies, in particular for hybrid protein which contains both the GGDEF and EAL domains. During my research experience at the Institute of Organic Chemistry at the Department of Chemistry of University of Hamburg (Germany), I was involved in a project comprising the stereoselective synthesis of new iso-carbocyclic nucleoside analogues for the further study of their bioactivity in the context of anti-HIV and anti-HCV inhibition. It is widely known that carbocyclic nucleoside analogues possess important biological advantages compared to natural nucleosides and they have received much attention as potential antiviral drugs over the last few decades. Carbovir, abacavir and carba-dT are examples of these efforts, resulting in efficient inhibitors of HIV's reverse transcriptase. A convergent approach was used for the synthesis, offering the formation of a variety of nucleoside analogues by the direct coupling of a chiral cyclopentanol with purine and pyrimidine nucleobases. These compounds are currently under investigation as potential antiviral drugs.